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the biogenesis of biological membranes that delineate the cell surface or act as intracellular space partition is a fundamental biological process and a remarkable evolutionary achievement . the hydrophobic barrier of membranes is comprised of fatty acid ( fa)-derived acyl - chains , typically 16 or 18 carbon atoms in length ; these fas are synthesized in a cyclic series of reactions by the multifunctional fa synthase ( fas ) complex by the addition of c2 units that are derived from malonyl - coenzyme a ( coa ) ( smith et al . , 2003 ) . remarkably , the fa chain length in membrane phospholipids ( pls ) is evolutionarily highly conserved ( daum et al . apparently provides maximum stability to the otherwise fluid bilayer structure through their hydrophobic interactions . at the same time , this acyl - chain composition permits rapid and significant adjustments by a single elongation step ( c16c18 ) and/or desaturation ( c16:0c16:1 ; c18:0c18:1 ) in response to alterations in environmental conditions such as temperature . furthermore , gradual increase in chain length and , subsequently , membrane thickness along the secretory pathway is an important determinant and provides sufficient differentiation for protein sorting ( bretscher and munro , 1993 ) . similar to mammalian cells , yeast membranes predominantly consist of mono- and di - unsaturated pls containing c16 and c18 acyl - chains to maintain the liquid crystalline state at physiological temperature ( de kroon et al . , 2013 ) . a decrease in the ambient temperature primarily triggers an increase in the c16/c18 ratio rather than a change in the degree of acyl - chain desaturation ( martin et al . , 2007 ) . the mechanisms that control the ratio between c16 versus c18 fas in cellular lipids are unknown . fas are synthesized de novo by the fas complex , which typically limits acyl - chain length extension to c16 and c18 carbon atoms , both in yeast and in mammals ( okuyama et al . , 1979 ; wakil et al . , 1983 ) ; fas are subsequently channeled through the common precursor phosphatidic acid ( pa ) into the synthesis of neutral and polar lipid species , i.e. , storage triacylglycerols ( tag ) and membrane pls , respectively . the substrate for the fas complex , malonyl - coa , is synthesized by the multifunctional enzyme , acetyl - coa carboxylase ( acc1 ) , the initial and rate - limiting step in cellular fa de novo synthesis in yeast ( al - feel et al . , 1992 ; chirala et al . , 1994 ; , 1993 ; woods et al . , 1994 ) and in mammals ( for a review , see wakil and abu - elheiga , 2009 ) . acc1 activity nutritional and metabolic signals , such as glucose limitation and salt stress , are transduced to acc1 by the snf1 kinase , the yeast ortholog of mammalian amp - activated protein kinase , ampk ( carling et al . , 1994 ; snf1 is the major energy - sensing kinase that phosphorylates and inactivates acc1 in vivo under conditions of low energy load ( woods et al . , 1994 ) . thus , yeast mutants lacking snf1 kinase display 3-fold elevated acc1 activity ; in addition , snf1 mutants are defective in transcription of multiple genes including ino1 , which encodes inositol-3-phosphate synthase and represents the most highly regulated gene of pl biosynthesis ( galdieri and vancura , 2012 ; jesch et al . , 2005 ; santiago and mamoun , 2003 ; shirra et al . , 2001 ) . inositol-3-phosphate synthase is indispensable for the endogenous production of inositol , which makes snf1 mutants dependent on inositol supplementation for growth ( ino phenotype ) . notably , transcription of acc1 and pl biosynthetic genes , including ino1 , is strongly regulated by inositol in the growth media , which is mediated by the uasino element in the promoter of these genes ( carman and han , 2009 ; henry et al . , 2012 , 2014 ) . full activation of ino1 transcription occurs in the absence of inositol and requires the ino2/ino4 transcriptional activator complex that binds to the uasino element ( ambroziak and henry , 1994 ; lopes and henry , 1991 ) . uasino - containing genes are repressed by opi1 ( white et al . , 1991 ) by its interaction with ino2 in the nucleus ( wagner et al . , 2001 ) . opi1 is a pa - binding protein and shuttles between the endoplasmic reticulum ( er ) and the nucleus , dependent on the level of pa and the presence of the yeast vamp - associated protein ( vap ) homolog scs2 , in the er . in the absence of inositol , pa levels are high , opi1 is bound to the er , and transcription of ino1 is enabled , leading to de novo production of inositol . in contrast , supplementation of inositol to the growth medium depletes pa by draining it into phosphatidylinositol ( pi ) synthesis , which promotes translocation of opi1 into the nucleus and repression of the ino1 gene ( loewen et al . , 2004 ; pa is a central lipid intermediate and precursor both for membrane pl and storage tag synthesis ; however , it is currently unclear how the metabolic flux either way is regulated in growing cells . defects in pl metabolism that lead to an increased steady - state concentration of pa in the er cause sequestration of the opi1 repressor to the er and elevated transcription of uasino - dependent genes . the cho2 and opi3 mutants , for instance , are defective in pl methylation and , thus , accumulate pa and overexpress ino1 leading to overproduction and excretion of inositol into the growth medium ( opi phenotype ; greenberg et al . , 1982 ; we previously showed that the inositol requirement of the snf1 mutant is suppressed by downregulation of acc1 , suggesting that its activity might be connected to the ino2/ino4/opi1 regulatory circuit ( shirra et al . however , due to the pleiotropic defects of the snf1 mutant , other regulatory mechanisms could not be ruled out . for instance , lack of ino1 expression in the snf1 mutant was suggested to be a result of defective chromatin modification ( lo et al . , 2001 ) . to uncouple acc1 activity from snf1-dependent phosphorylation , we have now characterized and mutated the specific phosphorylation site at position serine 1157 of acc1 . we provide evidence for a direct regulatory function of fa de novo synthesis in the transcriptional regulation of pl biosynthesis through the ino2/ino4/opi1 regulatory circuit . we show that acc1 activity is a critical determinant of the c16/c18 acyl - chain ratio in cellular lipids ; in conjunction with the specific binding preference of the opi1 repressor for c16-pa species , this describes a regulatory mechanism of gene transcription in response to altered acyl - chain length in cellular lipids and unveils a function of the snf1/amp - activated protein kinase in regulating lipid homeostasis . a large - scale phosphoproteome analysis identified a single phosphorylation site at serine 1157 of acc1 that is embedded in a mnravsvsdls sequence and matches the ampk consensus phosphorylation motif , r / k - x - x - s ( brinkworth et al . , 2006 ; dale et al . , 1995 ) . using avidin affinity - purified acc1 protein , we characterized and confirmed the phosphorylation site by qualitative and quantitative mass spectrometry analyses ( figure 1 ; figure s1 available online ; see supplemental experimental procedures for a detailed description ) . in wild - type , about 63% of the acc1 protein was phosphorylated at serine 1157 , suggesting that the majority of the enzyme is rather inactive in logarithmically growing yeast cells . however , in snf1 cells , the phosphorylation level at this residue in the acc1 protein decreased by half to about 35% , confirming serine 1157 as the target site of snf1 phosphorylation . alternative phosphorylation of serine 1159 , which was recently predicted to be a potential phosphorylation site ( oliveira et al . , 2012 ) , or phosphorylation of any other sites was not detected in our phosphoproteome analyses of log - phase yeast cells . to assess its in vivo function , we mutated serine 1157 to an alanine residue , thus locking the acc1 enzyme in a nonphosphorylatable state . as shown in figure 2a , the acc1 mutant , generated in this fashion , grew like wild - type on complete yeast extract peptone dextrose ( ypd ) media , but similarly to the snf1 mutant , it was resistant to the acc1-specific inhibitor , soraphen a ( sora ) ( shen et al . , 2004 ; vahlensieck et al . , in addition , acc1 mutants , like the snf1 mutant strain , were unable to grow on media lacking inositol ( i medium ) . this inositol auxotrophy ( ino phenotype ) could be suppressed either by decreasing acc1 activity with sora ( figure 2a ) or in the snf1 mutant by downregulation of acc1 transcription under control of the heterologous doxycycline - repressible tet07 promoter ( teto7-acc1 ; figure s2a ) . yeast snf1 mutants were originally identified by their inability to utilize exogenous sucrose ( snf , sucrose nonfermenting phenotype ) , resulting from their failure to express the suc2 gene ( encoding invertase ) on glucose depletion ( carlson et al . , 1981 ) . notably , the acc1 mutant was capable of growing on sucrose as the sole carbon source or on nonfermentable carbon sources like glycerol or ethanol , which distinguishes its phenotype from that of snf1 mutants with respect to carbon source utilization ( figures s2b and s2c ) . acc1 hyperactivity in both snf1 and acc1 mutants leads to highly elevated fa production and massive accumulation of tag and lipid droplets during logarithmic growth ( figures 2b and 2c ) ; these phenotypes are even more pronounced in stationary phase ( figure s2d ) . most notably , as a result of elevated malonyl - coa production by hyperactive acc1 in the acc1 mutant , fa acyl - chain distribution was significantly shifted toward longer acyl - chain lengths , increasing the c18:c16 acyl - chain ratio from 0.5 in wild - type to 2.0 in the acc1 mutant ( figure 2d ) . this shift is also reflected in the altered acyl - chain composition of virtually all glycerolipid classes for instance , tag ( figure s2e ) . taken together , the acc1 mutant displays similar phenotypes as the snf1 mutant with respect to fa overproduction , a shift to longer acyl - chains , and tag accumulation , as well as inositol auxotrophy . most notably , growth on nonfermentable carbon sources clearly distinguishes the acc1 mutant from the pleiotropic phenotypes that are associated with deletion of snf1 and uncouples acc1 activity from other roles of snf1 kinase in energy and carbon metabolism . the observed growth defect of acc1 mutants on solid media lacking inositol was reproduced in liquid cultures . when cultivated in the presence of inositol ( + i medium ) , the acc1 mutant grew at a rate identical to that of wild - type cells ( figure 3a ; figure s3a ) . after the shift to inositol - free medium , only a slightly reduced growth rate of the acc1 mutant strain was observed during the first 3 hr compared to wild - type , most likely due to intracellular inositol pools that are consumed during that period ( figure 3a ; see also figure s3b for detailed inositol titration experiment ) . however , a dramatic increase in doubling time of the acc1 mutant occurred between 3 and 6 hr after the shift to inositol - free medium ( figure 3a ) . notably , cells eventually reached wild - type - like final cell density after 20 hr , suggesting that cells are impaired in growth but remain viable ( figure s3a ) . this reduced growth of the acc1 mutant in the absence of inositol was cured by inhibition of acc1 by sora , clearly linking the requirement for inositol supplementation to acc1 activity . wild - type cells treated with the same concentration of sora , on the other hand , exhibited a severe growth defect in both the presence and absence of inositol , which is consistent with the essential role of acc1 in cellular metabolism ( figure 3a ) . we next asked whether the poor growth rate of the acc1 mutant in the absence of exogenous inositol was indeed associated with a reduced de novo production of that essential metabolite , i.e. , at the level of transcription of the ino1 gene . in wild - type , as expected , ino1 transcription was highly induced 3 hr following the shift to inositol - free medium ; in contrast , expression of ino1 was significantly attenuated in the acc1 mutant strain . consistent with the observed restoration of growth in the absence of inositol , inhibition of acc1 in the acc1 mutant by sora also restored wild - type - like ino1 expression kinetics ( figure 3b ) . these data support the notion that lack of growth of the acc1 ( and snf1 ) mutant on media lacking inositol is linked to a defect in ino1 expression as a consequence of acc1 hyperactivity . indeed , overexpression of ino1 from a high - copy - number plasmid or deletion of the opi1 gene fully restored growth of the acc1 strain in the absence of inositol ( figure 3c and figure s3c ) . in addition , downregulation of acc1 gene expression in the teto7acc1 strain by 0.53.0 g / ml doxycycline led to overproduction and secretion of inositol ( opi phenotype ; figure s4a ) , clearly demonstrating the inverse correlation between acc1 activity and the ability to express ino1 ( figure s4b ) . full repression of acc1 transcription ( > 5.0 g / ml doxycycline ) led to cell death of the teto7-acc1 strain , further supporting the essential function of acc1 . from previous studies , it is known that ino1 expression responds to the localization of opi1 , the transcription factor that senses the levels of the central lipid intermediate , pa , in the er ( loewen et al . , 2004 ) . consistent with the current model , opi1-green fluorescent protein ( gfp ) translocated in wild - type cells from the nucleus to the nuclear and peripheral er within 3 hr in inositol - free medium , leading to the observed rapid ino1 induction . in marked contrast , the acc1 mutant retained nuclear localization of opi1-gfp after the shift to inositol - free medium ( figure 3d ) ; this defect in opi1-gfp export from the nucleus parallels the reduced ino1 expression kinetics . notably , inhibition of acc1 activity in the acc1 mutant readily promoted export of opi1-gfp from the nucleus , leading to the observed relief of ino1 expression ( figures 3b and 3d ) . taken together , these analyses demonstrate that this growth deficit of the acc1 mutant is due to impaired ino1 expression through the ino2/ino4/opi1 regulatory circuit . since opi1 localization responds to the amount of pa in the er ( loewen et al . , 2004 ) , we hypothesized that overproduction of fa might lead to reduced cellular pa levels , perhaps due to its increased channeling into tag synthesis . this , however , was not the case : analysis of the lipid profile in the acc1 mutant showed that total glycerophospholipid content remained almost unaltered compared to wild - type . diacylglycerol ( dag ) and tag levels were raised about 3- to 4-fold , which was reversed by sora treatment within 4.5 hr ( figure 4 ) . thus , elevated de novo synthesized fas are preferentially channeled into tag synthesis , without affecting total pl content . we next analyzed to what extent cellular pa content was altered as a result of hyperactive acc1 in the acc1 mutant . surprisingly , the total pa pool was not decreased but rather slightly increased compared to wild - type ( figure 5 ) . , that reduced pa levels are responsible for translocation of opi1 from the er into the nucleus to repress ino2/ino4-dependent ino1 transcription ( loewen et al . , 2004 ) . since the molecular species distribution in all glycerolipids displayed a characteristic shift toward increased acyl - chain length in acc1 , we hypothesized that not the total amount of pa but rather its specific molecular acyl - chain composition may define its binding affinity to opi1 . thus , we next analyzed the molecular species composition of pa ( and of the major cellular pl phosphatidylcholine [ pc ] as a reference ) in wild - type and acc1 strains in greater detail . as shown in figure 5 , pa 32:1 ( containing c16:0 and c16:1 acyl - chains ) and pa 32:2 ( containing two c16:1 acyl - chains ) species were indeed greatly reduced in the acc1 mutant , whereas pa 36:1 ( containing c18:0 and c18:1 acyl - chains ) and pa 36:2 ( containing two c18:1 acyl - chains ) species were increased . this shift in acyl - chain composition was reversed by the addition of sora , a condition that also led to inositol prototrophy ( figures 2a and 3a ) , derepression of ino1 ( figure 3b ) , and nuclear export of opi1-gfp ( figure 3d ) in the acc1 strain . based on the hypothesis that an increase in the proportion of c18 versus c16 acyl - chains in pa is responsible for nuclear localization of opi1 , we next tested whether exogenous c18:1 supply and concomitant changes in the acyl - chain composition would phenocopy the effects of the hyperactive acc1 allele also in wild - type cells . as shown in figure 6a , addition of c18:1 indeed induced inositol auxotrophy in wild - type cells . this phenotype was not observed in the presence of palmitoleic acid ( c16:1 ) and , thus , can not be due to a general lipotoxic effect of supplied fa on inositol - free media . similarly , the ole1 mutant , which lacks the sole fa desaturase in yeast ( stukey et al . , 1989 ) , and which is dependent on unsaturated fa supplementation , displayed a strict inositol auxotrophy in the presence of c18:1 . notably , growth of wild - type , ole1 , and acc1 strains on media lacking inositol was fully rescued by ( co)supplementation with c16:1 , indicating that this fa reversed the inositol auxotrophy induced by excess c18:1 fa . this rescue of the acc1 mutant growth was not due to repression of acc1 activity by c16:1 , since total c18 fas remained at a high level , characteristic for acc1 hyperactivity ; rather , the ratio between c16 and c18 fas was restored by c16:1 supplementation ( figure 6b ) . the analysis of pa species in wild - type and ole1 strains supplemented with c18:1 showed a similar shift toward longer acyl - chain lengths as in the acc1 strain as compared to wild - type grown without c18:1 ( figure 6c ) : pa 32:1 and pa 32:2 species were significantly decreased , whereas pa species with longer fa chain length increased , especially 36:2 . to further confirm whether the acyl - chain composition of pa affects the interaction with opi1 , we performed in vitro binding assays with pc 36:2 liposomes as a matrix , containing 20 mol% of different pa species . indeed and in line with our hypothesis , we found significantly different binding affinities of opi1 for pa species dependent on the acyl - chain composition . liposomes containing saturated pa species ( pa 32:0 and pa 36:0 ) showed the highest affinity , followed by liposomes containing 32:1 pa ; liposomes containing pa 36:2 showed the lowest binding affinity for purified opi1 . liposomes , which consisted solely of pc 36:2 , showed no opi1 binding at all ( figure 7 ) . these results clearly demonstrate that the acyl - chain composition of pa is a major determinant of its interaction with the transcriptional repressor opi1 . this preferred binding affinity explains the observed phenotypes and significantly extends the current model of the transcriptional regulation of pl biosynthesis in yeast in response to deregulated fa de novo synthesis . throughout the eukaryotic kingdom , fas of 16 or 18 carbon atoms in length are the preferred acyl components in all glycerolipids that serve as bulk lipids in biological membranes or as storage lipids . whereas mechanisms controlling the level of fa desaturation in response to ambient temperature and membrane fluidity have been described in detail , much less is known about the mechanisms regulating chain length distribution ( de kroon et al . , 2013 ) . in addition to their fundamental roles as components of biological membranes , fas also serve essential functions as energy substrates , signaling molecules , or protein modifiers . cellular fa synthesis is also coordinated with nutritional supply , activation , and incorporation into complex lipids to support proper cell function and to prevent potentially detrimental fa - induced lipotoxic effects , which are believed to be causative for lipid - associated disorders , such as obesity and type 2 diabetes in humans ( kim and ye , 2013 ; unger , 2002 ; vidal - puig and unger , 2010 ) . thus , endogenous de novo synthesis of particular fas also plays an important role to counterbalance the physiological impact of nutritional fas that may be incorporated into cellular lipids . according to their central role in cellular lipid synthesis , both acc1 and fas have emerged as potential drug targets in obesity - related diseases , as well as in cancer , which are characterized by deregulated fa and lipid metabolism ( wakil and abu - elheiga , 2009 ; furuta et al . , 2010 ; pandey et al . , 2012 ; wang et al . , 2010 ; liu et al . , 2010 ; natter and kohlwein , 2013 ) . in yeast and mammalian cells , acyl - chain length is largely determined by the fas complex that limits acyl - chain length to 16 and 18 carbon atoms ( okuyama et al . , 1979 ) . we now show that the activity of acc1 , the initial and rate - limiting enzyme of de novo fa synthesis , is a major determinant of total cellular fa production and also has a fundamental impact on the relative distribution of c16 and c18 acyl - chains in cellular lipids . overproduction of fas leads to the accumulation of tag and proliferation of cytosolic lipid droplets , whereas membrane pl content remains unaltered . on the other hand , the shift in acyl - chains toward longer residues is reflected in all cellular glycerolipids , with consequences for the binding and sequestration of the transcriptional repressor opi1 to the er . the synthesis of fas is a major energy- and carbon - consuming pathway and , therefore , under several levels of control . one level involves the expression of the acc1 gene , which is coordinately regulated with pl synthesis by the pl precursors , inositol and choline , and the positive and negative transcription factors ino2/ino4 and opi1 , respectively ( hasslacher et al . , 1993 ; henry et al . , the regulation of acc1 expression in coordination with pl biosynthetic genes ( for review , see tehlivets et al . , 2007 ) suggests that fa production must also be balanced with tag formation in growing cells in order to sustain requirements for pl and membrane biogenesis . in addition , the activity of acc1 is strongly regulated by snf1-dependent phosphorylation ( mitchelhill et al . , 1994 ; woods et al . , 1994 ) , which , in turn , depends on the energy status of the cell ( wilson et al . , 1996 ) . here , we made use of a yeast mutant that lacks the single snf1 phosphorylation site in acc1 at serine 1157 ( acc1 ) and exhibits phenotypes very similar to snf1 mutants with respect to tag and lipid droplet accumulation as well as inositol auxotrophy , demonstrating that these snf1 phenotypes are caused by acc1 hyperactivity . the evidence we present here strongly supports the hypothesis that the inositol - requiring phenotype of the acc1 strain is due to decreased ino1 expression that is controlled by the well - characterized ino2/ino4/opi1regulatory circuit ( henry et al . , 2012 ; loewen et al . , 2004 ) , as both deletion of opi1 , which uncouples transcriptional repression of ino1 from cellular pa levels , and overexpression of ino1 fully restored inositol prototrophy to the acc1 mutant strain . we further suggest that pl synthesis may be limited by the coordinated repression of the uasino - containing pl biosynthetic genes in the presence of excess fa . the observed opi1 translocation to the nucleus concomitantly slows cell growth as pl and inositol production become limiting due to pa diversion into tag accumulation . most notably , neither increased endogenous fa production nor exogenous fa supply led to increased pl accumulation , despite the fact that an altered acyl - chain composition is reflected in all pl classes ; instead , excess fas are preferentially channeled into tag . in addition , the similarity of the cellular response to these two situations suggests that functional tag synthesis is crucial for accommodating these fas , thus avoiding the buildup of potential signal molecules , such as pa or dag , or free fas ( garbarino and sturley , 2009 ; kohlwein , 2010 ; kohlwein and petschnigg , 2007 ; petschnigg et al . , 2009 ; fakas et al . , importantly , our evidence significantly extends the current model that indeed the acyl - chain composition of pa is the critical determinant of opi1 sequestration to the er . we show that alterations in the acyl - chain composition of the acc1 strain or of wild - type and the ole1 mutant in the presence of oleic acid lead to inositol auxotrophy due to the reduced binding affinity of the opi1 repressor to pa molecular species containing longer acyl residues . we hypothesize that the recognition of the negatively charged head group of pa by the basic tract of opi1 might only represent the first level of interaction , whereas the second level is presumably determined by sensing the nature of the acyl - chains of pa within the er membrane . several other studies also support the importance of acyl - chain composition of pa for interaction with proteins ( kooijman and burger , 2009 ) ; for example , the binding specificity of human protein phosphatase 1 ( pp1c ) to pa species harboring a higher degree of acyl - chain unsaturation ( jones and hannun , 2002 ) . however , no consensus sequence motif for pa binding proteins has been identified yet ( stace and ktistakis , 2006 ) . it should be noted that the acidic pls pa and pi are exceptional regarding their membrane activity compared to other pls with respect to impact on membrane packing , electrostatics , and membrane curvature ( bigay and antonny , 2012 ) . pa is the only anionic pl with a pronounced cone shape , facilitating protein penetration into the membrane bilayer and recognition of the acyl moieties ( kooijman et al . . the actual concentration of pa in the yeast er membrane is not well defined . previous studies reported pa levels between 0.5% and 3% of total pls in isolated microsomal fractions ( zinser et al . , 1991 ; pichler et al . , 2001 ) , whereas a recent shotgun lipidomics study found about 10% pa of total lipids in whole cell extracts ( klose et al . these findings indicate that the short - lived intermediate pa may be metabolized during cell fractionation and may indeed be present in higher amounts than previously suggested . on the other hand , sole variation of the acyl - chain composition of pa in the liposome binding assay eliminated influences of membrane electrostatics and curvature as well as of proteins , such as the yeast vap scs2 , which is required for binding of opi1 to pa in vivo ( loewen et al . , 2004 ) . ( 1 ) currently , the mechanisms that regulate the total amount of cellular pls and their specific acyl - chain composition in response to altered fa supply are largely unclear . a mechanism , as described here , that responds not only to the content of critical lipid intermediates such as pa but also to its specific molecular composition provides an attractive regulatory circuit to integrate metabolic input parameters , such as availability of nutritional fas . ( 2 ) fa synthesis itself is under several levels of control and has a profound impact on metabolic and transcriptional regulation . ( 3 ) the major and highly conserved energy - sensing snf1 kinase operates through additional levels of regulation , namely , its metabolic downstream target acc1 . in conclusion , we demonstrate that acc1 hyperactivity leads to overproduction of fas and a shift in acyl - chain length distribution toward longer chains in all pl classes as well as in tag . the acyl - chain distribution of pa is critical for its interaction with the opi1 repressor , which displays reduced affinity for pa molecular species containing longer and more unsaturated acyl - chains . in the context of the snf1 mutant scenario , acc1 hyperactivity is a potent player in sequestering carbon into fat production and regulating cell growth and proliferation . this unveils an impact of the major energy - sensing kinase on membrane lipid composition and function and may explain some of the seemingly unrelated pleiotropic consequences of its deficiency . s. cerevisiae strains used in this study , except for the aid strain , are congenic with by , a derivative of s288c , and are listed in the supplemental experimental procedures . for liquid media experiments , cells were cultivated on a rotary shaker at 30c and 180 rpm using threonine - free minimal synthetic defined media with ( + i ) or without ( i ) 75 m inositol ( villa - garca et al . , 2011 ) . solid ypd ( 1% yeast extract , 2% peptone and 2% glucose , 2% agar ) and yeast extract peptone ( 1% yeast extract , 2% peptone , 2% agar ) with 2% of the nonfermentable carbon sources sucrose , glycerol , ethanol , or 1% glycerol and 1% ethanol were additionally used for plate tests . cell cultivation if not described differently was performed as follows ; a single colony of the desired strains was taken from a ypd plate and grown for 16 hr in + i medium ( 50 ml ) to midlogarithmic growth phase , rediluted to optical density 600 ( od600 ) = 0.1/ml in 100 ml of fresh + i medium , and grown for an additional 4.5 hr maintaining midlogarithmical growth . a cell aliquot of od600 = 20 was then shifted by filtration ( pore size , 0.45 m ) to 40 ml of fresh + i and i media , respectively , resulting in an initial od600 = 0.5/ml ( 0 hr time point ) . cell growth was monitored during growth , and doubling times were determined from the od600 readings as described elsewhere ( gaspar et al . , 2011 ) . for cultivation of the fa auxotrophic ole1 strain , the media for the 16 hr precultivation and the 4.5 hr growth after redilution contained 0.01% oleic acid and 1% tergitol . additionally , 20 ml of a 1 m stock solution of mes , ph 6.0 , was added per liter medium , resulting in an initial ph of 5.7 . cells were then shifted to fresh i media containing 20 mm mes with 0.01% oleic acid and 1% tergitol or with solely 1% tergitol , as a control , and grown for 6 hr . acc1 activity was inhibited in vivo by adding 1 g / ml sora to the culture medium where indicated : sora is highly specific for the eukaryotic form of acetyl - coa carboxylase , and by its binding to the biotin carboxylase dimer interface , it inhibits oligomerization , which is required for enzyme activity ( shen et al . , 2004 ) . for plate tests , a single colony of the desired strains was taken from a ypd plate and grown overnight in ypd medium ( for the ole1 strain , 0.01% oleic acid was supplemented ) to reach late - logarithmic growth phase . a cell aliquot of od600 = 10 was harvested and washed twice with sterile water . five microliters of serial 1:10 dilutions starting with od600 = 1/ml were spotted on to the indicated media plates . plates were analyzed after 2 days of growth at 30c . to determine overproduction and excretion of inositol ( opi phenotype ) , 5 l od600 ( 1 aliquot of wild - type , teto7acc1 , and the control strains opi1 and cho2 ) were spotted on i medium , containing the indicated concentration of doxycycline . cells were grown for 2 days at 30c and sprayed with a suspension of the inositol auxotrophic ( ino ) tester strain ( aid ) , which grows as a halo only around strains excreting inositol . for microscopic analysis , cultures were grown as described above and analyzed 3 hr after the shift or in stationary phase . labeling of neutral lipids with bodipy 493/503 or nile red was performed by adding the fluorescence dye directly to the cell culture for 10 min at room temperature in the dark ( final concentration , 1 g / ml ) . cells were grown as described above , and a cell aliquot of od600 = 20 was harvested at the indicated time points after the media shift . lipids were extracted according to a slightly modified folch method ( folch et al . , 1957 ; schneiter and daum , 2006 ) and analyzed on a uplc - synapt qtof hdms system ( waters ) , as described by knittelfelder et al . an aliquot of the lipid extract was used for methyl ester production and gas chromatography - mass spectrometry measurements . refer to supplemental experimental procedures for lipid extraction , chromatography , and mass spectrometry parameters . cultures were grown as described above and harvested 1.5 hr and 3 hr after the shift . real - time pcr was conducted as described elsewhere ( gaspar et al . , 2011 ) . ino1 values were normalized to wild - type grown on + i media for 1.5 hr following the shift ( ino1-repressing conditions ) . refer to the supplemental experimental procedures for rna isolation , reverse transcription , and real - time pcr parameters . for opi1 binding assays , 150 l of liposome suspension and 150 l glutathione s - transferase ( gst)-opi1 were incubated for 30 min at 22c and centrifuged at 30,000 rpm for 10 min at 22c in an optima tlx ultracentrifuge using a tla-100.3 fixed angle rotor ( beckmann ) . pellets were washed once with elution buffer without glutathione , resuspended in 150 l elution buffer without glutathione , and precipitated with 600 l acetone at 20c overnight . additionally , 150 l gst - opi1 were precipitated with 600 l acetone at 20c overnight as a loading control . precipitated gst - opi1 protein was pelleted at 13,200 rpm for 10 min at 4c in an eppendorf centrifuge ( type 5415r ) . pellets were washed once in 600 l acetone and dissolved in protein loading buffer for sds - page . for detection of gst - opi1 fusion protein , western blot analysis was performed using an anti - gst antibody from goat ( sigma - aldrich , 1:10.000 ) and an anti - goat antibody conjugated with horseradish peroxidase ( pierce , 1:7.500 ) for detection with supersignal west pico chemiluminescent substrate ( thermo scientific ) . additional experimental information in expanded form on acc1 in vitro mutagenesis , phospho - mass spectrometry , fluorescence microscopy , lipid analytics , ino1 expression , opi1 overexpression and purification , liposome studies , and pa 32:1 synthesis is available in the supplemental experimental procedures .

summarymembrane phospholipids typically contain fatty acids ( fas ) of 16 and 18 carbon atoms . this particular chain length is evolutionarily highly conserved and presumably provides maximum stability and dynamic properties to biological membranes in response to nutritional or environmental cues . here , we show that the relative proportion of c16 versus c18 fas is regulated by the activity of acetyl - coa carboxylase ( acc1 ) , the first and rate - limiting enzyme of fa de novo synthesis . acc1 activity is attenuated by ampk / snf1-dependent phosphorylation , which is required to maintain an appropriate acyl - chain length distribution . moreover , we find that the transcriptional repressor opi1 preferentially binds to c16 over c18 phosphatidic acid ( pa ) species : thus , c16-chain containing pa sequesters opi1 more effectively to the er , enabling ampk / snf1 control of pa acyl - chain length to determine the degree of derepression of opi1 target genes . these findings reveal an unexpected regulatory link between the major energy - sensing kinase , membrane lipid composition , and transcription .

Introduction Results Discussion Experimental Procedures

the hydrophobic barrier of membranes is comprised of fatty acid ( fa)-derived acyl - chains , typically 16 or 18 carbon atoms in length ; these fas are synthesized in a cyclic series of reactions by the multifunctional fa synthase ( fas ) complex by the addition of c2 units that are derived from malonyl - coenzyme a ( coa ) ( smith et al . remarkably , the fa chain length in membrane phospholipids ( pls ) is evolutionarily highly conserved ( daum et al . apparently provides maximum stability to the otherwise fluid bilayer structure through their hydrophobic interactions . at the same time , this acyl - chain composition permits rapid and significant adjustments by a single elongation step ( c16c18 ) and/or desaturation ( c16:0c16:1 ; c18:0c18:1 ) in response to alterations in environmental conditions such as temperature . similar to mammalian cells , yeast membranes predominantly consist of mono- and di - unsaturated pls containing c16 and c18 acyl - chains to maintain the liquid crystalline state at physiological temperature ( de kroon et al . a decrease in the ambient temperature primarily triggers an increase in the c16/c18 ratio rather than a change in the degree of acyl - chain desaturation ( martin et al . the mechanisms that control the ratio between c16 versus c18 fas in cellular lipids are unknown . fas are synthesized de novo by the fas complex , which typically limits acyl - chain length extension to c16 and c18 carbon atoms , both in yeast and in mammals ( okuyama et al . , 1983 ) ; fas are subsequently channeled through the common precursor phosphatidic acid ( pa ) into the synthesis of neutral and polar lipid species , i.e. the substrate for the fas complex , malonyl - coa , is synthesized by the multifunctional enzyme , acetyl - coa carboxylase ( acc1 ) , the initial and rate - limiting step in cellular fa de novo synthesis in yeast ( al - feel et al . acc1 activity nutritional and metabolic signals , such as glucose limitation and salt stress , are transduced to acc1 by the snf1 kinase , the yeast ortholog of mammalian amp - activated protein kinase , ampk ( carling et al . , 1994 ; snf1 is the major energy - sensing kinase that phosphorylates and inactivates acc1 in vivo under conditions of low energy load ( woods et al . thus , yeast mutants lacking snf1 kinase display 3-fold elevated acc1 activity ; in addition , snf1 mutants are defective in transcription of multiple genes including ino1 , which encodes inositol-3-phosphate synthase and represents the most highly regulated gene of pl biosynthesis ( galdieri and vancura , 2012 ; jesch et al . notably , transcription of acc1 and pl biosynthetic genes , including ino1 , is strongly regulated by inositol in the growth media , which is mediated by the uasino element in the promoter of these genes ( carman and han , 2009 ; henry et al . full activation of ino1 transcription occurs in the absence of inositol and requires the ino2/ino4 transcriptional activator complex that binds to the uasino element ( ambroziak and henry , 1994 ; lopes and henry , 1991 ) . opi1 is a pa - binding protein and shuttles between the endoplasmic reticulum ( er ) and the nucleus , dependent on the level of pa and the presence of the yeast vamp - associated protein ( vap ) homolog scs2 , in the er . in the absence of inositol , pa levels are high , opi1 is bound to the er , and transcription of ino1 is enabled , leading to de novo production of inositol . in contrast , supplementation of inositol to the growth medium depletes pa by draining it into phosphatidylinositol ( pi ) synthesis , which promotes translocation of opi1 into the nucleus and repression of the ino1 gene ( loewen et al . defects in pl metabolism that lead to an increased steady - state concentration of pa in the er cause sequestration of the opi1 repressor to the er and elevated transcription of uasino - dependent genes . , 1982 ; we previously showed that the inositol requirement of the snf1 mutant is suppressed by downregulation of acc1 , suggesting that its activity might be connected to the ino2/ino4/opi1 regulatory circuit ( shirra et al . to uncouple acc1 activity from snf1-dependent phosphorylation , we have now characterized and mutated the specific phosphorylation site at position serine 1157 of acc1 . we provide evidence for a direct regulatory function of fa de novo synthesis in the transcriptional regulation of pl biosynthesis through the ino2/ino4/opi1 regulatory circuit . we show that acc1 activity is a critical determinant of the c16/c18 acyl - chain ratio in cellular lipids ; in conjunction with the specific binding preference of the opi1 repressor for c16-pa species , this describes a regulatory mechanism of gene transcription in response to altered acyl - chain length in cellular lipids and unveils a function of the snf1/amp - activated protein kinase in regulating lipid homeostasis . as shown in figure 2a , the acc1 mutant , generated in this fashion , grew like wild - type on complete yeast extract peptone dextrose ( ypd ) media , but similarly to the snf1 mutant , it was resistant to the acc1-specific inhibitor , soraphen a ( sora ) ( shen et al . this inositol auxotrophy ( ino phenotype ) could be suppressed either by decreasing acc1 activity with sora ( figure 2a ) or in the snf1 mutant by downregulation of acc1 transcription under control of the heterologous doxycycline - repressible tet07 promoter ( teto7-acc1 ; figure s2a ) . notably , the acc1 mutant was capable of growing on sucrose as the sole carbon source or on nonfermentable carbon sources like glycerol or ethanol , which distinguishes its phenotype from that of snf1 mutants with respect to carbon source utilization ( figures s2b and s2c ) . most notably , as a result of elevated malonyl - coa production by hyperactive acc1 in the acc1 mutant , fa acyl - chain distribution was significantly shifted toward longer acyl - chain lengths , increasing the c18:c16 acyl - chain ratio from 0.5 in wild - type to 2.0 in the acc1 mutant ( figure 2d ) . this shift is also reflected in the altered acyl - chain composition of virtually all glycerolipid classes for instance , tag ( figure s2e ) . taken together , the acc1 mutant displays similar phenotypes as the snf1 mutant with respect to fa overproduction , a shift to longer acyl - chains , and tag accumulation , as well as inositol auxotrophy . wild - type cells treated with the same concentration of sora , on the other hand , exhibited a severe growth defect in both the presence and absence of inositol , which is consistent with the essential role of acc1 in cellular metabolism ( figure 3a ) . from previous studies , it is known that ino1 expression responds to the localization of opi1 , the transcription factor that senses the levels of the central lipid intermediate , pa , in the er ( loewen et al . notably , inhibition of acc1 activity in the acc1 mutant readily promoted export of opi1-gfp from the nucleus , leading to the observed relief of ino1 expression ( figures 3b and 3d ) . since opi1 localization responds to the amount of pa in the er ( loewen et al . , 2004 ) , we hypothesized that overproduction of fa might lead to reduced cellular pa levels , perhaps due to its increased channeling into tag synthesis . , that reduced pa levels are responsible for translocation of opi1 from the er into the nucleus to repress ino2/ino4-dependent ino1 transcription ( loewen et al . since the molecular species distribution in all glycerolipids displayed a characteristic shift toward increased acyl - chain length in acc1 , we hypothesized that not the total amount of pa but rather its specific molecular acyl - chain composition may define its binding affinity to opi1 . thus , we next analyzed the molecular species composition of pa ( and of the major cellular pl phosphatidylcholine [ pc ] as a reference ) in wild - type and acc1 strains in greater detail . as shown in figure 5 , pa 32:1 ( containing c16:0 and c16:1 acyl - chains ) and pa 32:2 ( containing two c16:1 acyl - chains ) species were indeed greatly reduced in the acc1 mutant , whereas pa 36:1 ( containing c18:0 and c18:1 acyl - chains ) and pa 36:2 ( containing two c18:1 acyl - chains ) species were increased . this shift in acyl - chain composition was reversed by the addition of sora , a condition that also led to inositol prototrophy ( figures 2a and 3a ) , derepression of ino1 ( figure 3b ) , and nuclear export of opi1-gfp ( figure 3d ) in the acc1 strain . based on the hypothesis that an increase in the proportion of c18 versus c16 acyl - chains in pa is responsible for nuclear localization of opi1 , we next tested whether exogenous c18:1 supply and concomitant changes in the acyl - chain composition would phenocopy the effects of the hyperactive acc1 allele also in wild - type cells . similarly , the ole1 mutant , which lacks the sole fa desaturase in yeast ( stukey et al . , 1989 ) , and which is dependent on unsaturated fa supplementation , displayed a strict inositol auxotrophy in the presence of c18:1 . this rescue of the acc1 mutant growth was not due to repression of acc1 activity by c16:1 , since total c18 fas remained at a high level , characteristic for acc1 hyperactivity ; rather , the ratio between c16 and c18 fas was restored by c16:1 supplementation ( figure 6b ) . the analysis of pa species in wild - type and ole1 strains supplemented with c18:1 showed a similar shift toward longer acyl - chain lengths as in the acc1 strain as compared to wild - type grown without c18:1 ( figure 6c ) : pa 32:1 and pa 32:2 species were significantly decreased , whereas pa species with longer fa chain length increased , especially 36:2 . to further confirm whether the acyl - chain composition of pa affects the interaction with opi1 , we performed in vitro binding assays with pc 36:2 liposomes as a matrix , containing 20 mol% of different pa species . indeed and in line with our hypothesis , we found significantly different binding affinities of opi1 for pa species dependent on the acyl - chain composition . liposomes containing saturated pa species ( pa 32:0 and pa 36:0 ) showed the highest affinity , followed by liposomes containing 32:1 pa ; liposomes containing pa 36:2 showed the lowest binding affinity for purified opi1 . these results clearly demonstrate that the acyl - chain composition of pa is a major determinant of its interaction with the transcriptional repressor opi1 . this preferred binding affinity explains the observed phenotypes and significantly extends the current model of the transcriptional regulation of pl biosynthesis in yeast in response to deregulated fa de novo synthesis . throughout the eukaryotic kingdom , fas of 16 or 18 carbon atoms in length are the preferred acyl components in all glycerolipids that serve as bulk lipids in biological membranes or as storage lipids . whereas mechanisms controlling the level of fa desaturation in response to ambient temperature and membrane fluidity have been described in detail , much less is known about the mechanisms regulating chain length distribution ( de kroon et al . cellular fa synthesis is also coordinated with nutritional supply , activation , and incorporation into complex lipids to support proper cell function and to prevent potentially detrimental fa - induced lipotoxic effects , which are believed to be causative for lipid - associated disorders , such as obesity and type 2 diabetes in humans ( kim and ye , 2013 ; unger , 2002 ; vidal - puig and unger , 2010 ) . thus , endogenous de novo synthesis of particular fas also plays an important role to counterbalance the physiological impact of nutritional fas that may be incorporated into cellular lipids . in yeast and mammalian cells , acyl - chain length is largely determined by the fas complex that limits acyl - chain length to 16 and 18 carbon atoms ( okuyama et al . we now show that the activity of acc1 , the initial and rate - limiting enzyme of de novo fa synthesis , is a major determinant of total cellular fa production and also has a fundamental impact on the relative distribution of c16 and c18 acyl - chains in cellular lipids . on the other hand , the shift in acyl - chains toward longer residues is reflected in all cellular glycerolipids , with consequences for the binding and sequestration of the transcriptional repressor opi1 to the er . one level involves the expression of the acc1 gene , which is coordinately regulated with pl synthesis by the pl precursors , inositol and choline , and the positive and negative transcription factors ino2/ino4 and opi1 , respectively ( hasslacher et al . in addition , the activity of acc1 is strongly regulated by snf1-dependent phosphorylation ( mitchelhill et al . , 1994 ) , which , in turn , depends on the energy status of the cell ( wilson et al . here , we made use of a yeast mutant that lacks the single snf1 phosphorylation site in acc1 at serine 1157 ( acc1 ) and exhibits phenotypes very similar to snf1 mutants with respect to tag and lipid droplet accumulation as well as inositol auxotrophy , demonstrating that these snf1 phenotypes are caused by acc1 hyperactivity . the evidence we present here strongly supports the hypothesis that the inositol - requiring phenotype of the acc1 strain is due to decreased ino1 expression that is controlled by the well - characterized ino2/ino4/opi1regulatory circuit ( henry et al . , 2004 ) , as both deletion of opi1 , which uncouples transcriptional repression of ino1 from cellular pa levels , and overexpression of ino1 fully restored inositol prototrophy to the acc1 mutant strain . most notably , neither increased endogenous fa production nor exogenous fa supply led to increased pl accumulation , despite the fact that an altered acyl - chain composition is reflected in all pl classes ; instead , excess fas are preferentially channeled into tag . in addition , the similarity of the cellular response to these two situations suggests that functional tag synthesis is crucial for accommodating these fas , thus avoiding the buildup of potential signal molecules , such as pa or dag , or free fas ( garbarino and sturley , 2009 ; kohlwein , 2010 ; kohlwein and petschnigg , 2007 ; petschnigg et al . , importantly , our evidence significantly extends the current model that indeed the acyl - chain composition of pa is the critical determinant of opi1 sequestration to the er . we show that alterations in the acyl - chain composition of the acc1 strain or of wild - type and the ole1 mutant in the presence of oleic acid lead to inositol auxotrophy due to the reduced binding affinity of the opi1 repressor to pa molecular species containing longer acyl residues . we hypothesize that the recognition of the negatively charged head group of pa by the basic tract of opi1 might only represent the first level of interaction , whereas the second level is presumably determined by sensing the nature of the acyl - chains of pa within the er membrane . several other studies also support the importance of acyl - chain composition of pa for interaction with proteins ( kooijman and burger , 2009 ) ; for example , the binding specificity of human protein phosphatase 1 ( pp1c ) to pa species harboring a higher degree of acyl - chain unsaturation ( jones and hannun , 2002 ) . these findings indicate that the short - lived intermediate pa may be metabolized during cell fractionation and may indeed be present in higher amounts than previously suggested . on the other hand , sole variation of the acyl - chain composition of pa in the liposome binding assay eliminated influences of membrane electrostatics and curvature as well as of proteins , such as the yeast vap scs2 , which is required for binding of opi1 to pa in vivo ( loewen et al . ( 1 ) currently , the mechanisms that regulate the total amount of cellular pls and their specific acyl - chain composition in response to altered fa supply are largely unclear . a mechanism , as described here , that responds not only to the content of critical lipid intermediates such as pa but also to its specific molecular composition provides an attractive regulatory circuit to integrate metabolic input parameters , such as availability of nutritional fas . ( 3 ) the major and highly conserved energy - sensing snf1 kinase operates through additional levels of regulation , namely , its metabolic downstream target acc1 . in conclusion , we demonstrate that acc1 hyperactivity leads to overproduction of fas and a shift in acyl - chain length distribution toward longer chains in all pl classes as well as in tag . the acyl - chain distribution of pa is critical for its interaction with the opi1 repressor , which displays reduced affinity for pa molecular species containing longer and more unsaturated acyl - chains . this unveils an impact of the major energy - sensing kinase on membrane lipid composition and function and may explain some of the seemingly unrelated pleiotropic consequences of its deficiency . acc1 activity was inhibited in vivo by adding 1 g / ml sora to the culture medium where indicated : sora is highly specific for the eukaryotic form of acetyl - coa carboxylase , and by its binding to the biotin carboxylase dimer interface , it inhibits oligomerization , which is required for enzyme activity ( shen et al . to determine overproduction and excretion of inositol ( opi phenotype ) , 5 l od600 ( 1 aliquot of wild - type , teto7acc1 , and the control strains opi1 and cho2 ) were spotted on i medium , containing the indicated concentration of doxycycline . refer to the supplemental experimental procedures for rna isolation , reverse transcription , and real - time pcr parameters .

the online version of this article ( doi:10.1007/s10822 - 011 - 9519 - 9 ) contains supplementary material , which is available to authorized users . life expectancy of man , and especially man in the western world , increased by more than 2 days per week for the whole previous century . much of this dramatic increase is to the credit of hygiene , but medicines , and especially antibiotics and vaccines , have contributed significantly too . in the first world war , for example , almost as many soldiers died of disease as of bullets . during the second world war this unfortunate situation got medical doctors around the world can write prescriptions for tens of thousands of medicines , and an even larger number is available of herbal medicines , homeopathic wonder - cures , and other preparations for which the medicinal value has not been proven . most medicines function by interacting with proteins in the body . of the more than twenty thousand protein types in our body this , of course , gives hope for the future of drug design because most proteins are still available as a target for which a blockbuster drug can be designed . despite massive , world - wide efforts the number of new molecular entities ( nmes ) that the fda approves per year for use as medicines certainly is nt growing , while the amount of money involved goes up much faster than inflation even when we include obama s troubled asset relief program this journal ( jcamd ) has published many , many articles on techniques that according to the authors of these articles were the holy grail for drug design , and that in today s reality are just good tools used in this process . following a path familiar in science others follow suit and publish improvement after improvement , after which yet others start testing all similar methods . this was introduced in 2000 and only 3 years of improvements were needed before the first comparison methods were published . . the ever increasing costs mainly result from development and marketing and , unfortunately for us , not from research . this might explain why each time a new drug design research tool gets published pharmaceutical industries immediately jump on it and give it a hype status.fig . munos summarises these numbers eloquently in his 2009 review , but you are also encouraged to read the commentary by firestone amount of money spent in billion us$/nme ( after munos ) . munos summarises these numbers eloquently in his 2009 review , but you are also encouraged to read the commentary by firestone the first hype in drug design was born out of the famous article by hol in which he coined the name rational drug design for all protein - structure based techniques , thereby implicitly calling all methods that actually worked , such as screening or luck , irrational ; see fig . next to a monochrome picture showing this same fit , hol wrote : as to whether a drug can actually reach its target , e.g. the active center of an enzyme , is primarily a spatial problem . assuming that the structures of both components are known as example , the structure of the complex formed between a bacterial dihydrofolate - reductase , nadp and the anticancer drug methotrexate ( gray dots ) is shown on the right . next to a monochrome picture showing this same fit , hol wrote : as to whether a drug can actually reach its target , e.g. the active center of an enzyme , is primarily a spatial problem . assuming that the structures of both components are known as example , the structure of the complex formed between a bacterial dihydrofolate - reductase , nadp and the anticancer drug methotrexate ( gray dots ) is shown on the right . it is not by eye that we can determine either the fitness of a ligand for a pocket , or the safety or efficacy of a drug . it does not seem illogical to assume that the founders of jcamd were at least subconsciously dealing with the oversimplification implied by fig . we believe that most articles published in jcamd have dealt with aspects of drug design left out of fig . 2 . the advent of faster computers like first the vax / vms , then supercomputers such as the cray , and finally the pc , have allowed scientist to numerically solve chemical problems of ever increasing size and complexity . semi - empirical quantum calculation methods have been devised to calculate the chemically relevant aspects of the electronic wave - functions associated with small organic molecules and thus compute their 3d dimensional structures as well as the energy of their conformers [ 1519 ] . all the techniques derived in this domain are referred to as ligand - based drug design . in parallel , the development of molecular mechanics force fields combined with the fact that newton s equations of motion could be solved for entire proteins in their aqueous environments were true innovations in the investigation of the structure function relationships [ 2028 ] . thus , not only the geometry and the potential energy surface of macromolecular assemblies could be calculated but also their dynamic and thermodynamic properties [ 29 , 30 ] . for the early computational chemists this opened the perspective of testing at will the energy of interactions between protein targets and large collections of small molecule ligands [ 29 , 31 , 32 ] . the original thoughts that this would replace experimental validation processes , though , have long been shown to be a nice dream at best . the perception that the underlying mechanism of protein ligand recognition would be unravelled and would thus allow what ever since has been called structure - based drug design has never looked so clear and promising as at that particular moment in 1986 . with the exception of a very small fraction of ligands that are purely rigid the values of the torsion angles in ligands are determined by the valence electrons of the atoms . the development of empirical molecular mechanics force field in the late 1970s [ 33 , 34 ] have allowed for the in silico determination of the geometries ( low energy conformers ) of ligands in vacuo . application of these methods relies on two underlying assumptions : ( 1 ) that the conformation of the dissolved ligand corresponds closely to its gas - phase conformation ; and ( 2 ) that the biologically active conformation of the ligand is likely to be found among the set of low energy conformers of the isolated ligand [ 36 , 37 ] . the combined knowledge of the ligand structure ( determined by nmr or x - ray ) , the measured binding affinities , and the spatial overlay of the low energy conformations should then be sufficient to establish a structure activity relationship and pinpoint the spatial organization of the recurrent chemical features correlated with activity ( pharmacophore ) . this paved the way for a series of successes for ligand - based drug design [ e.g. 39 , 40 , 16 ] . however , although it seems fairly reasonable at first sight , both assumptions in practice proved to be incomplete and/or insufficient [ 4150 ] . the computational process by which the complementary aspects between a ligand and a receptor binding site can be ascertained has been explored with the design of specifically dedicated docking programmes . early docking methods were based uniquely on assessing the shape complementarity between a pocket in the 3d structure of a protein and low energy conformers of a ligand . the approach was computationally cumbersome due to the need to systematically search all possible ligand orientations within the pocket and scoring each of these poses by its steric hindrance . subsequent developments have taken place in several directions : improved scoring functions [ 5263 ] different ways to deal with ligand flexibility [ 60 , 6472 ] , and most recently also ways to deal with receptor flexibility [ 7378 ] . fundamental research has been performed into directions such as desolvation energies [ 7983 ] , or other aspects of the force fields used for scoring docking poses [ 66 , 78 , 8496 ] . the idea to calculate from first principles all atomic motions occurring in an active enzyme in its aqueous environment has attracted many scientists to computer aided molecular design . starting with the atomic loci obtained from the x - ray structure of en enzyme a series of snapshots describing the trajectory of the enzyme over time could thus be produced and ensemble average properties calculated based on boltzmann s ergodic hypothesis . the near infinite computer time needed for such experiments muted this field till concepts from alchemy could be embraced . in silico , one is not bound by the sequential order of events that govern paths between states , and hence so - called thermodynamic cycle methods could be developed that replaced chemical steps with alchemical steps that in principle should lead to the same outcome [ 29 , 30 , 97 , 98 ] . comparative molecular field analysis ( comfa ) is based on the overlay of active ligands [ 99 , 100102 ] . initially , the technique was more a concept than an effective tool as computer power was very limited and molecular descriptors as well as dedicated algorithms needed to be developed . the underlying idea that the 3d dimensional steric / non - bonded ( van der waals ) and electrostatic potential fields generated by the spatial organization of the chemical features around the scaffold of a ligand ( fig . 3 ) play a fundamental role in the biomolecular receptor recognition was so intuitively right and the technique made a break - through in 1988 . about 15% of all articles in jcamd refer to the use of this technique , refined and applied in all sorts of ways to produce the overly famous quantitative structure activity relationship ( qsar ) equations . however , comfa suffers from three drawbacks : ( 1 ) the alignment of the ligands in the pocket must be either known or gambled correctly ; ( 2 ) the method has been established for rigid or quasi - rigid classes of molecules ( e.g. steroids ) ; and ( 3 ) the detailed influence of the protein pocket is not known which means that any feature that is not implicitly present in the training set will be missed [ 104109 ] . these nearly fatal drawbacks prevented the generalization of the method as a standalone solution to rational drug design . certainly , the best way to apply comfa is to combine it with a pharmacophore model and a carefully conducted conformational study of the ligands .fig . 3contour representation of key features from a comfa analysis of a series of coumarin substrates and inhibitors of cytochrome p4502a5 [ poso et al , adapted from the publicly available ucla chemistry 125 course ] . the red and blue regions indicate positions where it would be favourable , respectively unfavourable to place a negative charge and the green / yellow regions where it would be favourable / unfavourable to locate bulky groups contour representation of key features from a comfa analysis of a series of coumarin substrates and inhibitors of cytochrome p4502a5 [ poso et al , adapted from the publicly available ucla chemistry 125 course ] . the red and blue regions indicate positions where it would be favourable , respectively unfavourable to place a negative charge and the green / yellow regions where it would be favourable / unfavourable to locate bulky groups many drug design projects include at some stage knowledge of the 3d structure of the target protein , and homology modelling is normally used when neither x - ray nor nmr derived coordinates are available [ 111118 ] . many computer programs were written for this purpose [ 119123 ] and the casp competition illustrates every 2 years where the field stands . presently , yasara seems to be performing very well , but many labs are working hard so this situation might change again in the future . for example , methods are under development that use plim to provide a first fix on the ligand docking site where - after steered molecular dynamics is used to continue the trajectory to convergence . similar to the casp competitions , the gpcr - dock [ 126 , 127 ] competitions have evaluated the quality of docking software , but with the additional complexity that the target structures needed to be modelled before docking could be attempted . in recent years a whole series of studies have been published in which homology modelling , combined with other tools , proved a viable replacement for the cumbersome experimental determination of target structures [ 111 , 114 , 128 , 129 ] . the good performance of two dutch teams in the recent gpcr dock competition beautifully illustrates the often mentioned fact that even the best tools only perform well in the hands of good scientists . in this latter article we find the interesting quote interestingly enough , it is the model built with most human intervention which proves to be the best . in the early 1990s the radical new idea emerged that instead of the virtual and/or real screening of large libraries of already existing molecules to identify new bioactive hits , one could rather attempt to construct entirely new synthesizable molecular entities solely based on the knowledge of the active site of the pharmaceutical target enzyme . to do so , small organic fragments composed of few atoms only must be assembled in silico inside the binding sites of enzymes in such a way that optimal protein ligand , steric , and electronic complementarity is achieved [ 84 , 125 , 135141 ] . the major problem of this approach arises from the complexity of the active site landscape and the combinatorial vastness of all possible arrangements of fragments in the volume delineated by an enzyme active site [ 66 , 142144 ] . how to choose the first fragment and where should it be positioned and oriented with respect to the inner surface of the binding pocket or cleft [ 143 , 145 , 146 ] ? which next fragment should be attached to it ? ? the genetic algorithms [ 66 , 142 , 148150 ] have been invented which allow this concept to be realized within a tractable amount of computer time by performing random transformations on a ligand collection . these transformations are selection , mutation , and crossover , and are reminiscent of the corresponding evolutionary processes in biology underlying the optimisation of genes , hence their name genetic algorithms. experience shows that these algorithms provide solutions that nicely fit the objective function , although it often is difficult to understand exactly why . randomly screening very large libraries containing up to 10 or even 10 chemical entities in in vitro enzymatic assays to produce leads has been the central paradigm of the pharmaceutical industry across the 1990s . however , after years of operating very expensive screening facilities , it has been realized that the hits produced were not of the expected quality . for example , often a bias is observed toward too lipophilic compounds that are impossible to optimize . compared to the actual number of chemically entities ( ~infinite ) the any amount of compounds that can be screened via this process is essentially zero [ 151 , 152 ] . in parallel , computational chemists had inferred that screening could be successfully operated virtually throughout computers at all stages in the drug design process from hit identification via hit optimization to lead optimization [ 153162 ] . in each of these three stages virtual libraries can be created and filtered either using chemometrics to exclude molecules that obviously are nt drug - like because of their predicted solubility or adme / tox properties , or using 3d chemical molecular descriptors ( pharmacophores ) , or using docking results . thus , libraries of compound that do not actually exist can be screened and a much smaller , manageable number of compounds selected . this is of particular advantage at the stage of lead optimization , when only few compounds are left . scaffolds of lead compounds usually carry a number of branching points were chemical variation is allowed . the in silico creation of combinatorial libraries of all the variant compounds is a dramatically faster process than its in vitro counterpart [ 163165 ] . one of the main difficulties in establishing reliable and/or transferrable qsar equations is that , even within a class of chemical analogues , ligand affinities may not respond linearly to the variation of one or several of the molecular descriptors that have been identified as related to activity . for instance , across a series of chemical substituents sorted by increasing polarity the measured affinity may respond linearly only for a restricted number of them because steric hindrance or global effect such as desolvation may penalize the binding of slightly larger groups . the modification of a branched group at another point around the scaffold may however allow some of the previously excluded ligands to become highly active . indeed the mere addition of one methyl group may result in a sudden tenfold leap in potency , dramatically increasing ligand efficiency [ 166 , 167 ] . it was demonstrated that these problems could be circumvented using artificial intelligence methods ( neural network , support vector machine , etc . ) that are insensitive to the spatial alignment of the ligand scaffolds and that are able to recognize particular combinations of properties distributed around the scaffold of a set of active ligands [ 168170 ] . artificial intelligence can be ubiquitously implemented at various stages in the rational drug design process to improve results that can be otherwise be more uncertainly obtained with classical methods , especially when assessing general properties that are the result of the subtle combination of many different factors in relation to others such as drug - likeness . various examples of artificial intelligence applications and their limitations have been published in jcamd [ 172175 ] . notwithstanding the utility of artificial intelligence , normal intelligence remains useful in avoiding some of the all too common pitfalls in the derivation and application of qsar models . we apologise to the many authors of methods that did nt make it into the above list ( see esm table 1 ) . much good work has been done that the editors certainly would nt allow us to include because citing all 1,200 articles published in jcamd in the first 25 years would perhaps be a bit excessive . we could have mentioned the work by che on privilege structures , or by lotta et al . on multivariate pls modelling of data sets . the recent work by zhou et al . on the use of dft calculation to accurately assess the existence of intermolecular h - bonds in docking instances . sarmah et al s work on solvent effects also added significantly to the drug - designers toolbox , but the methods described in these articles did nt achieve hype status . the rapid increase in costs of developing and marketing new medicines is not leaving the pharmaceutical industry untouched . recent years have seen a strong concentration of activities in terms of mergers , buy - outs , and closures . it may simply be , that a research - intensive industry like the pharmaceutical industry does not lend itself to the type of management that is common in consumer goods , fashion and footwear . it seems a paradox , though , that the high costs associated with drug design are caused by development , marketing , and legal fees , but when it comes to cost - reduction research departments are , euphemistically called , consolidated . jcamd has published a large series of articles in which multiple methods have been combined . [ 22 , 128 , 129 , 181185 ] . all these pipelines and otherwise combined methods speed up the use of the existing tools , and allow them to be applied to ever larger numbers of small molecules in ever shorter times . actually , there is a new hype raging at the moment , and it is called translational science. in the wikipedia we find under translational research : in the field of medicine , for example , it is used to translate the findings in basic research more quickly and efficiently into medical practice and , thus , meaningful health outcomes , whether those are physical , mental , or social outcomes . in a sense , the recent spate of articles on combining existing techniques into more easily applicable super - tools fit nicely to this translational paradigm . it must be stated , though , that the translation science hype is feeling stiff competition from systems biology and modelling pharmacokinetics and pharmacodynamics . between the lines we read in translational science that the pharmaceutical industry has finally realized that our deep lack of understanding of all aspects of the interaction of a medicine with a human being is the main cause for luck still being the most determining factor in the drug design process . consequently , we see the out - sourcing budgets of the large pharmaceutical industries go up , and more and more fundamental research performed in academia is finding its way to small and medium size enterprises ( smes ) where it can be incorporated in their lean and mean research machines . big pharma will at some time buy either their products or the whole smes and convert validated targets and leads or even phase i products into new medicines . this new paradigm will probably also be proven a hype soon ; only time can tell if translational research will rescue the pharmaceutical industry , or that it will only better illustrate what it all is that we do nt know yet . it remains a fact that better understanding the underlying biology , better treatment of all available data , and more intelligent combinations of data , information , and knowledge must be beneficial for the drug design process and thus , on the long run , for all of us . if the pharmaceutical industry wants academia more involved in the drug design process they could themselves make a giant first step by making available all ( or at least very many ) x - ray structures of protein ligand complexes . we estimate that the number of pdb [ 189 , 190 ] files collecting computer dust in the pharmaceutical industry is considerably bigger than the 75,000 structures now in the pdb . we have discussed this possibility with industrial crystallographers who realized that they were sometimes sitting on thousands of structure files for which secrecy was no longer an issue . they remained nevertheless hesitant to even consider discussing with their management the release of these data in fear of paranoia based rejection . another often heard rejection criterion is that they are a bit ashamed for these data because often these files have not been refined any further than was needed to answer the biological or pharmaceutical question at hand . we offer to set up a database for these files , and we offer to re - refine all industrial structures of protein ligand complexes . we will then only release those coordinates to the wider public that pass certain minimal validation criteria . if one day deposition of coordinate files into the pdb becomes significantly easier , we can consider depositing all files in the pdb on behalf of the original depositors . it might seem a bold promise to re - refine perhaps even 100,000 structures , but the pdb_redo experiment [ 192196 ] shows that today this can be done . in pdb_redo we significantly improved 85% of all presently available pdb files that were solved by x - ray . it seems likely that structures that often have been minimally refined can be improved even easier . one can even envisage that industries would like to look back at their own coordinates after we went through the elaborate and time consuming refinement process for them ; in management speak that would be the ultimate win win situation . despite massive efforts in the design of tools , databases , robotic techniques , and management innovations , luck seems to be at the basis of the discovery of most new medicines . the blockbuster viagra is probably the best illustration of the opportunism that we tend to call serendipity . in 1997 , i.e. , long before the first gpcr structure became available , kuipers et al . performed a massive literature search for aryloxypropanolamines and similar compounds binding to the serotonin 5ht-1a receptor and a series of sequence similar amine receptors . a correlation analysis revealed that only one residue s presence / absence showed a perfect correlation with binding / non - binding of a series of compounds . a mutational study validated the hypothesis that this correlation indicated a direct hydrogen bond between an alcohol group in the aminergic ligand and asparagine 719 . when the structure of the human 2 adrenoceptor bound to carazolol was solved by x - ray [ pdbid 2rh1 ; 202 ] , it showed indeed two hydrogen bonds between asn-719 and this similar ligand ( see fig 4 ) . by the way , in none of the gpcr homology models available in 199 , left : ( part of ) the x - ray structure of the 2 adrenoceptor bound to an inverse agonist that is somewhat similar to ( s)-penbutolol . right : ( s)-penbutolol binding of asn-386 in serotonin 5ht-1a predicted long before the first gpcr structure data became available ligand binding by asn-386 . left : ( part of ) the x - ray structure of the 2 adrenoceptor bound to an inverse agonist that is somewhat similar to ( s)-penbutolol . right : ( s)-penbutolol binding of asn-386 in serotonin 5ht-1a predicted long before the first gpcr structure data became available in another gpcr related project aimed at using as much heterogeneous data as can possibly be combined , oliveira et al . predicted the role of all active site residues in gpcrs , the pivotal role of arg-340 , and even a series of residue interactions involved in the activation process , and the presence and location of helix viii . the recent flurry of articles on gpcr xray structures [ 206209 ] , and especially the structure with a covalently agonist - bound g protein showed all these predictions to be conceptually right . these two gpcr - related examples make clear that there is a lot to be gained from using experimental data . but these examples also taught us how hard it is to actually get access to those data . with the gpcrdb [ 211213 ] we have started a trend to make molecular class specific information systems ( mcsis ) . and a small company , bio - prodict ( www.bio-prodict.nl ) recently caught on and is now making mcsises for a wide variety of commercially interesting molecules [ 214218 ] . their systems ( some of which are freely accessible from their website ) revolve around a structure based , and thus very accurate multiple sequence alignment ( msa ) for a whole protein super - family . this msa then functions as the anchor on which to position all kinds of data that can range from 3d structures to genome related data , from mutation studies to ligand binding constants , or from sequence correlation patterns to the prediction of mutations that enhance the protein s stability . as the most powerful information tends to be carefully hidden in the literature , an extensive set of literature - mining scripts aids with the extraction of , for example , mutation information . in fact , it was shown that the suite of mutation data extracting scripts reaches a much better coverage than can be obtained by human experts [ 214218 ] . a recent development that will aid the drug hunters of the future . showed how this programmable pdf reader could be used to directly couple data in articles on gpcrs to the gpcrdb . first , the residue numbering problem gets solved because the reader can ask the gpcrdb for the position in the gpcr msa of any residue mentioned in the article , and it can even modify or correct the sequence numbers in the article if needed . much good gpcr mutation data was published in the pre - gpcr - structure era that ended with the opsin structure article , and often these data were misinterpreted because of the poor quality of the available homology models . the utopia - gpcr pdf reader can correct those interpretations thereby salvaging old , high quality experimental data for future use . figure 5 shows an image from an old mutation study in which the authors describe several ground - breaking mutations in the guinea pig histamine h1 receptor , building and validating a homology model using these data , and arguing , for example , that residue trp161 plays an important role in receptor - ligand binding . this assumption was based on the effect of the mutation on receptor function , leading to a model in which trp161 was modelled in the ligand - binding site . by contrast , the gpcrdb generated annotation listed in the sidebar of the reader indicates that this residue , located in tm iv , points towards the membrane and possibly interacts with cholesterol . looking at the model provided by the gpcrdb , based on the latest crystal structures , it can be seen that a direct role of trp161 in receptor - ligand binding is highly unlikely.fig . 5left , one page from the histamine h1 article by wieland et al . in which trp161 is suggested to interact with the ligand while the pdf reader sidebar shows today s interpretation that this tryptophan is facing outwards towards the lipid or a dimer partner . the original picture of the modelled active site is shown enlarged in the middle panel while the right hand side figure is a plot of the gpcrdb - derived model of this receptor . the gpcrdb does not ( yet ) dock ligands , so the ligand is represented by a hand - added gray ball left , one page from the histamine h1 article by wieland et al . in which trp161 is suggested to interact with the ligand while the pdf reader sidebar shows today s interpretation that this tryptophan is facing outwards towards the lipid or a dimer partner . the original picture of the modelled active site is shown enlarged in the middle panel while the right hand side figure is a plot of the gpcrdb - derived model of this receptor . the gpcrdb does not ( yet ) dock ligands , so the ligand is represented by a hand - added gray ball folkerstma et al . combined with manually curated multiple sequence alignments , key positions in the ligand binding pocket were identified that had specific interactions with functionally diverse compounds . this analysis required a substantial amount of work : categorizing structures and compounds , creating multiple sequence alignments , analyzing ligand contacts , and transferring the results into a homogeneous residue numbering scheme ( the so - called 3d numbers ) . with the 3 dm information system [ 223 ; see the help movie ] , these analyses can today be performed in a matter of minutes [ 215 , 217 , 224].fig . 6bargraph showing the number of ligand contacts per residue extracted from 776 nuclear receptor ligand binding domain structures plotted as function of their 3d numbers . the residue with 3d number 26 is only bound to antagonistic compounds bargraph showing the number of ligand contacts per residue extracted from 776 nuclear receptor ligand binding domain structures plotted as function of their 3d numbers . the residue with 3d number 26 is only bound to antagonistic compounds more than 100 articles were found that discuss the effects of mutating this residue on the ligand binding of the receptor . in all these articles the use of a common 3d numbering scheme enables transfer of heterogeneous information between protein family members . figure 7 shows 40 antagonists in red and 70 agonists in blue . in this example , a hundred articles had to be read to extract all available mutation information for this single position mutated in 22 different receptor species combinations . that these 100 articles had to be found among 100,000 pubmed entries that contain nr information 7cartoon representation of two superposed representative nr structures ( one bound to an agonist ; one bound to an antagonist ) . the ligands were placed in the same orientation as found in their native pdb file . residue 26 , for which the antagonist interaction had been mentioned in the literature , is shown in yellow , as is residue for which fig . what if suite cartoon representation of two superposed representative nr structures ( one bound to an agonist ; one bound to an antagonist ) . the ligands were placed in the same orientation as found in their native pdb file . residue 26 , for which the antagonist interaction had been mentioned in the literature , is shown in yellow , as is residue for which fig . what if suite if , one day , all structures of nr - ligand complexes that now are scattered over inaccessible industrial hard disks could be concentrated in one system , then we could consider asking much more elaborate questions . we could consider correlating aspects of ligands with protein atom characteristics , or we could analyse if residues not contacting the ligand have an influence on binding or activation , etcetera . it is not only important to get as much information as possible stored in systems amenable to scrutiny , but it is also important to realize that for every one bioinformatician or drug hunter there are one hundred scientists who do not use molecular software regularly . project hope aims to predict the molecular phenotype of point mutations that were shown causally related to human disease states . this system attempts in all stages of user interaction to cater for human geneticists who typically do not use molecular software at all . hope only asks the user to cut - n - paste the sequence , and click the residue mutated and the mutation residue type . it then builds a homology model if needed , calls dozens of servers and services in seven countries , combines all possible information and writes a final report that can be directly used in publications , but , more importantly , that is written without using any bioinformatics jargon and even has a build - in dictionary that explains terms such as active site , salt - bridge , or hope thus is the ultimate translation machine because in doing translational research it even translates between the researchers . we believe that the recent spate of consolidations in the pharmaceutical industry is not a problem but an opportunity . mankind needs medicines , and now that pushing ones luck is slowly becoming a less successful technique , only research can find them . this research can progress rapidly if the thousands and thousands of x - ray structures of protein ligand complexes would find their way from hard - disks behind pharmaceutical industry firewalls to the public domain . drug design research in the next 25 years will revolve around ever broader collaborations , ever more holistic understanding of the drug human interactions , and ever better use of the available data , information , and knowledge . supplementary material 1 ( docx 89 kb ) supplementary material 1 ( docx 89 kb )

in its first 25 years jcamd has been disseminating a large number of techniques aimed at finding better medicines faster . these include genetic algorithms , comfa , qsar , structure based techniques , homology modelling , high throughput screening , combichem , and dozens more that were a hype in their time and that now are just a useful addition to the drug - designers toolbox . despite massive efforts throughout academic and industrial drug design research departments , the number of fda - approved new molecular entities per year stagnates , and the pharmaceutical industry is reorganising accordingly . the recent spate of industrial consolidations and the concomitant move towards outsourcing of research activities requires better integration of all activities along the chain from bench to bedside . the next 25 years will undoubtedly show a series of translational science activities that are aimed at a better communication between all parties involved , from quantum chemistry to bedside and from academia to industry . this will above all include understanding the underlying biological problem and optimal use of all available data.electronic supplementary materialthe online version of this article ( doi:10.1007/s10822 - 011 - 9519 - 9 ) contains supplementary material , which is available to authorized users .

Electronic supplementary material Introduction A brief history of tools Where do we stand today? Dealing with data, information, and knowledge: from hype to hope? Electronic supplementary material

the online version of this article ( doi:10.1007/s10822 - 011 - 9519 - 9 ) contains supplementary material , which is available to authorized users . life expectancy of man , and especially man in the western world , increased by more than 2 days per week for the whole previous century . much of this dramatic increase is to the credit of hygiene , but medicines , and especially antibiotics and vaccines , have contributed significantly too . during the second world war this unfortunate situation got medical doctors around the world can write prescriptions for tens of thousands of medicines , and an even larger number is available of herbal medicines , homeopathic wonder - cures , and other preparations for which the medicinal value has not been proven . of the more than twenty thousand protein types in our body this , of course , gives hope for the future of drug design because most proteins are still available as a target for which a blockbuster drug can be designed . despite massive , world - wide efforts the number of new molecular entities ( nmes ) that the fda approves per year for use as medicines certainly is nt growing , while the amount of money involved goes up much faster than inflation even when we include obama s troubled asset relief program this journal ( jcamd ) has published many , many articles on techniques that according to the authors of these articles were the holy grail for drug design , and that in today s reality are just good tools used in this process . this might explain why each time a new drug design research tool gets published pharmaceutical industries immediately jump on it and give it a hype status.fig . munos summarises these numbers eloquently in his 2009 review , but you are also encouraged to read the commentary by firestone the first hype in drug design was born out of the famous article by hol in which he coined the name rational drug design for all protein - structure based techniques , thereby implicitly calling all methods that actually worked , such as screening or luck , irrational ; see fig . assuming that the structures of both components are known as example , the structure of the complex formed between a bacterial dihydrofolate - reductase , nadp and the anticancer drug methotrexate ( gray dots ) is shown on the right . assuming that the structures of both components are known as example , the structure of the complex formed between a bacterial dihydrofolate - reductase , nadp and the anticancer drug methotrexate ( gray dots ) is shown on the right . we believe that most articles published in jcamd have dealt with aspects of drug design left out of fig . the advent of faster computers like first the vax / vms , then supercomputers such as the cray , and finally the pc , have allowed scientist to numerically solve chemical problems of ever increasing size and complexity . in parallel , the development of molecular mechanics force fields combined with the fact that newton s equations of motion could be solved for entire proteins in their aqueous environments were true innovations in the investigation of the structure function relationships [ 2028 ] . the perception that the underlying mechanism of protein ligand recognition would be unravelled and would thus allow what ever since has been called structure - based drug design has never looked so clear and promising as at that particular moment in 1986 . with the exception of a very small fraction of ligands that are purely rigid the values of the torsion angles in ligands are determined by the valence electrons of the atoms . the combined knowledge of the ligand structure ( determined by nmr or x - ray ) , the measured binding affinities , and the spatial overlay of the low energy conformations should then be sufficient to establish a structure activity relationship and pinpoint the spatial organization of the recurrent chemical features correlated with activity ( pharmacophore ) . this paved the way for a series of successes for ligand - based drug design [ e.g. the computational process by which the complementary aspects between a ligand and a receptor binding site can be ascertained has been explored with the design of specifically dedicated docking programmes . the approach was computationally cumbersome due to the need to systematically search all possible ligand orientations within the pocket and scoring each of these poses by its steric hindrance . subsequent developments have taken place in several directions : improved scoring functions [ 5263 ] different ways to deal with ligand flexibility [ 60 , 6472 ] , and most recently also ways to deal with receptor flexibility [ 7378 ] . fundamental research has been performed into directions such as desolvation energies [ 7983 ] , or other aspects of the force fields used for scoring docking poses [ 66 , 78 , 8496 ] . the idea to calculate from first principles all atomic motions occurring in an active enzyme in its aqueous environment has attracted many scientists to computer aided molecular design . starting with the atomic loci obtained from the x - ray structure of en enzyme a series of snapshots describing the trajectory of the enzyme over time could thus be produced and ensemble average properties calculated based on boltzmann s ergodic hypothesis . in silico , one is not bound by the sequential order of events that govern paths between states , and hence so - called thermodynamic cycle methods could be developed that replaced chemical steps with alchemical steps that in principle should lead to the same outcome [ 29 , 30 , 97 , 98 ] . initially , the technique was more a concept than an effective tool as computer power was very limited and molecular descriptors as well as dedicated algorithms needed to be developed . the underlying idea that the 3d dimensional steric / non - bonded ( van der waals ) and electrostatic potential fields generated by the spatial organization of the chemical features around the scaffold of a ligand ( fig . 3 ) play a fundamental role in the biomolecular receptor recognition was so intuitively right and the technique made a break - through in 1988 . about 15% of all articles in jcamd refer to the use of this technique , refined and applied in all sorts of ways to produce the overly famous quantitative structure activity relationship ( qsar ) equations . however , comfa suffers from three drawbacks : ( 1 ) the alignment of the ligands in the pocket must be either known or gambled correctly ; ( 2 ) the method has been established for rigid or quasi - rigid classes of molecules ( e.g. certainly , the best way to apply comfa is to combine it with a pharmacophore model and a carefully conducted conformational study of the ligands .fig . 3contour representation of key features from a comfa analysis of a series of coumarin substrates and inhibitors of cytochrome p4502a5 [ poso et al , adapted from the publicly available ucla chemistry 125 course ] . the red and blue regions indicate positions where it would be favourable , respectively unfavourable to place a negative charge and the green / yellow regions where it would be favourable / unfavourable to locate bulky groups contour representation of key features from a comfa analysis of a series of coumarin substrates and inhibitors of cytochrome p4502a5 [ poso et al , adapted from the publicly available ucla chemistry 125 course ] . the red and blue regions indicate positions where it would be favourable , respectively unfavourable to place a negative charge and the green / yellow regions where it would be favourable / unfavourable to locate bulky groups many drug design projects include at some stage knowledge of the 3d structure of the target protein , and homology modelling is normally used when neither x - ray nor nmr derived coordinates are available [ 111118 ] . many computer programs were written for this purpose [ 119123 ] and the casp competition illustrates every 2 years where the field stands . similar to the casp competitions , the gpcr - dock [ 126 , 127 ] competitions have evaluated the quality of docking software , but with the additional complexity that the target structures needed to be modelled before docking could be attempted . in recent years a whole series of studies have been published in which homology modelling , combined with other tools , proved a viable replacement for the cumbersome experimental determination of target structures [ 111 , 114 , 128 , 129 ] . the good performance of two dutch teams in the recent gpcr dock competition beautifully illustrates the often mentioned fact that even the best tools only perform well in the hands of good scientists . in the early 1990s the radical new idea emerged that instead of the virtual and/or real screening of large libraries of already existing molecules to identify new bioactive hits , one could rather attempt to construct entirely new synthesizable molecular entities solely based on the knowledge of the active site of the pharmaceutical target enzyme . to do so , small organic fragments composed of few atoms only must be assembled in silico inside the binding sites of enzymes in such a way that optimal protein ligand , steric , and electronic complementarity is achieved [ 84 , 125 , 135141 ] . the major problem of this approach arises from the complexity of the active site landscape and the combinatorial vastness of all possible arrangements of fragments in the volume delineated by an enzyme active site [ 66 , 142144 ] . how to choose the first fragment and where should it be positioned and oriented with respect to the inner surface of the binding pocket or cleft [ 143 , 145 , 146 ] ? the genetic algorithms [ 66 , 142 , 148150 ] have been invented which allow this concept to be realized within a tractable amount of computer time by performing random transformations on a ligand collection . these transformations are selection , mutation , and crossover , and are reminiscent of the corresponding evolutionary processes in biology underlying the optimisation of genes , hence their name genetic algorithms. randomly screening very large libraries containing up to 10 or even 10 chemical entities in in vitro enzymatic assays to produce leads has been the central paradigm of the pharmaceutical industry across the 1990s . however , after years of operating very expensive screening facilities , it has been realized that the hits produced were not of the expected quality . for example , often a bias is observed toward too lipophilic compounds that are impossible to optimize . compared to the actual number of chemically entities ( ~infinite ) the any amount of compounds that can be screened via this process is essentially zero [ 151 , 152 ] . in parallel , computational chemists had inferred that screening could be successfully operated virtually throughout computers at all stages in the drug design process from hit identification via hit optimization to lead optimization [ 153162 ] . thus , libraries of compound that do not actually exist can be screened and a much smaller , manageable number of compounds selected . scaffolds of lead compounds usually carry a number of branching points were chemical variation is allowed . for instance , across a series of chemical substituents sorted by increasing polarity the measured affinity may respond linearly only for a restricted number of them because steric hindrance or global effect such as desolvation may penalize the binding of slightly larger groups . that are insensitive to the spatial alignment of the ligand scaffolds and that are able to recognize particular combinations of properties distributed around the scaffold of a set of active ligands [ 168170 ] . artificial intelligence can be ubiquitously implemented at various stages in the rational drug design process to improve results that can be otherwise be more uncertainly obtained with classical methods , especially when assessing general properties that are the result of the subtle combination of many different factors in relation to others such as drug - likeness . we apologise to the many authors of methods that did nt make it into the above list ( see esm table 1 ) . much good work has been done that the editors certainly would nt allow us to include because citing all 1,200 articles published in jcamd in the first 25 years would perhaps be a bit excessive . the recent work by zhou et al . on the use of dft calculation to accurately assess the existence of intermolecular h - bonds in docking instances . sarmah et al s work on solvent effects also added significantly to the drug - designers toolbox , but the methods described in these articles did nt achieve hype status . the rapid increase in costs of developing and marketing new medicines is not leaving the pharmaceutical industry untouched . recent years have seen a strong concentration of activities in terms of mergers , buy - outs , and closures . it may simply be , that a research - intensive industry like the pharmaceutical industry does not lend itself to the type of management that is common in consumer goods , fashion and footwear . it seems a paradox , though , that the high costs associated with drug design are caused by development , marketing , and legal fees , but when it comes to cost - reduction research departments are , euphemistically called , consolidated . jcamd has published a large series of articles in which multiple methods have been combined . all these pipelines and otherwise combined methods speed up the use of the existing tools , and allow them to be applied to ever larger numbers of small molecules in ever shorter times . actually , there is a new hype raging at the moment , and it is called translational science. in a sense , the recent spate of articles on combining existing techniques into more easily applicable super - tools fit nicely to this translational paradigm . between the lines we read in translational science that the pharmaceutical industry has finally realized that our deep lack of understanding of all aspects of the interaction of a medicine with a human being is the main cause for luck still being the most determining factor in the drug design process . consequently , we see the out - sourcing budgets of the large pharmaceutical industries go up , and more and more fundamental research performed in academia is finding its way to small and medium size enterprises ( smes ) where it can be incorporated in their lean and mean research machines . this new paradigm will probably also be proven a hype soon ; only time can tell if translational research will rescue the pharmaceutical industry , or that it will only better illustrate what it all is that we do nt know yet . it remains a fact that better understanding the underlying biology , better treatment of all available data , and more intelligent combinations of data , information , and knowledge must be beneficial for the drug design process and thus , on the long run , for all of us . if the pharmaceutical industry wants academia more involved in the drug design process they could themselves make a giant first step by making available all ( or at least very many ) x - ray structures of protein ligand complexes . we estimate that the number of pdb [ 189 , 190 ] files collecting computer dust in the pharmaceutical industry is considerably bigger than the 75,000 structures now in the pdb . we offer to set up a database for these files , and we offer to re - refine all industrial structures of protein ligand complexes . we will then only release those coordinates to the wider public that pass certain minimal validation criteria . in pdb_redo we significantly improved 85% of all presently available pdb files that were solved by x - ray . despite massive efforts in the design of tools , databases , robotic techniques , and management innovations , luck seems to be at the basis of the discovery of most new medicines . performed a massive literature search for aryloxypropanolamines and similar compounds binding to the serotonin 5ht-1a receptor and a series of sequence similar amine receptors . a correlation analysis revealed that only one residue s presence / absence showed a perfect correlation with binding / non - binding of a series of compounds . right : ( s)-penbutolol binding of asn-386 in serotonin 5ht-1a predicted long before the first gpcr structure data became available in another gpcr related project aimed at using as much heterogeneous data as can possibly be combined , oliveira et al . predicted the role of all active site residues in gpcrs , the pivotal role of arg-340 , and even a series of residue interactions involved in the activation process , and the presence and location of helix viii . the recent flurry of articles on gpcr xray structures [ 206209 ] , and especially the structure with a covalently agonist - bound g protein showed all these predictions to be conceptually right . their systems ( some of which are freely accessible from their website ) revolve around a structure based , and thus very accurate multiple sequence alignment ( msa ) for a whole protein super - family . this msa then functions as the anchor on which to position all kinds of data that can range from 3d structures to genome related data , from mutation studies to ligand binding constants , or from sequence correlation patterns to the prediction of mutations that enhance the protein s stability . a recent development that will aid the drug hunters of the future . showed how this programmable pdf reader could be used to directly couple data in articles on gpcrs to the gpcrdb . first , the residue numbering problem gets solved because the reader can ask the gpcrdb for the position in the gpcr msa of any residue mentioned in the article , and it can even modify or correct the sequence numbers in the article if needed . much good gpcr mutation data was published in the pre - gpcr - structure era that ended with the opsin structure article , and often these data were misinterpreted because of the poor quality of the available homology models . the utopia - gpcr pdf reader can correct those interpretations thereby salvaging old , high quality experimental data for future use . figure 5 shows an image from an old mutation study in which the authors describe several ground - breaking mutations in the guinea pig histamine h1 receptor , building and validating a homology model using these data , and arguing , for example , that residue trp161 plays an important role in receptor - ligand binding . by contrast , the gpcrdb generated annotation listed in the sidebar of the reader indicates that this residue , located in tm iv , points towards the membrane and possibly interacts with cholesterol . the original picture of the modelled active site is shown enlarged in the middle panel while the right hand side figure is a plot of the gpcrdb - derived model of this receptor . this analysis required a substantial amount of work : categorizing structures and compounds , creating multiple sequence alignments , analyzing ligand contacts , and transferring the results into a homogeneous residue numbering scheme ( the so - called 3d numbers ) . 6bargraph showing the number of ligand contacts per residue extracted from 776 nuclear receptor ligand binding domain structures plotted as function of their 3d numbers . the residue with 3d number 26 is only bound to antagonistic compounds bargraph showing the number of ligand contacts per residue extracted from 776 nuclear receptor ligand binding domain structures plotted as function of their 3d numbers . in all these articles the use of a common 3d numbering scheme enables transfer of heterogeneous information between protein family members . the ligands were placed in the same orientation as found in their native pdb file . what if suite if , one day , all structures of nr - ligand complexes that now are scattered over inaccessible industrial hard disks could be concentrated in one system , then we could consider asking much more elaborate questions . project hope aims to predict the molecular phenotype of point mutations that were shown causally related to human disease states . hope only asks the user to cut - n - paste the sequence , and click the residue mutated and the mutation residue type . we believe that the recent spate of consolidations in the pharmaceutical industry is not a problem but an opportunity . mankind needs medicines , and now that pushing ones luck is slowly becoming a less successful technique , only research can find them . this research can progress rapidly if the thousands and thousands of x - ray structures of protein ligand complexes would find their way from hard - disks behind pharmaceutical industry firewalls to the public domain . drug design research in the next 25 years will revolve around ever broader collaborations , ever more holistic understanding of the drug human interactions , and ever better use of the available data , information , and knowledge . supplementary material 1 ( docx 89 kb ) supplementary material 1 ( docx 89 kb )

our current picture of cell nuclear function is shifting from the idea of fully assembled molecular complexes towards molecules freely roaming in nuclear space ( pombo and cook 1996 ; phair and misteli 2000 ; bentley 2002 ) . here , transient interactions ( protein protein , protein dna , and protein rna ) result in a stochastic but directional progression of multi - scale and multi - player processes ( elf et al . 2007 ; zenklusen et al . 2008 ; darzacq et al . 2009 ) . while this change in paradigm challenges our view of the composition of multi - protein complexes , it also raises questions of how the nuclear space is organized , how the availability of factors is regulated and how interaction sites are found by the right interaction partners . while no membrane - bound compartments ( such as in the cytoplasm ) have been identified specific areas can be labeled , including the nucleolus , pcg bodies , nuclear speckles , cajal bodies , gems , cleavage bodies , perinucleolar compartments , the sam68 nuclear body , pml bodies , etc . ( spector 2001 ) forming a patterned space . as a result , besides crowding effects also found in the cytoplasm , molecules in the nucleus encounter a highly complex , anisotropic landscape ( richter et al . 2007 ) . to probe the dynamic functions of this landscape , an ensemble of technologies ( such as frap , flip , confocal imaging etc . ) have been applied to elucidate the dynamics of protein factors and rna particles ( becker et al . many functional protein factors have been found to vary in their immobile versus mobile states , as well as in their average off rates from potential binding sites ( misteli and spector 1999 ; grunwald et al . similarly , a wide range of mobility distributions exist in rna studies ( politz et al . even nuclear bodies , that appear stationary in imaging , are actually dynamic complexes undergoing a steady exchange of their building blocks ( politz et al . these data draw a picture of a highly mobile environment with changing fractions of immobilized populations and a continual change in composition . experimental access to probe this environment and its embedded processes at the molecular scale is limited as : nuclear organization is actively maintained and so can only be studied in living cells ; ensemble technologies , being diffraction limited , fail when challenged by molecular interactions on the nanometer length scale ; and the high dynamics of these often transient interactions demand collection of data in the milliseconds time scale , limiting the achievable signal - to - noise ratios . while multiple strategies are employed today to challenge the resolution criteria ( hell 2003 ; betzig et al . 2006 ) , imaging of single - molecule signals can provide the position of the observed molecule with an accuracy that can overcome the resolution limit by a factor of ten in the living cell , and is in principle capable of providing nanometer precision1 ( bobroff 1986 ; schmidt et al . continuous image acquisition also enables following a single protein or rna over an extended amount of time utilizing the high localization accuracy for tracking rather than construction of a high resolution image . an immediate advantage is that no synchronization of cellular processes is needed as single - molecule tracking ( smt ) is fast enough to observe the sequence of events in real time and synchronization is achieved during data analysis . finally , by providing superb time and spatial information simultaneously , smt is perhaps the tool of choice when it comes to investigating the dynamic landscape of molecular interactions in the living cell . the question however is how fast smt can be , or in other words , what is the fastest process observable by this technology ? smt has been shown to be fast enough to even follow freely moving molecules in an aqueous solution ( grunwald et al . measurements of nucleocytoplasmic transport have shown definitively that transient interactions in the living cell can be resolved on the nanometer length- and milliseconds time scales using smt . in the same way , single protein factors and rnas in the nucleus have been measured using inert test probes and functional protein factors ( goulian and simon 2000 ; kubitscheck et al . compared with fluorescence correlation spectroscopy ( fcs ) or fluorescence recovery after photobleaching ( frap ) , smt provides the means to analyze multiple forms of mobility in heterogeneous environments . even more , smt enables observation of the molecular behavior over the whole field of view and not only within either the confocal volume of an fcs spot or the bleached area in frap . this makes the technique very valuable for studying intranuclear processes of proteins and rnas in relation to defined nuclear structures . however , observation of single molecules in living cells needs to overcome the problem of having too many overlapping signals from the labeled population of molecules . adjustment of the number of labeled molecules is usually done by : providing small amounts of recombinant fluorescently labeled proteins ( e.g. , by microinjection ) , by careful titration of labeled ligand concentrations , by adjusting expression levels of fluorescent reporter molecules ( e.g. , using leaky promoters ) , or by signal amplification ( e.g. , dna probes , molecular beacons or the ms2 system ) ( bertrand et al . the resulting experimental design is then driven by optimizing the signal - to - noise ratio during data acquisition , usually by a combination of background reduction , signal multiplication on the detector ( e.g. , apds or emccds ) and very sensitive detection at low read noise conditions . finally , the observation time needs to be maximized , taking photobleaching rates , desirable signal intensity and applicable amounts of light into consideration . while many pioneering studies used excitation energies in the range of several kilo watts per square centimeter , cells and especially nuclear structures and processes are sensitive to high - photon fluxes ( dobrucki et al . 2007 ; 2009 ) . in this context , calibrating the amount of light delivered to the cell is of crucial importance and can be done for broadband light sources as well as for laser based imaging ( grunwald et al . 2008b ) . depending on the mobility of the observed molecules and the acquisition speed of the microscope system used , the acquired data is then analyzed based on the tracking of individual signals ( see ( grunwald et al . 2008c ) for a detailed review ) . figure 1 details simple modifications that can be applied to commercially available equipment to improve performance for the detection of single molecules in living cells at appropriate time scales . in short , a laser illumination module ( here shown in a customized home - built version , but also commercially available ) is combined with a fast switching mechanism and a fast intensity regulator for the laser light and fiber coupled to the microscope . a stable stage with high precision and small drift is used to position samples above the objective lens and to allow for cell manipulation ( e.g. , microinjection ) if needed . lastly , a sensitive , fast camera , typically an emccd , is used for recording the single - molecule signal and is mounted as directly as possible onto the microscope to increase photon transmission . either sequential imaging or a second detector is used to monitor a cellular reference structure . a conventional wide - field epifluorescence microscope is custom - modified to optimize single molecule detection in biological samples . the excitation ( laser ) light is merged using dichromatic beam splitters and mirrors and coupled into an optical mono - mode fiber to deliver the light to the microscope . an acousto - optical tunable filter is used to attenuate laser power and switch illumination on and off with microsecond resolution . the fiber output can either be delivered as a collimated beam to the excitation tube lens or be imaged via a conjugated image plane into the objective s back focal plane . emitted fluorescence light , collected by a high numerical aperture objective , is separated from the excitation light by an appropriate dichromatic beam splitter , which reflects the three excitation lines into the sample , while the three fluorescence bands are transmitted to the detection system . the microscope is mounted on a steel scaffold fixed to the optical table to allow easy access to the base port , where an emccd for single molecule tracking is mounted directly onto the microscope stand collecting signal in the primary image plane . a second camera , located at a side port , a microinjection system allows delivery of fluorescently labeled probes into a living cell an smt microscope setup . a conventional wide - field epifluorescence microscope is custom - modified to optimize single molecule detection in biological samples . single - mode , low - noise lasers are the preferred choice . the excitation ( laser ) light is merged using dichromatic beam splitters and mirrors and coupled into an optical mono - mode fiber to deliver the light to the microscope . an acousto - optical tunable filter is used to attenuate laser power and switch illumination on and off with microsecond resolution . the fiber output can either be delivered as a collimated beam to the excitation tube lens or be imaged via a conjugated image plane into the objective s back focal plane . emitted fluorescence light , collected by a high numerical aperture objective , is separated from the excitation light by an appropriate dichromatic beam splitter , which reflects the three excitation lines into the sample , while the three fluorescence bands are transmitted to the detection system . the microscope is mounted on a steel scaffold fixed to the optical table to allow easy access to the base port , where an emccd for single molecule tracking is mounted directly onto the microscope stand collecting signal in the primary image plane . a second camera , located at a side port , is used to detect reference images . a microinjection system allows delivery of fluorescently labeled probes into a living cell to summarize , smt and fcs provide the means to study single molecules or very small , locally resolved , ensembles of fluorescently labeled molecules in the living cell . in the following , we discuss our current view of the nuclear landscape , dynamical aspects of the nucleus and point out controversial areas that remain . the road to live cell imaging was laid down by confocal microscopy and today s fcs implementations are building upon this technology ( eigen and rigler 1994 ) . the first experiment to provide a mobility profile of the nuclear space used fcs and fluorescein - labeled oligodeoxynucleotides ( oligos ) to measure intranuclear diffusion coefficients in living cells ( politz et al . 1998 ) . labeled poly - thymidine oligos , that are passively taken up by living cells , formed hybrids with the poly - adenylated tails of rnas ( poly(a)rnas ) in the nucleus , and two major fractions of diffusion rates were observed . besides the free oligonucleotide diffusion , a slow moving fraction , interpreted as the labeled diffusive rnas ( d = 10 m / s ) , was detected . a third minor fraction was also detected with a 10-fold slower mobility rate ( see table 1 ) . in another experiment , the effect of nuclear compartments on the movement of poly(a)rnas in the nucleoplasm was compared to poly(a)rna mobility in speckles ( politz et al . these values are similar to the mobility of the slower class reported in the first study ( table 1 ) . interestingly , temperature reduction from 37c to 22c had no detectable effect on poly(a)rna mobility , and was interpreted as meaning that movement between the nucleoplasm and speckles does not require metabolic energy , which is in stark contrast to a previous report ( calapez et al . table 1overview of nuclear diffusion coefficients measured by smt and fcsreferenceconstructmethoddiffusion coefficientsubcompartment(grunwald et al . 2006b)u1 snrnp bioactivesmtd = 0.5 to 8 m / snucleoplasm , speckles(grunwald et al . 2008a)streptavidin - nls inertsmtd = 0.8 to 5 m / snucleoplasm , nucleolus , heterochromatin(speil and kubitscheck 2010)ovalbumin inertsmtd = 0.5 to 12 m / snucleoplasm , nucleolus(bancaud et al . 2009)gfp - monomerfcsd1 = 29 m / seuchromatingfp - monomerd1 = 87 m / saqueous solutiongfp - dimerd2 = 17 m / seuchromatingfp - dimerd2 = 55 m / saqueous solutiongfp - pentamerd3 = 7.7 m / snucleoplasm(politz et al . 1998)oligo(dt):poly(a ) rnafcsd = 1 to 10 m / snucleoplasm(politz et al . 2006)oligo(dt):poly(a ) rnafcsd1 = 0.65specklesd2 = 0.7nucleoplasm(shav - tal et al . 2004)yfp - ms2 mrnpfrapd = 0.09 m / snucleoplasmsmtd = 0.04 m / snucleoplasm(van den bogaard and tyagi 2009)gfp - mrna-96-mersmtd = 0.033 m / snucleus(siebrasse et al . 2008)dna - labeled br mrnpsmtd = 0.24 to 4.0 m / ssalivary - gland cell nucleus(mor et al . 2005)hiv - infcsd = 10.5 m / snucleusin these studies , diffusion coefficients were fixed to compare the relative amounts of the different mobility classes in different compartments . for details , see original literature overview of nuclear diffusion coefficients measured by smt and fcs in these studies , diffusion coefficients were fixed to compare the relative amounts of the different mobility classes in different compartments . for details , see original literature probing the nuclear space with inert probe molecules , initially described by grunwald et al . 2008a using smt , was then used to explore the effects of molecular crowding on diffusion and binding of nuclear proteins in heterochromatin using fcs technology ( bancaud et al . volume exclusion , diffusive barriers in more dense nuclear compartments and transient trapping in heterochromatin , as observed by grunwald et al . photoactivation experiments were carried out , and in euchromatin , diffusion and binding parameters were well explained by a diffusion limited model . however , when the same probes were tested in heterochromatin , where slower kinetics were expected ( dependent on the heterochromatin to euchromatin abundance ratio ) , an unexpected biphasic mobility was observed that could not be explained by a diffusion reaction model or a random crowding model ( bancaud et al . based on these results the authors deduced fractal geometry of chromatin as previously suggested ( wachsmuth et al . circumventing the small active measurement area and time averaging inherent to fcs , improvements in camera technology , and the development of rna labeling systems , made imaging of messenger ribonucleoprotein particles ( mrnps ) in living cells a possibility . one such labeling system is the ms2 system that consists of two components , i.e. , a sequence of ms2 rna stem - loops introduced into the rna sequence of interest and a coat protein to which a fluorescent protein is fused . the coat protein binds to the rna sequence with nanomolar affinity thereby generating a multiplexed signal sufficient for single molecule tracking ( stockley et al . lago et al . 2001 ) . to detect mrnps in real time in living mammalian cells , 24 ms2 stem - loops were inserted downstream of a target rna ( shav - tal et al . movements of individual mrnps were followed for 100 frames using exposure times of 250 and 333 ms , respectively . the tracking criteria for mrnps was set to more than eight frames of continuous observation , and the mean diffusion coefficient , in stark contrast to the fcs data cited above , was found to be 0.04 m / s at 37c . this diffusion was also temperature dependent , as at room temperature the diffusion coefficient was reduced indicating that mrnps released after transcription moved probabilistically through the nucleoplasm following simple laws of diffusion ( shav - tal et al . when very large mrnps were tracked in the nucleus ( dys - mrnp , ranging from 4.8 to 14 kb ) , an even lower mobility of mrnps was found ( d = 0.005 m / s ) ( mor et al . 2010 ) . interestingly , in this report , mrnps were found to travel in chromatin free zones in a channeled diffusion manner with the accumulation of mobile particles in distinct chromatin free zones ( mor et al . 2010 ) . while there is evidence of open spaces in the lamin network as revealed by three - dimensional structured illumination microscopy ( 3d - sim ) ( schermelleh et al . 2008 ) , it is unclear whether these spaces are indeed channels and if they exist over the entire nucleus as predicted ( mor et al . 2010 ) . another labeling approach uses molecular beacons for fluorescent labeling of mrnp ( van den bogaard and tyagi 2009 ) . a molecular beacon is composed of a stem hybrid that keeps the fluorophore close to a quencher rendering the probe initially non - fluorescent . when the probe hybridizes to a target sequence , the fluorophore separates from the quencher due to a conformational change , and becomes detectable . sequence specific multiplexing of probes on a target rna is possible , reaching up to 100 probe copies / rna , resulting in signal intensities of single mrnps that can be detected as diffraction - limited spots in living cells . in an experiment where imaging was performed at 300 ms integration time , an average diffusion coefficient of 0.033 m / s was found for one half of the observed mrnps , while the other half displayed a stationary behavior with a calculated diffusion coefficient of 0.0006 m / s . when the single mrnps positions were mapped to a chromatin density image the stationary particles were located in high - density chromatin regions . atp depletion increased the number of stalled particles but no strong effect on diffusion was found , again suggesting that mrnp movement is an atp - independent process ( van den bogaard and tyagi 2009 ) . all of these results are 10100-fold lower in their diffusion constants ( see table 1 ) compared to the fcs based mobility profile ( politz et al . in contrast to the above studies , however , when an insect model system ( instead of a mammalian cell system ) was used , i.e. , balbiani ring ( br2 ) mrnas ( extremely large mrnas found in the salivary glands of the chironomus midge ) , four different mobile fractions , widely varying , were found approaching diffusion coefficients comparable to those found by fcs ( table 1 ) ( siebrasse et al . interestingly , the distribution of diffusion coefficients is better explained as a probability distribution of mobile states that an mrna molecule can adopt , rather than by the existence of distinct classes of differently mobile mrnas , a first indication of which was already seen in protein mobility ( see fig . 2 and ( grunwald et al . 2008a ) ) . fluorescent labeling here was done either by microinjection of oligos complementary to br2 mrna or with a labeled - hrp36 protein that specifically binds br2 mrna ( siebrasse et al . 2nuclear protein mobility as determined by smt . a displacement - dependent trace analysis of streptavidin in the nucleoplasm . jump distances were binned into three groups of 080 nm , 80240 nm , and larger than 240 nm displacement . no difference in observation frequency and decay time is observable between the three binned classes . b the same three classes are used to analyze jump distances of streptavidin molecules inside mecp2-induced heterochromatin . a clear decrease in observation frequency for longer jump distances c diffusion coefficients are a convenient method to extract a mean mobility value , however , it is a rather broad parameter . in cells two or three diffusion coefficients single - molecule observation suggests that individual molecules can adopt different mobile states at different times . here , we present the mean square displacement of a single streptavidin trace ( complete trace black ) , showing that indeed within one observation interval the molecule changes from a trapped immobile state ( blue ) to a mobile , diffusive state ( cyan ) and back to an immobile state ( red ) . d time projection of the trace analyzed in c projected onto the reference image from the cell nucleus . e overview of individual nls - streptavidin - cy5 traces in a mecp2-gfp - labeled cell nucleus . different colors indicate distinguishable tracks in the nucleoplasm ( yellow ) , in pericentric heterochromatin ( green ) , and the transition between nucleoplasm and heterochromatin ( blue ) . f the diffusion coefficient was estimated from the mean square displacement versus time of the mobility of the red trace shown in ( e ) nuclear protein mobility as determined by smt . a displacement - dependent trace analysis of streptavidin in the nucleoplasm . jump distances were binned into three groups of 080 nm , 80240 nm , and larger than 240 nm displacement . no difference in observation frequency and decay time is observable between the three binned classes . b the same three classes are used to analyze jump distances of streptavidin molecules inside mecp2-induced heterochromatin . a clear decrease in observation frequency for longer jump distances c diffusion coefficients are a convenient method to extract a mean mobility value , however , it is a rather broad parameter . in cells two or three diffusion coefficients single - molecule observation suggests that individual molecules can adopt different mobile states at different times . here , we present the mean square displacement of a single streptavidin trace ( complete trace black ) , showing that indeed within one observation interval the molecule changes from a trapped immobile state ( blue ) to a mobile , diffusive state ( cyan ) and back to an immobile state ( red ) . d time projection of the trace analyzed in c projected onto the reference image from the cell nucleus . e overview of individual nls - streptavidin - cy5 traces in a mecp2-gfp - labeled cell nucleus . different colors indicate distinguishable tracks in the nucleoplasm ( yellow ) , in pericentric heterochromatin ( green ) , and the transition between nucleoplasm and heterochromatin ( blue ) . f the diffusion coefficient was estimated from the mean square displacement versus time of the mobility of the red trace shown in ( e ) as there are such dramatic differences between the fcs data and tracking data of one group versus another , it is very clear that controversy exists in this field , which might be explained by clear technical differences in how the experiments were performed . as we have seen above that rna tracking is possible , it would be interesting to also track single proteins as proteins are the facilitators of many major cellular processes . one such study looked at the mobility of fluorescently labeled uridine - rich small nuclear ribonucleoproteins ( u1 snrnps ) , biologically active splicing factors ( grunwald et al . gfp - labeled asf / sf2 was used to mark nuclear speckles allowing direct comparison of u1 snrnp dynamics inside and outside of the nuclear speckles . using high speed fluorescence microscopy , with frame rates of up to 200 hz , no significant mobility was found for 80% of u1 snrnps , possibly caused by molecular trapping in nuclear structures as well as immobilization in spliceosomes and post - splicing processes . a continuous range of mobility for u1 snrnps , ranging from 0.5 to 8 m / s was found . the diffusion coefficient of 0.5 m / s corresponds to impeded uncomplexed single u1 snrnps or higher organized spliceosome - complexes . correspondingly , using frap experiments , a three to five fold reduction of the diffusion coefficient of larger molecules in the nuclei was also found ( gorski et al . from here we conclude that there is not just one kinetic condition for association and dissociation events of biologically active proteins in the nucleus . how does the large immobile fraction of u1 snrnps compare , however , to inert proteins ? tagged streptavidin coupled to a nuclear localization sequence ( nls ) with a size of about 60 kda , and a second probe , ovalbumin with a size of 45 kda , were tracked in living cell nuclei ( grunwald et al . while streptavidin is not translocated by nuclear pores , primarily due to its size , ovalbumin likely passively transports into the nucleus . using high speed fluorescence imaging at frame rates of up to 200 hz , the streptavidin experiment succeeded in capturing even single traces of probe molecules and deduced a diffusion rate comparable to that of the inert gfp protein as seen by fcs ( see table 1 and fig . 2 ) ( grunwald et al . 2008a ; bancaud et al . 2009 ) . different nuclear compartments affect the movement of inert proteins differently , but even in the nucleoplasm , two kinetic components ( mobile and trapped ) were observed , and the mobile fraction is widely spread over a wide range of diffusion coefficients ( the data could not be fitted satisfactorily assuming only one or two diffusing components , table 1 ) . compared with the nucleoplasm ( defined as space neither labeled by mecp2 or asf1 exclusion ) , proteins seemed to become trapped predominantly in pericentric heterochromatin resulting in fewer proteins moving freely ( see fig . 2 ) . even more exciting , and in contrast with the fcs study discussed above ( bancaud et al . 2009 ) , proteins were trapped less frequently in the nucleolus compared to the nucleoplasm . while confocal imaging implies that the nucleolus excludes the test protein , the mobility data suggest minimal trapping in this compartment and combined with no retention of proteins at the compartment interface , exclusion turns out to be a dynamic effect , where mobile proteins leave the nucleolus frequently and fast ( grunwald et al . 2008a ; speil and kubitscheck 2010 ) . when ovalbumin was used , an inert protein that does not contain an nls , comparable observations were made demonstrating the limited , if any , impact of the sv40 nls - related electric charge on protein mobility in the nucleus . an important question that can be addressed using single molecule tracking is the mobility of hiv ( human immunodeficiency virus ) related factors , their interaction times and regulation of cellular distribution . we first provide a brief summary of the hiv lifecycle , including import and export processes of hiv genetic material through the nuclear membrane , which is a very elegant symphony between cytoplasmic and nuclear viral and host cell factors . single molecule tracking techniques might enable deeper understanding of the spatial - temporal dynamics of these processes thereby not only providing key information on very basic biological processes , but we surmise , also exposing vulnerabilities that could be targeted in the fight against this devastating disease . the hiv virus is an rna lentivirus with a genome comprising two single - rna strands containing the full genetic information needed for viral replication in host cells ( luciw et al . 1996 ; fields et al . 2007 ; levy 2007 ) . through the process of fusion and endocytosis 3 ) , by binding its trimeric surface gp120 to a cd4 molecule present on the host cell ( reviewed in ( gallo et al . 2003 ) ) . this binding induces a conformational change to a specific chemokine receptor ( predominantly ccr5 or cxcr4 ( berger et al . 1999 ) ) , and this in turn induces a conformational change in gp41 , another viral surface factor . this fusion of the viral and infected cell s membranes results in the release of the hiv viral core into the cytoplasm ( chan et al . 2010 ) and the viral reverse transcription complex , comprising viral and host proteins , is assembled and cdna synthesis begins . the result of reverse transcription is an hiv preintegration complex ( pic ) , which is then challenged with importing itself into the nucleus . it is still unclear how the pic imports into the nucleus ( dvorin et al . 2002 ; bukrinsky 2004 ; yamashita and emerman 2005 ; yamashita and emerman 2006 ) . 1 the hiv-1 virus binds via its gp120 protein to a host cell s cd4 molecule and a chemokine receptor leading to gp41-mediated viral - host cell membrane fusion . 2 viral and host cell factors mediate the uncoating , reverse transcription and import of the viral preintegration complex through the nuclear pore ( 3 ) . 4 upon arrival in the nucleus , viral genome integration into the host cell s chromosome occurs ( again , mediated by viral and host cell factors ) and transcription invariably begins . 5 while fully spliced rnas are exported as usual to the cytoplasm , singly spliced or unspliced rnas are also exported through the nuclear pore ( 6 ) via a rev - mediated mechanism , again assisted by host proteins . 7 packaging of new virions occurs at the host cell s membrane . 8 in a final fusion event , the newly formed virion is released to continue the infection cycle in a new cell . a future promise to enhance our understanding of these steps in more detail is to investigate interactions between selected components by super - registration dual color smt the life cycle of hiv . 1 the hiv-1 virus binds via its gp120 protein to a host cell s cd4 molecule and a chemokine receptor leading to gp41-mediated viral - host cell membrane fusion . 2 viral and host cell factors mediate the uncoating , reverse transcription and import of the viral preintegration complex through the nuclear pore ( 3 ) . 4 upon arrival in the nucleus , viral genome integration into the host cell s chromosome occurs ( again , mediated by viral and host cell factors ) and transcription invariably begins . 5 while fully spliced rnas are exported as usual to the cytoplasm , singly spliced or unspliced rnas are also exported through the nuclear pore ( 6 ) via a rev - mediated mechanism , again assisted by host proteins . 7 packaging of new virions occurs at the host cell s membrane . 8 in a final fusion event , the newly formed virion is released to continue the infection cycle in a new cell . a future promise to enhance our understanding of these steps in more detail is to investigate interactions between selected components by super - registration dual color smt following transport across the nuclear pore complex viral and host proteins again participate in integration of the viral genome into the host cell genome ( greene and peterlin 2002 ) . while this is primarily mediated by integrase ( in ) , which binds to the ends of the viral dna , host proteins are also involved and required , though their precise functions remain unknown ( kalpana et al . 1994 ; lee and craigie 1994 ; farnet and bushman 1997 ; li et al . 2000 ; suzuki and craigie 2002 ; beitzel and bushman 2003 ; lin and engelman 2003 ) . ledgf / p75 also associates with in and has also been implicated in participating in its nuclear import and/or the integration process ( cherepanov et al . 2003 ; maertens et al . 2003 ) . as the hiv provirus can integrate into many different chromosomal locations in the cell it is tempting to explain viral latency ( vs. transcriptional activity ) as integration into repressed heterochromatin . while this is at least in part true , it is not the whole story ( for reviews on hiv viral latency , see ( han et al . 2007 ; coiras et al . 2009 ; graci et al . 2009 ) ) . in the case of a transcriptionally active integrated provirus , the 5 ltr ( long terminal repeat containing promoter elements ( taube et al . 1999 ) ) positions rna polymerase ii ( rnapii ) at the site of initiation of transcription and is responsible for the assembly of the pre - initiation complex . while transcription can begin with these minimal components , rnapii invariably fails to elongate efficiently ( kao et al . is required which associates with host cyclin t1 , which in turn recruits host cdk9 . tat binds the 5 bulge region of tar ( a 59-nucleotide stem - loop rna element in the ltr ) via its arginine - rich motif and recruitment of p - tef - b ( the complex formed by cyclin t and cdk9 ) results in hyper - phosphorylation of the c - terminal domain of rna polymerase ii , thereby stimulating efficient transcriptional elongation ( zhou et al . the integrated provirus , while only 9 kb long , then successfully expresses 15 distinct viral proteins , facilitated by an elegant and complex splicing mechanism that involves both complete and incomplete splicing ( frankel and young 1998 ) . when the mrna is completely spliced ( encoding for nef , tat , and rev ) it is rapidly transported into the cytoplasm and transcribed ( cullen 1998 ) . when the mrna , however , is singly spliced or unspliced , viral transcripts remain in the nucleus and are relatively stable ( luo et al . 1994 ; powell et al . 1997 ) . while the export of unspliced ( or partially spliced ) rna in eukaryotic cells is usually prohibited , the virus overcomes this block by utilizing the viral protein rev ( custodio et al . the rev protein binds to an rev response element ( rre ) encoded in the viral rna sequence and together with recruited host proteins manages to export the unspliced or partially spliced rna in what is referred to as the rev - rre - crm1 export mechanism ( reviewed in ( hope 1999 ) ) . while the partially ( or singly ) spliced viral transcripts encode the structural enzymatic accessory proteins , the unspliced rna species constitute the genome of newly formed progeny virions , and export of these rnas depend heavily on rev s leucine - rich nes ( nuclear - export sequence ) as well as on host proteins , predominantly ran gtpase ( fornerod et al . 1997 ) . as the viral components needed for new virion formation are assembled in the cytoplasm , shuttling to the plasma membrane ( where budding will occur ) takes place ( reviewed in ( kaplan 2002 ; li and wild 2005 ; mazze and degreve 2006 ) ) . this process is highly reliant on the viral gag polyprotein , but involves , once again , a number of host cell factors , without which the process does not occur ( dussupt et al . the terminal step in the budding reaction involves a second membrane fusion event for the virus , and this too is an orchestrated effort between the viral gag polyprotein and host proteins ( kaplan 2002 ; li and wild 2005 ; mazze and degreve 2006 ) . the virion is then released and free to continue the vicious cycle of infection . where to from here ? as becomes evident from the wealth of information already available , the past several decades of research have defined a large number of host cell proteins that influence every step of the viral life cycle ( ptak et al . 2008 ; fu et al . 2009 ; pinney et al . 2009 ) . initial imaging results on the localization of , for example cd4 and ccr5 on the cellular membrane ( steffens and hope 2003 ; steffens and hope 2004 ) , rna distribution in virions ( chen et al . 2009 ) , and the biogenesis of hiv virions at the plasma membrane ( jouvenet et al . however , a report that investigated in mobility within the nucleus by fcs , also demonstrates the current limitations at the nanometer length- and milliseconds time - scale ( maertens et al . clear examples of how smt could be applied to the study of hiv include analysis of the import and export of viral rna and proteins , e.g. the pic complex , the interaction durations and sites for the rev - rre - crm1 complex , the distribution of genome integration sites or the interplay of tat and rnapii . while the above are good examples of what can be achieved with single - color smt , investigating the interaction times and complex formation of , for instance hiv - rna and rev or in protein , will require simultaneous multi - color smt . the problem with this approach is that while in each individual color single molecules will be localized with high precision , the colocalization of these single - molecule positions will remain diffraction limited , hence in the range of more than 200 nm . while single - molecule motors , imaged directly on the cover glass in solution have been double labeled and registered with sub - diffraction - limited precision ( churchman et al . 2005 ) , this virtually renders multi - color smt in the living cell impossible , as in the living cell specific problems with aberrations and chromatic effects inherent to the cellular environment need to be overcome . this problem appears to be overcome by the recent development of a super - registration technique for single molecules with high spatial precision between spectral channels . using a nuclear pore fluorescent stain it was possible to achieve a 10-nm local registration of nuclear pores and endogenous mrna , labeled with the ms2 system . super - registration allowed following interactions of single mrnas and pores during export and resolving individual transient steps of the export process and their respective rate constants , which were previously undefined ( grunwald and singer 2010 ) . in conclusion , the hiv life cycle demonstrates how important it is to gather a complete picture in time and space of cellular and viral mechanisms that , without microscopy , might not be possible . the horizon for the future should include single - molecule studies on the virus dynamic equilibrium of processes in living cells that , until now , have been out of reach . even more , the perspective of investigating individual complexes and molecular interactions in the living cell at the single - molecule level , e.g. , through developments like super - registration , guides the way towards a quantitative picture of the dynamic equilibrium of cellular life .

cellular life can be described as a dynamic equilibrium of a highly complex network of interacting molecules . for this reason , it is no longer sufficient to only know the identity of the participants in a cellular process , but questions such as where , when , and for how long also have to be addressed to understand the mechanism being investigated . additionally , ensemble measurements may not sufficiently describe individual steps of molecular mobility , spatial - temporal resolution , kinetic parameters , and geographical mapping . it is vital to investigate where individual steps exactly occur to enhance our understanding of the living cell . the nucleus , home too many highly complex multi - order processes , such as replication , transcription , splicing , etc . , provides a complicated , heterogeneous landscape . its dynamics were studied to a new level of detail by fluorescence correlation spectroscopy ( fcs ) . single - molecule tracking , while still in its infancy in cell biology , is becoming a more and more attractive method to deduce key elements of this organelle . here we discuss the potential of tracking single rnas and proteins in the nucleus . their dynamics , localization , and interaction rates will be vital to our understanding of cellular life . to demonstrate this , we provide a review of the hiv life cycle , which is an extremely elegant balance of nuclear and cytoplasmic functions and provides an opportunity to study mechanisms deeply integrated within the structure of the nucleus . in summary , we aim to present a specific , dynamic view of nuclear cellular life based on single molecule and fcs data and provide a prospective for the future .

Introduction Observing single molecules in the nucleus, a possibility? Nuclear mobility as seen by fluorescence correlation spectroscopy Single RNA tracking in the nucleus Single protein tracking in the nucleus Imaging HIV in the living cella perspective The life cycle of HIV

here , transient interactions ( protein protein , protein dna , and protein rna ) result in a stochastic but directional progression of multi - scale and multi - player processes ( elf et al . while this change in paradigm challenges our view of the composition of multi - protein complexes , it also raises questions of how the nuclear space is organized , how the availability of factors is regulated and how interaction sites are found by the right interaction partners . while no membrane - bound compartments ( such as in the cytoplasm ) have been identified specific areas can be labeled , including the nucleolus , pcg bodies , nuclear speckles , cajal bodies , gems , cleavage bodies , perinucleolar compartments , the sam68 nuclear body , pml bodies , etc . as a result , besides crowding effects also found in the cytoplasm , molecules in the nucleus encounter a highly complex , anisotropic landscape ( richter et al . to probe the dynamic functions of this landscape , an ensemble of technologies ( such as frap , flip , confocal imaging etc . ) these data draw a picture of a highly mobile environment with changing fractions of immobilized populations and a continual change in composition . 2006 ) , imaging of single - molecule signals can provide the position of the observed molecule with an accuracy that can overcome the resolution limit by a factor of ten in the living cell , and is in principle capable of providing nanometer precision1 ( bobroff 1986 ; schmidt et al . an immediate advantage is that no synchronization of cellular processes is needed as single - molecule tracking ( smt ) is fast enough to observe the sequence of events in real time and synchronization is achieved during data analysis . finally , by providing superb time and spatial information simultaneously , smt is perhaps the tool of choice when it comes to investigating the dynamic landscape of molecular interactions in the living cell . measurements of nucleocytoplasmic transport have shown definitively that transient interactions in the living cell can be resolved on the nanometer length- and milliseconds time scales using smt . in the same way , single protein factors and rnas in the nucleus have been measured using inert test probes and functional protein factors ( goulian and simon 2000 ; kubitscheck et al . compared with fluorescence correlation spectroscopy ( fcs ) or fluorescence recovery after photobleaching ( frap ) , smt provides the means to analyze multiple forms of mobility in heterogeneous environments . adjustment of the number of labeled molecules is usually done by : providing small amounts of recombinant fluorescently labeled proteins ( e.g. finally , the observation time needs to be maximized , taking photobleaching rates , desirable signal intensity and applicable amounts of light into consideration . depending on the mobility of the observed molecules and the acquisition speed of the microscope system used , the acquired data is then analyzed based on the tracking of individual signals ( see ( grunwald et al . figure 1 details simple modifications that can be applied to commercially available equipment to improve performance for the detection of single molecules in living cells at appropriate time scales . in short , a laser illumination module ( here shown in a customized home - built version , but also commercially available ) is combined with a fast switching mechanism and a fast intensity regulator for the laser light and fiber coupled to the microscope . lastly , a sensitive , fast camera , typically an emccd , is used for recording the single - molecule signal and is mounted as directly as possible onto the microscope to increase photon transmission . the fiber output can either be delivered as a collimated beam to the excitation tube lens or be imaged via a conjugated image plane into the objective s back focal plane . emitted fluorescence light , collected by a high numerical aperture objective , is separated from the excitation light by an appropriate dichromatic beam splitter , which reflects the three excitation lines into the sample , while the three fluorescence bands are transmitted to the detection system . the microscope is mounted on a steel scaffold fixed to the optical table to allow easy access to the base port , where an emccd for single molecule tracking is mounted directly onto the microscope stand collecting signal in the primary image plane . a second camera , located at a side port , a microinjection system allows delivery of fluorescently labeled probes into a living cell an smt microscope setup . emitted fluorescence light , collected by a high numerical aperture objective , is separated from the excitation light by an appropriate dichromatic beam splitter , which reflects the three excitation lines into the sample , while the three fluorescence bands are transmitted to the detection system . the microscope is mounted on a steel scaffold fixed to the optical table to allow easy access to the base port , where an emccd for single molecule tracking is mounted directly onto the microscope stand collecting signal in the primary image plane . a second camera , located at a side port , is used to detect reference images . a microinjection system allows delivery of fluorescently labeled probes into a living cell to summarize , smt and fcs provide the means to study single molecules or very small , locally resolved , ensembles of fluorescently labeled molecules in the living cell . in the following , we discuss our current view of the nuclear landscape , dynamical aspects of the nucleus and point out controversial areas that remain . the first experiment to provide a mobility profile of the nuclear space used fcs and fluorescein - labeled oligodeoxynucleotides ( oligos ) to measure intranuclear diffusion coefficients in living cells ( politz et al . labeled poly - thymidine oligos , that are passively taken up by living cells , formed hybrids with the poly - adenylated tails of rnas ( poly(a)rnas ) in the nucleus , and two major fractions of diffusion rates were observed . in another experiment , the effect of nuclear compartments on the movement of poly(a)rnas in the nucleoplasm was compared to poly(a)rna mobility in speckles ( politz et al . these values are similar to the mobility of the slower class reported in the first study ( table 1 ) . interestingly , temperature reduction from 37c to 22c had no detectable effect on poly(a)rna mobility , and was interpreted as meaning that movement between the nucleoplasm and speckles does not require metabolic energy , which is in stark contrast to a previous report ( calapez et al . for details , see original literature overview of nuclear diffusion coefficients measured by smt and fcs in these studies , diffusion coefficients were fixed to compare the relative amounts of the different mobility classes in different compartments . 2008a using smt , was then used to explore the effects of molecular crowding on diffusion and binding of nuclear proteins in heterochromatin using fcs technology ( bancaud et al . the coat protein binds to the rna sequence with nanomolar affinity thereby generating a multiplexed signal sufficient for single molecule tracking ( stockley et al . the tracking criteria for mrnps was set to more than eight frames of continuous observation , and the mean diffusion coefficient , in stark contrast to the fcs data cited above , was found to be 0.04 m / s at 37c . when very large mrnps were tracked in the nucleus ( dys - mrnp , ranging from 4.8 to 14 kb ) , an even lower mobility of mrnps was found ( d = 0.005 m / s ) ( mor et al . 2008 ) , it is unclear whether these spaces are indeed channels and if they exist over the entire nucleus as predicted ( mor et al . a molecular beacon is composed of a stem hybrid that keeps the fluorophore close to a quencher rendering the probe initially non - fluorescent . when the probe hybridizes to a target sequence , the fluorophore separates from the quencher due to a conformational change , and becomes detectable . in an experiment where imaging was performed at 300 ms integration time , an average diffusion coefficient of 0.033 m / s was found for one half of the observed mrnps , while the other half displayed a stationary behavior with a calculated diffusion coefficient of 0.0006 m / s . in contrast to the above studies , however , when an insect model system ( instead of a mammalian cell system ) was used , i.e. , balbiani ring ( br2 ) mrnas ( extremely large mrnas found in the salivary glands of the chironomus midge ) , four different mobile fractions , widely varying , were found approaching diffusion coefficients comparable to those found by fcs ( table 1 ) ( siebrasse et al . a clear decrease in observation frequency for longer jump distances c diffusion coefficients are a convenient method to extract a mean mobility value , however , it is a rather broad parameter . in cells two or three diffusion coefficients single - molecule observation suggests that individual molecules can adopt different mobile states at different times . here , we present the mean square displacement of a single streptavidin trace ( complete trace black ) , showing that indeed within one observation interval the molecule changes from a trapped immobile state ( blue ) to a mobile , diffusive state ( cyan ) and back to an immobile state ( red ) . e overview of individual nls - streptavidin - cy5 traces in a mecp2-gfp - labeled cell nucleus . different colors indicate distinguishable tracks in the nucleoplasm ( yellow ) , in pericentric heterochromatin ( green ) , and the transition between nucleoplasm and heterochromatin ( blue ) . a displacement - dependent trace analysis of streptavidin in the nucleoplasm . a clear decrease in observation frequency for longer jump distances c diffusion coefficients are a convenient method to extract a mean mobility value , however , it is a rather broad parameter . in cells two or three diffusion coefficients single - molecule observation suggests that individual molecules can adopt different mobile states at different times . here , we present the mean square displacement of a single streptavidin trace ( complete trace black ) , showing that indeed within one observation interval the molecule changes from a trapped immobile state ( blue ) to a mobile , diffusive state ( cyan ) and back to an immobile state ( red ) . e overview of individual nls - streptavidin - cy5 traces in a mecp2-gfp - labeled cell nucleus . different colors indicate distinguishable tracks in the nucleoplasm ( yellow ) , in pericentric heterochromatin ( green ) , and the transition between nucleoplasm and heterochromatin ( blue ) . f the diffusion coefficient was estimated from the mean square displacement versus time of the mobility of the red trace shown in ( e ) as there are such dramatic differences between the fcs data and tracking data of one group versus another , it is very clear that controversy exists in this field , which might be explained by clear technical differences in how the experiments were performed . correspondingly , using frap experiments , a three to five fold reduction of the diffusion coefficient of larger molecules in the nuclei was also found ( gorski et al . from here we conclude that there is not just one kinetic condition for association and dissociation events of biologically active proteins in the nucleus . tagged streptavidin coupled to a nuclear localization sequence ( nls ) with a size of about 60 kda , and a second probe , ovalbumin with a size of 45 kda , were tracked in living cell nuclei ( grunwald et al . different nuclear compartments affect the movement of inert proteins differently , but even in the nucleoplasm , two kinetic components ( mobile and trapped ) were observed , and the mobile fraction is widely spread over a wide range of diffusion coefficients ( the data could not be fitted satisfactorily assuming only one or two diffusing components , table 1 ) . while confocal imaging implies that the nucleolus excludes the test protein , the mobility data suggest minimal trapping in this compartment and combined with no retention of proteins at the compartment interface , exclusion turns out to be a dynamic effect , where mobile proteins leave the nucleolus frequently and fast ( grunwald et al . when ovalbumin was used , an inert protein that does not contain an nls , comparable observations were made demonstrating the limited , if any , impact of the sv40 nls - related electric charge on protein mobility in the nucleus . an important question that can be addressed using single molecule tracking is the mobility of hiv ( human immunodeficiency virus ) related factors , their interaction times and regulation of cellular distribution . we first provide a brief summary of the hiv lifecycle , including import and export processes of hiv genetic material through the nuclear membrane , which is a very elegant symphony between cytoplasmic and nuclear viral and host cell factors . single molecule tracking techniques might enable deeper understanding of the spatial - temporal dynamics of these processes thereby not only providing key information on very basic biological processes , but we surmise , also exposing vulnerabilities that could be targeted in the fight against this devastating disease . the hiv virus is an rna lentivirus with a genome comprising two single - rna strands containing the full genetic information needed for viral replication in host cells ( luciw et al . this binding induces a conformational change to a specific chemokine receptor ( predominantly ccr5 or cxcr4 ( berger et al . this fusion of the viral and infected cell s membranes results in the release of the hiv viral core into the cytoplasm ( chan et al . the result of reverse transcription is an hiv preintegration complex ( pic ) , which is then challenged with importing itself into the nucleus . it is still unclear how the pic imports into the nucleus ( dvorin et al . 1 the hiv-1 virus binds via its gp120 protein to a host cell s cd4 molecule and a chemokine receptor leading to gp41-mediated viral - host cell membrane fusion . 2 viral and host cell factors mediate the uncoating , reverse transcription and import of the viral preintegration complex through the nuclear pore ( 3 ) . 4 upon arrival in the nucleus , viral genome integration into the host cell s chromosome occurs ( again , mediated by viral and host cell factors ) and transcription invariably begins . 8 in a final fusion event , the newly formed virion is released to continue the infection cycle in a new cell . a future promise to enhance our understanding of these steps in more detail is to investigate interactions between selected components by super - registration dual color smt the life cycle of hiv . 1 the hiv-1 virus binds via its gp120 protein to a host cell s cd4 molecule and a chemokine receptor leading to gp41-mediated viral - host cell membrane fusion . 2 viral and host cell factors mediate the uncoating , reverse transcription and import of the viral preintegration complex through the nuclear pore ( 3 ) . 4 upon arrival in the nucleus , viral genome integration into the host cell s chromosome occurs ( again , mediated by viral and host cell factors ) and transcription invariably begins . 8 in a final fusion event , the newly formed virion is released to continue the infection cycle in a new cell . a future promise to enhance our understanding of these steps in more detail is to investigate interactions between selected components by super - registration dual color smt following transport across the nuclear pore complex viral and host proteins again participate in integration of the viral genome into the host cell genome ( greene and peterlin 2002 ) . while this is primarily mediated by integrase ( in ) , which binds to the ends of the viral dna , host proteins are also involved and required , though their precise functions remain unknown ( kalpana et al . as the hiv provirus can integrate into many different chromosomal locations in the cell it is tempting to explain viral latency ( vs. transcriptional activity ) as integration into repressed heterochromatin . while this is at least in part true , it is not the whole story ( for reviews on hiv viral latency , see ( han et al . in the case of a transcriptionally active integrated provirus , the 5 ltr ( long terminal repeat containing promoter elements ( taube et al . 1999 ) ) positions rna polymerase ii ( rnapii ) at the site of initiation of transcription and is responsible for the assembly of the pre - initiation complex . tat binds the 5 bulge region of tar ( a 59-nucleotide stem - loop rna element in the ltr ) via its arginine - rich motif and recruitment of p - tef - b ( the complex formed by cyclin t and cdk9 ) results in hyper - phosphorylation of the c - terminal domain of rna polymerase ii , thereby stimulating efficient transcriptional elongation ( zhou et al . when the mrna is completely spliced ( encoding for nef , tat , and rev ) it is rapidly transported into the cytoplasm and transcribed ( cullen 1998 ) . when the mrna , however , is singly spliced or unspliced , viral transcripts remain in the nucleus and are relatively stable ( luo et al . the terminal step in the budding reaction involves a second membrane fusion event for the virus , and this too is an orchestrated effort between the viral gag polyprotein and host proteins ( kaplan 2002 ; li and wild 2005 ; mazze and degreve 2006 ) . as becomes evident from the wealth of information already available , the past several decades of research have defined a large number of host cell proteins that influence every step of the viral life cycle ( ptak et al . however , a report that investigated in mobility within the nucleus by fcs , also demonstrates the current limitations at the nanometer length- and milliseconds time - scale ( maertens et al . clear examples of how smt could be applied to the study of hiv include analysis of the import and export of viral rna and proteins , e.g. while the above are good examples of what can be achieved with single - color smt , investigating the interaction times and complex formation of , for instance hiv - rna and rev or in protein , will require simultaneous multi - color smt . the problem with this approach is that while in each individual color single molecules will be localized with high precision , the colocalization of these single - molecule positions will remain diffraction limited , hence in the range of more than 200 nm . while single - molecule motors , imaged directly on the cover glass in solution have been double labeled and registered with sub - diffraction - limited precision ( churchman et al . 2005 ) , this virtually renders multi - color smt in the living cell impossible , as in the living cell specific problems with aberrations and chromatic effects inherent to the cellular environment need to be overcome . this problem appears to be overcome by the recent development of a super - registration technique for single molecules with high spatial precision between spectral channels . using a nuclear pore fluorescent stain it was possible to achieve a 10-nm local registration of nuclear pores and endogenous mrna , labeled with the ms2 system . super - registration allowed following interactions of single mrnas and pores during export and resolving individual transient steps of the export process and their respective rate constants , which were previously undefined ( grunwald and singer 2010 ) . in conclusion , the hiv life cycle demonstrates how important it is to gather a complete picture in time and space of cellular and viral mechanisms that , without microscopy , might not be possible . the horizon for the future should include single - molecule studies on the virus dynamic equilibrium of processes in living cells that , until now , have been out of reach . even more , the perspective of investigating individual complexes and molecular interactions in the living cell at the single - molecule level , e.g. , through developments like super - registration , guides the way towards a quantitative picture of the dynamic equilibrium of cellular life .

liver cancer is a significant worldwide health problem and is the sixth most frequently diagnosed cancer in the world . infection with hepatitis b or c virus is the major risk factor for liver cancer , which accounts for more than 85% of cases in developing countries . the incidence rates of liver cancer are increasing in many parts of the world including the usa and central europe , possibly because of the obesity epidemic and the rise in hepatitis c virus infection 1 . a significant proportion of cases of liver cancer are accompanied by serious cirrhosis or liver dysfunction . liver transplantation ( lt ) is considered to be the optimal therapy for small - sized hepatic carcinomas in patients with decompensated liver cirrhosis . to date , the milan criteria have been adopted by the united network of organ sharing ( unos ) as the standard lt selection criteria for patients with hepatocellular carcinoma ( hcc ) 2,3 . recently , it has been heavily investigated whether we can expand the milan criteria to enable more patients to qualify as transplant candidates . indeed , previous studies 49 have shown that moderate expansion of the milan criteria could yield favorable outcomes . -fetoprotein ( afp ) has been widely accepted in the screening of hcc and in the identification of high - risk populations 10 , and carbohydrate antigen 19 - 9 ( ca19 - 9 ) , also called sialylated lewis ( a ) antigen , is a tumor marker for screening of different human cancers in the digestive system 11 . moreover , our own experience with long - term follow - up of hepatic carcinoma patients also confirmed that elevated preoperative levels of afp or ca19 - 9 predicted a poor prognosis in such patients after lt . thus , in the current study , we assessed presurgical serum levels of afp and ca19 - 9 as prognostic markers in the prediction of overall survival ( os ) and relapse - free survival ( rfs ) for patients with hepatic carcinoma after lt . thereafter we tried to add more lt selection criteria for such patients , in addition to the milan criteria . from january 2007 to june 2010 , a total of 237 consecutive patients with histologically proven primary hepatic carcinoma underwent lt at the department of liver surgery , ren ji hospital ( shanghai , china ) . eleven patients were excluded from the current study because of the following reasons : ( i ) seven patients had possible metastatic disease before lt ; ( ii ) two patients had coexistence of hcc and gallbladder carcinoma , confirmed pathologically after lt ; ( iii ) one patient had undergone additional left nephrectomy for concurrent renal carcinoma ; and ( iv ) one patient had undergone combined liver kidney transplantation . ultimately , 226 patients met the eligibility criteria and were enrolled in this study . the clinicopathological data from our prospective lt database were retrospectively reviewed . salvage lt was performed in patients who developed recurrent hepatic carcinoma after the primary liver resection . preoperative downstaging treatment for tumor size reduction included transcatheter arterial chemoembolization , radiofrequency ablation , percutaneous ethanol injection , and stereotactic body radiation therapy ( gamma knife ) . tumor size was measured as the maximal diameter of the largest tumor in the resected specimens . steiner criteria 12 ( grade i , well - differentiated ; grade ii , moderately differentiated ; and grade iii , poorly differentiated ) . the latest measurement results of afp and ca19 - 9 before lt were recorded in the database , and in most patients both results were obtained within 7 days before surgery . a serum ca19 - 9 level greater than 500 u / ml was rare , thus higher values were truncated at this threshold . all the surgical procedures were performed by specialists with experience in the lt technique at the department of liver surgery , ren ji hospital . all patients undergoing living donor lt were operated upon using right liver grafts without the middle hepatic vein . organ donation or transplantation in the study was strictly implemented under the regulation of shanghai organ transplant committee and the declaration of helsinki . all of the living organs were donated with informed consent , and cadaveric donors involved in the study were brain - dead donors or those with no heart beat . after lt , a triple - drug regimen of tacrolimus or cyclosporine ( csa ) combined with methylprednisolone and/or mycophenolate mofetil ( mmf ) was used . immunosuppression was started during surgery with 500 mg methylprednisolone ; this dose was tapered from 240 mg on postoperative day 1 to 40 mg on postoperative day 6 . maintenance prednisone at an initial dose of 20 mg daily was gradually reduced every week and was withdrawn 3 months after transplantation . the initial dose of tacrolimus was 0.060.15 mg / kg / day with a target trough level of 810 ng / ml during the first 30 days . if tacrolimus did not reach the target level , it would be replaced by csa at 610 mg / kg / day . the target c0 and c2 levels for csa were 150200 and 8001200 ng / ml , respectively . the surviving patients were regularly followed up at the clinic : monthly during the first 6 months after lt , every 3 months from the seventh to the 18th month , and every 6 months thereafter . serum levels of afp and ca19 - 9 , chest radiographs , and abdominal ultrasound scans were routinely assessed at each follow - up visit , and abdominal contrast - enhanced computed tomography was performed every 6 months during the first 2 years and annually thereafter . an increased afp or ca19 - 9 level alone was not identified as being indicative of tumor recurrence , but once tumor recurrence had been confirmed , the date at which the afp or ca19 - 9 level began to increase was taken as the date of recurrence . os was calculated from the time of lt until death or the last follow - up contact ; the cut - off date of follow - up was 1 september 2013 . rfs was defined as the duration from lt to the date of a suspected tumor recurrence in patients with eventually confirmed tumor recurrence or to the last follow - up contact in patients without tumor recurrence . the follow - up period ranged from 1 to 78 months , with a median of 36 months . statistical analysis was carried out using spss for windows version 13.0 ( spss inc . , wilk method , and meansd was used to describe the central tendency and dispersion of the measurement data with a normal distribution , whereas median ( range ) was applied to the data without a normal distribution . the equality of survival distributions among different patient groups was tested using the log - rank method . univariate analysis was used to analyze each factor that might have influenced the prognosis of patients with hepatic carcinoma after lt , and any variable identified as statistically significant ( p<0.05 ) in univariate analysis was subjected to multivariate cox analysis , which assessed the independent predictors for os and rfs . from january 2007 to june 2010 , a total of 237 consecutive patients with histologically proven primary hepatic carcinoma underwent lt at the department of liver surgery , ren ji hospital ( shanghai , china ) . eleven patients were excluded from the current study because of the following reasons : ( i ) seven patients had possible metastatic disease before lt ; ( ii ) two patients had coexistence of hcc and gallbladder carcinoma , confirmed pathologically after lt ; ( iii ) one patient had undergone additional left nephrectomy for concurrent renal carcinoma ; and ( iv ) one patient had undergone combined liver kidney transplantation . ultimately , 226 patients met the eligibility criteria and were enrolled in this study . the clinicopathological data from our prospective lt database were retrospectively reviewed . salvage lt was performed in patients who developed recurrent hepatic carcinoma after the primary liver resection . preoperative downstaging treatment for tumor size reduction included transcatheter arterial chemoembolization , radiofrequency ablation , percutaneous ethanol injection , and stereotactic body radiation therapy ( gamma knife ) . tumor size was measured as the maximal diameter of the largest tumor in the resected specimens . steiner criteria 12 ( grade i , well - differentiated ; grade ii , moderately differentiated ; and grade iii , poorly differentiated ) . the latest measurement results of afp and ca19 - 9 before lt were recorded in the database , and in most patients both results were obtained within 7 days before surgery . a serum ca19 - 9 level greater than 500 u / ml was rare , thus higher values were truncated at this threshold . all the surgical procedures were performed by specialists with experience in the lt technique at the department of liver surgery , ren ji hospital . all patients undergoing living donor lt were operated upon using right liver grafts without the middle hepatic vein . organ donation or transplantation in the study was strictly implemented under the regulation of shanghai organ transplant committee and the declaration of helsinki . all of the living organs were donated with informed consent , and cadaveric donors involved in the study were brain - dead donors or those with no heart beat . after lt , a triple - drug regimen of tacrolimus or cyclosporine ( csa ) combined with methylprednisolone and/or mycophenolate mofetil ( mmf ) was used . immunosuppression was started during surgery with 500 mg methylprednisolone ; this dose was tapered from 240 mg on postoperative day 1 to 40 mg on postoperative day 6 . maintenance prednisone at an initial dose of 20 mg daily was gradually reduced every week and was withdrawn 3 months after transplantation . the initial dose of tacrolimus was 0.060.15 mg / kg / day with a target trough level of 810 ng / ml during the first 30 days . if tacrolimus did not reach the target level , it would be replaced by csa at 610 mg / kg / day . the target c0 and c2 levels for csa were 150200 and 8001200 ng / ml , respectively . the surviving patients were regularly followed up at the clinic : monthly during the first 6 months after lt , every 3 months from the seventh to the 18th month , and every 6 months thereafter . serum levels of afp and ca19 - 9 , chest radiographs , and abdominal ultrasound scans were routinely assessed at each follow - up visit , and abdominal contrast - enhanced computed tomography was performed every 6 months during the first 2 years and annually thereafter . an increased afp or ca19 - 9 level alone was not identified as being indicative of tumor recurrence , but once tumor recurrence had been confirmed , the date at which the afp or ca19 - 9 level began to increase was taken as the date of recurrence . os was calculated from the time of lt until death or the last follow - up contact ; the cut - off date of follow - up was 1 september 2013 . rfs was defined as the duration from lt to the date of a suspected tumor recurrence in patients with eventually confirmed tumor recurrence or to the last follow - up contact in patients without tumor recurrence . the follow - up period ranged from 1 to 78 months , with a median of 36 months . statistical analysis was carried out using spss for windows version 13.0 ( spss inc . , wilk method , and meansd was used to describe the central tendency and dispersion of the measurement data with a normal distribution , whereas median ( range ) was applied to the data without a normal distribution . the equality of survival distributions among different patient groups was tested using the log - rank method . univariate analysis was used to analyze each factor that might have influenced the prognosis of patients with hepatic carcinoma after lt , and any variable identified as statistically significant ( p<0.05 ) in univariate analysis was subjected to multivariate cox analysis , which assessed the independent predictors for os and rfs . specifically , there were 192 male ( 85.0% ) and 34 female ( 15.0% ) patients , with a mean age of 50.2 ( 8.8 ) years . two hundred and twenty - three ( 98.7% ) patients were confirmed to have had liver cirrhosis during lt . eighty - five ( 37.6% ) patients had a preoperative serum afp level higher than 400 ng / ml and 32 ( 14.2% ) patients had a preoperative ca19 - 9 level greater than 100 u / ml . further , according to the child pugh classification , 86 ( 38.1% ) patients were of child s class a , 100 ( 44.2% ) patients were of child s class b , and 40 ( 17.7% ) patients were of child s class c. the majority of the patients ( 54.9% ) had a model for end - stage liver disease score of 1019 . the most common etiology of cirrhosis was hepatitis b virus infection , which accounted for 217 of 226 cases ( 96.0% ) . there were two ( 0.9% ) patients with hepatitis c infection , one ( 0.4% ) with hepatitis b and c coinfection , two ( 0.9% ) with alcoholic liver disease , one ( 0.4% ) with autoimmune hepatitis , and three ( 1.3% ) with idiopathic liver cirrhosis . baseline characteristics of patients ( n=226 ) table 2 shows the details of the entire cohort s histopathologic features . there were 119 ( 52.7% ) patients who did not fulfill the milan criteria . fifty - four ( 23.9% ) patients and 25 ( 11.1% ) patients had a maximum tumor size of 510 cm and greater than 10 cm , respectively , whereas 40 ( 17.7% ) patients were identified as having a vascular invasion and 21 ( 9.3% ) patients as having an extrahepatic invasion . one - year and 5-year os rates among these 226 patients were 79.0 and 58.0% , respectively , whereas rfs rates were 70.3 and 62.2% , respectively . histopathologic features of patients ( n=226 ) to define tmt , we assessed os and rfs of these patients with different preoperative serum levels of afp or ca19 - 9 by interaction between preoperative afp and ca19 - 9 levels and os and rfs rates . we first generated a receiver operating characteristic ( roc ) curve for ca19 - 9 levels . 1 , the area under the roc curve ( area=0.613 , p=0.005 ) showed that an elevated preoperative serum level of ca19 - 9 was a useful predictor for high mortality of hepatic carcinoma patients within 3 years after lt . to prevent false - positive results in such a high - risk population of tumor recurrence , we used 100 u / ml as the cut - off value for the preoperative serum level of ca19 - 9 in this study . the data showed that those patients with a preoperative ca19 - 9 level greater than 100 u / ml had a significantly worse prognosis than those with a ca19 - 9 level of 100 u / ml or lower ( 5-year os : 32.4 vs. 62.2% , p<0.001 ; 5-year rfs : 35.1 vs. 66.1% , p<0.001 ; fig . 2 ) . however , 26 of 32 patients ( 81.3% ) with an elevated ca19 - 9 level fell outside the milan criteria . further , there were seven non - hcc patients enrolled in this study , including five patients with intrahepatic cholangiocarcinoma ( icc ) and two patients with combined hcc - cholangiocarcinoma ( chcc - cc ) . the mean levels of preoperative ca19 - 9 in hcc and non - hcc ( n=219 vs. 7 ) patients were 65.9 and 259.8 u / ml , respectively ( p=0.052 ) . in addition , we adopted a cut - off value of 400 ng / ml for the afp level . serum levels of preoperative afp of 400 ng / ml or lower versus greater than 400 ng / ml showed a significant survival benefit on both 5-year os ( 69.5 vs. 38.7% , p<0.001 ) and rfs ( 76.1 vs. 40.3% , p<0.001 ; fig . , we analyzed os and rfs of these patients according to afp and ca19 - 9 levels , which led to their segregation into four groups : group 1 patients ( n=13 ) had elevated levels of both preoperative afp ( > 400 ng / ml ) and ca19 - 9 ( > 100 u / ml ) ; group 2 ( n=72 ) had a preoperative afp level of greater than 400 ng / ml but a ca19 - 9 level of 100 u / ml or lower ; group 3 ( n=19 ) had a preoperative afp level of 400 ng / ml or lower , but a high level ( > 100 u / ml ) of preoperative ca19 - 9 ; and group 4 ( n=122 ) had low levels of both afp and ca19 - 9 ( 400 ng / ml and 100 u / ml , respectively ) . the data on os and rfs of these four groups of patients are summarized in table 3 and fig . 4 . patients in group 4 reached 1-year and 5-year os rates of 89.9 and 74.6% , respectively , whereas the rfs rates were 84.9 and 78.5% , respectively . the data showed that these patients had significant survival advantages compared with those in each of the other three groups ( os : p14<0.001 , p24<0.001 , p34<0.001 ; rfs : p14<0.001 , p24<0.001 , p34=0.021 ; table 3 ) . moreover , both 5-year os ( 41.2 vs. 36.8% , p=0.118 ) and rfs ( 45.3 vs. 56.1% , p=0.649 ) rates were equivalent between groups 2 and 3 . however , patients in group 1 ( 5-year os and rfs rates=25.4 and 15.4% ) showed the worst prognosis among these four groups of patients . thus , on the basis of these data , tmt was defined , and hepatic carcinoma patients could be classified on the basis of tmt as follows : type negative ( n ) , 122 patients ( group 4 ) ; type single positive ( sp ) , 91 patients ( groups 2 and 3 ) ; and type double positive ( dp ) , 13 patients ( group 1 ) . the os and rfs rates of patients in the n , sp , and dp groups are shown in fig . 5 . the roc curve of the serum level of preoperative ca19 - 9 and patient survival within 3 years after lt . the area under the roc curve ( area=0.613 , p=0.005 ) showed that an elevated preoperative ca19 - 9 level was a significant predictor for high mortality of patients within 3 years after lt . ca19 - 9 , carbohydrate antigen 19 - 9 ; lt , liver transplantation ; roc , receiver operating characteristic . comparison of os and rfs between patients with a preoperative ca19 - 9 level 100 u / ml and those with a preoperative ca19 - 9 level > 100 u / ml . ( a ) the os rates ( 5-year : 62.2 vs. 32.4% , p<0.001 ) ; ( b ) the rfs rates ( 5-year : 66.1 vs. 35.1% , p<0.001 ) . ca19 - 9 , carbohydrate antigen 19 - 9 ; os , overall survival ; rfs , relapse - free survival . comparison of os and rfs between patients with a preoperative afp level 400 ng / ml and those with a preoperative afp level > 400 ng / ml . ( a ) the os rates ( 5-year : 69.5 vs. 38.7% , p<0.001 ) ; ( b ) the rfs rates ( 5-year : 76.1 vs. 40.3% , p<0.001 ) . afp , -fetoprotein ; os , overall survival ; rfs , relapse - free survival . os and rfs rates of group 1 to 4 of patients comparison of os and rfs among patients of groups 1 to 4 . ( a ) the os rates ( p12=0.120 , p23=0.118 , p13=0.922 , p34<0.001 , p14<0.001 , p24<0.001 ) ; ( b ) the rfs rates ( p12=0.037 , p23=0.649 , p13=0.071 , p34=0.021 , p14<0.001 , p24<0.001 ) . group 1 ( n=13 ) , afp of > 400 ng / ml+ca19 - 9 of > 100 u / ml ; group 2 ( n=72 ) , afp of > 400 ng / ml+ca19 - 9 of 100 u / ml ; group 3 ( n=19 ) , afp of 400 ng / ml+ca19 - 9 of > 100 u / ml ; group 4 ( n=122 ) , afp of 400 ng / ml+ca19 - 9 of 100 u / ml . afp , -fetoprotein ; ca19 - 9 , carbohydrate antigen 19 - 9 ; os , overall survival ; rfs , relapse - free survival . comparison of os and rfs among patients in the n , sp , and dp groups . ( a ) the os rates ( 5-year , 74.6 vs. 40.5 vs. 25.4% ; pn dp=0.233 ) ; ( b ) the rfs rates ( 5-year , 78.5 vs. 47.1 vs. 15.4% ; pn sp<0.001 , pn dp , double positive ; n , negative ; os , overall survival ; rfs , relapse - free survival ; sp , single positive . we entered these tmt and clinicopathological data into multiple cox regression models of os and rfs as covariates . a total of 14 variables that might affect the os or rfs of patients with hepatic carcinoma were subjected to univariate analysis , including age ( 50 or > 50 years ) , sex ( male or female ) , child pugh class ( a , b , or c ) , model for end - stage liver disease score ( < 10 , 1019 , or 20 ) , primary or salvage lt , preoperative downstaging treatment ( yes or no ) , surgical technique ( living donor lt or deceased donor lt ) , tmt ( type n , sp , or dp ) , tumor pathological type ( hcc or non - hcc ) , tumor size ( 5 , 510 , or > 10 cm ) , tumor number ( single or multiple ) , extrahepatic invasion ( presence or absence ) , vascular invasion ( presence or absence ) , and histopathologic grading ( i ii or iii ) . we found that six variables were significant predictors for both os and rfs , including tmt ( type n , sp , or dp ) , tumor size ( 5 , 510 , or > 10 cm ) , tumor number ( single or multiple ) , extrahepatic invasion ( presence or absence ) , vascular invasion ( presence or absence ) , and histopathologic grading ( i ii or iii ) . in addition , tumor pathological type ( hcc or non - hcc ) was also a significant predictor for os ( table 4 ) . the multivariate cox analysis showed that tmt , tumor size , and extrahepatic invasion were all independent predictors for os and rfs of these patients , whereas vascular invasion was an independent predictor for rfs ( table 5 ) . univariate analysis of variables that significantly affected os or rfs independent variables in the multivariate analysis for os and rfs to propose lt eligibility criteria for patients with hepatic carcinoma , in addition to the milan criteria , using our current data , we further exploited tmt as a prognostic predictor for survival in these patients and found that the n - group patients were a special subgroup with a favorable prognosis . therefore , we proposed additional criteria for lt eligibility for hepatic carcinoma patients who did not meet the milan criteria , which could consist of those within the n group , who were free from vascular invasion and extrahepatic metastasis , regardless of tumor size and number . the os rates of patients meeting the milan criteria ( n=107 ) and those exceeding the milan criteria but fulfilling the proposed criteria ( n=30 ) were 87.7 versus 96.6% for 1 year and 77.8 versus 79.3% for 5 years , respectively ( p=0.862 ) , whereas the rfs rates were 90.7 versus 86.0% for 1 year and 85.5 versus 75.1% for 5 years , respectively ( p=0.210 ) . in contrast , patients who did not fulfill both criteria ( n=89 ) showed poor prognostic outcomes ( 1-year os and rfs rates=61.7 and 39.8% , and 5-year os and rfs rates=23.6 and 28.8% ) . the median tumor size in patients exceeding the milan criteria but fulfilling the proposed criteria was 5.8 cm ( range from 1.0 to 14.0 cm ) , and multiple tumor lesions occurred in 18 ( 60% ) patients . only two patients with a tumor greater than 10 cm were found among these 30 newly proposed eligible patients for lt , with one patient achieving long - term survival and the other dying of tumor recurrence 19 months after lt . specifically , there were 192 male ( 85.0% ) and 34 female ( 15.0% ) patients , with a mean age of 50.2 ( 8.8 ) years . two hundred and twenty - three ( 98.7% ) patients were confirmed to have had liver cirrhosis during lt . eighty - five ( 37.6% ) patients had a preoperative serum afp level higher than 400 ng / ml and 32 ( 14.2% ) patients had a preoperative ca19 - 9 level greater than 100 u / ml . further , according to the child pugh classification , 86 ( 38.1% ) patients were of child s class a , 100 ( 44.2% ) patients were of child s class b , and 40 ( 17.7% ) patients were of child s class c. the majority of the patients ( 54.9% ) had a model for end - stage liver disease score of 1019 . the most common etiology of cirrhosis was hepatitis b virus infection , which accounted for 217 of 226 cases ( 96.0% ) . there were two ( 0.9% ) patients with hepatitis c infection , one ( 0.4% ) with hepatitis b and c coinfection , two ( 0.9% ) with alcoholic liver disease , one ( 0.4% ) with autoimmune hepatitis , and three ( 1.3% ) with idiopathic liver cirrhosis . baseline characteristics of patients ( n=226 ) table 2 shows the details of the entire cohort s histopathologic features . there were 119 ( 52.7% ) patients who did not fulfill the milan criteria . fifty - four ( 23.9% ) patients and 25 ( 11.1% ) patients had a maximum tumor size of 510 cm and greater than 10 cm , respectively , whereas 40 ( 17.7% ) patients were identified as having a vascular invasion and 21 ( 9.3% ) patients as having an extrahepatic invasion . one - year and 5-year os rates among these 226 patients were 79.0 and 58.0% , respectively , whereas rfs rates were 70.3 and 62.2% , respectively . to define tmt , we assessed os and rfs of these patients with different preoperative serum levels of afp or ca19 - 9 by interaction between preoperative afp and ca19 - 9 levels and os and rfs rates . we first generated a receiver operating characteristic ( roc ) curve for ca19 - 9 levels . 1 , the area under the roc curve ( area=0.613 , p=0.005 ) showed that an elevated preoperative serum level of ca19 - 9 was a useful predictor for high mortality of hepatic carcinoma patients within 3 years after lt . to prevent false - positive results in such a high - risk population of tumor recurrence , we used 100 u / ml as the cut - off value for the preoperative serum level of ca19 - 9 in this study . the data showed that those patients with a preoperative ca19 - 9 level greater than 100 u / ml had a significantly worse prognosis than those with a ca19 - 9 level of 100 u / ml or lower ( 5-year os : 32.4 vs. 62.2% , p<0.001 ; 5-year rfs : 35.1 vs. 66.1% , p<0.001 ; fig . 2 ) . however , 26 of 32 patients ( 81.3% ) with an elevated ca19 - 9 level fell outside the milan criteria . further , there were seven non - hcc patients enrolled in this study , including five patients with intrahepatic cholangiocarcinoma ( icc ) and two patients with combined hcc - cholangiocarcinoma ( chcc - cc ) . the mean levels of preoperative ca19 - 9 in hcc and non - hcc ( n=219 vs. 7 ) patients were 65.9 and 259.8 u / ml , respectively ( p=0.052 ) . in addition , we adopted a cut - off value of 400 ng / ml for the afp level . serum levels of preoperative afp of 400 ng / ml or lower versus greater than 400 ng / ml showed a significant survival benefit on both 5-year os ( 69.5 vs. 38.7% , p<0.001 ) and rfs ( 76.1 vs. 40.3% , p<0.001 ; fig . thereafter , we analyzed os and rfs of these patients according to afp and ca19 - 9 levels , which led to their segregation into four groups : group 1 patients ( n=13 ) had elevated levels of both preoperative afp ( > 400 ng / ml ) and ca19 - 9 ( > 100 u / ml ) ; group 2 ( n=72 ) had a preoperative afp level of greater than 400 ng / ml but a ca19 - 9 level of 100 u / ml or lower ; group 3 ( n=19 ) had a preoperative afp level of 400 ng / ml or lower , but a high level ( > 100 u / ml ) of preoperative ca19 - 9 ; and group 4 ( n=122 ) had low levels of both afp and ca19 - 9 ( 400 ng / ml and 100 u / ml , respectively ) . the data on os and rfs of these four groups of patients are summarized in table 3 and fig . 4 . patients in group 4 reached 1-year and 5-year os rates of 89.9 and 74.6% , respectively , whereas the rfs rates were 84.9 and 78.5% , respectively . the data showed that these patients had significant survival advantages compared with those in each of the other three groups ( os : p14<0.001 , p24<0.001 , p34<0.001 ; rfs : p14<0.001 , p24<0.001 , p34=0.021 ; table 3 ) . moreover , both 5-year os ( 41.2 vs. 36.8% , p=0.118 ) and rfs ( 45.3 vs. 56.1% , p=0.649 ) rates were equivalent between groups 2 and 3 . however , patients in group 1 ( 5-year os and rfs rates=25.4 and 15.4% ) showed the worst prognosis among these four groups of patients . thus , on the basis of these data , tmt was defined , and hepatic carcinoma patients could be classified on the basis of tmt as follows : type negative ( n ) , 122 patients ( group 4 ) ; type single positive ( sp ) , 91 patients ( groups 2 and 3 ) ; and type double positive ( dp ) , 13 patients ( group 1 ) . the os and rfs rates of patients in the n , sp , and dp groups are shown in fig . the roc curve of the serum level of preoperative ca19 - 9 and patient survival within 3 years after lt . the area under the roc curve ( area=0.613 , p=0.005 ) showed that an elevated preoperative ca19 - 9 level was a significant predictor for high mortality of patients within 3 years after lt . ca19 - 9 , carbohydrate antigen 19 - 9 ; lt , liver transplantation ; roc , receiver operating characteristic . comparison of os and rfs between patients with a preoperative ca19 - 9 level 100 u / ml and those with a preoperative ca19 - 9 level > 100 u / ml . ( a ) the os rates ( 5-year : 62.2 vs. 32.4% , p<0.001 ) ; ( b ) the rfs rates ( 5-year : 66.1 vs. 35.1% , p<0.001 ) . ca19 - 9 , carbohydrate antigen 19 - 9 ; os , overall survival ; rfs , relapse - free survival . comparison of os and rfs between patients with a preoperative afp level 400 ng / ml and those with a preoperative afp level > 400 ng / ml . ( a ) the os rates ( 5-year : 69.5 vs. 38.7% , p<0.001 ) ; ( b ) the rfs rates ( 5-year : 76.1 vs. 40.3% , p<0.001 ) . afp , -fetoprotein ; os , overall survival ; rfs , relapse - free survival . os and rfs rates of group 1 to 4 of patients comparison of os and rfs among patients of groups 1 to 4 . ( a ) the os rates ( p12=0.120 , p23=0.118 , p13=0.922 , p34<0.001 , p14<0.001 , p24<0.001 ) ; ( b ) the rfs rates ( p12=0.037 , p23=0.649 , p13=0.071 , p34=0.021 , p14<0.001 , p24<0.001 ) . group 1 ( n=13 ) , afp of > 400 ng / ml+ca19 - 9 of > 100 u / ml ; group 2 ( n=72 ) , afp of > 400 ng / ml+ca19 - 9 of 100 u / ml ; group 3 ( n=19 ) , afp of 400 ng / ml+ca19 - 9 of > 100 u / ml ; group 4 ( n=122 ) , afp of 400 ng / ml+ca19 - 9 of 100 u / ml . afp , -fetoprotein ; ca19 - 9 , carbohydrate antigen 19 - 9 ; os , overall survival ; rfs , relapse - free survival . comparison of os and rfs among patients in the n , sp , and dp groups . ( a ) the os rates ( 5-year , 74.6 vs. 40.5 vs. 25.4% ; pn dp=0.233 ) ; ( b ) the rfs rates ( 5-year , 78.5 vs. 47.1 vs. 15.4% ; pn sp<0.001 , pn dp , double positive ; n , negative ; os , overall survival ; rfs , relapse - free survival ; sp , single positive . we entered these tmt and clinicopathological data into multiple cox regression models of os and rfs as covariates . a total of 14 variables that might affect the os or rfs of patients with hepatic carcinoma were subjected to univariate analysis , including age ( 50 or > 50 years ) , sex ( male or female ) , child pugh class ( a , b , or c ) , model for end - stage liver disease score ( < 10 , 1019 , or 20 ) , primary or salvage lt , preoperative downstaging treatment ( yes or no ) , surgical technique ( living donor lt or deceased donor lt ) , tmt ( type n , sp , or dp ) , tumor pathological type ( hcc or non - hcc ) , tumor size ( 5 , 510 , or > 10 cm ) , tumor number ( single or multiple ) , extrahepatic invasion ( presence or absence ) , vascular invasion ( presence or absence ) , and histopathologic grading ( i ii or iii ) . we found that six variables were significant predictors for both os and rfs , including tmt ( type n , sp , or dp ) , tumor size ( 5 , 510 , or > 10 cm ) , tumor number ( single or multiple ) , extrahepatic invasion ( presence or absence ) , vascular invasion ( presence or absence ) , and histopathologic grading ( i ii or iii ) . in addition , tumor pathological type ( hcc or non - hcc ) was also a significant predictor for os ( table 4 ) . the multivariate cox analysis showed that tmt , tumor size , and extrahepatic invasion were all independent predictors for os and rfs of these patients , whereas vascular invasion was an independent predictor for rfs ( table 5 ) . univariate analysis of variables that significantly affected os or rfs independent variables in the multivariate analysis for os and rfs to propose lt eligibility criteria for patients with hepatic carcinoma , in addition to the milan criteria , using our current data , we further exploited tmt as a prognostic predictor for survival in these patients and found that the n - group patients were a special subgroup with a favorable prognosis . therefore , we proposed additional criteria for lt eligibility for hepatic carcinoma patients who did not meet the milan criteria , which could consist of those within the n group , who were free from vascular invasion and extrahepatic metastasis , regardless of tumor size and number . the os rates of patients meeting the milan criteria ( n=107 ) and those exceeding the milan criteria but fulfilling the proposed criteria ( n=30 ) were 87.7 versus 96.6% for 1 year and 77.8 versus 79.3% for 5 years , respectively ( p=0.862 ) , whereas the rfs rates were 90.7 versus 86.0% for 1 year and 85.5 versus 75.1% for 5 years , respectively ( p=0.210 ) . in contrast , patients who did not fulfill both criteria ( n=89 ) showed poor prognostic outcomes ( 1-year os and rfs rates=61.7 and 39.8% , and 5-year os and rfs rates=23.6 and 28.8% ) . the median tumor size in patients exceeding the milan criteria but fulfilling the proposed criteria was 5.8 cm ( range from 1.0 to 14.0 cm ) , and multiple tumor lesions occurred in 18 ( 60% ) patients . only two patients with a tumor greater than 10 cm were found among these 30 newly proposed eligible patients for lt , with one patient achieving long - term survival and the other dying of tumor recurrence 19 months after lt . in 1996 , mazzaferro et al . 2 introduced the milan criteria ( i.e. single nodule 5 cm , or no more than three nodules , with each measuring 3 cm or less ) on the basis of a retrospective study of 48 patients who underwent lt for hcc , and the milan criteria have been used thereafter as a guideline for candidate selection for lt in many transplant centers worldwide . thus , patients with liver cancer who meet the milan criteria are expected to have a low rate of tumor recurrence . it is true that more stringent selection criteria for lt could achieve a lower tumor recurrence rate , but at the expense of excluding more patients from receiving lt . a previous multicenter study conducted at seven us transplant centers showed that only 65% of hcc patients who underwent lt met the milan criteria in the usa 13 . moreover , japanese studies had expanded the criteria to include hcc patients with more than three lesions 6,7 , and more than half of the hcc patients who underwent lt exceeded the milan criteria in japan 14 . in the current study , 119 ( 52.7% ) 9 ( hangzhou criteria ) on the basis of lt for hepatitis b - related hcc patients in mainland china . dilute effect that is , a majority of hcc patients who fulfilled the milan criteria were included in the hangzhou group , and a separate comparison between the newly proposed eligible patients for lt and the milan group of patients was absent in the study . indeed , our previous study 15 verified both the expanded criteria in patients with hepatitis b - related hcc using a prospectively collected database , and the data suggested that the 1- , 3- , and 5-year recurrence rates of the newly eligible patients selected using the shanghai or hangzhou criteria were significantly higher than those among patients fulfilling the milan criteria . thus , it is important to efficiently and accurately identify the patients with favorable prognosis from those outside the milan criteria . toward this , the serum level of afp is the most commonly used biomarker to assist in hcc diagnosis and is used as a screening tool for hcc in patients with chronic liver disease 16,17 . a previous study showed that a persistently elevated afp level is a risk factor for hcc development and helps identify high - risk populations 10 . however , afp lacks specificity in hcc diagnosis because its levels may also be high in patients with liver cirrhosis 18 . a higher cut - off value of afp may increase its specificity , whereas the sensitivity drops remarkably 16 . thus , use of the serum afp level alone is not recommended for the diagnosis of hcc , whereas the afp level has been demonstrated to have a predictive value for prognosis in patients with liver cancer . in the prognostic scoring system proposed by the cancer of the liver italian program group ( clip ) on the basis of retrospective evaluation of 435 italian hcc patients , afp was used as an important prognostic factor for hcc patients and was included in the clip scoring system 19 . it was advised by the recent easl eortc clinical practice guidelines to test the level of afp for poor prognosis of hcc patients using greater than 400 ng / ml as a predictor 20 . thus , in the current study , we used the cut - off value of 400 ng / ml and our data further confirm the prognostic value of afp . further , ca19 - 9 has important diagnostic value in the detection of cholangiocarcinoma in primary sclerosing cholangitis 2123 , and similar data have also been obtained for patients without primary sclerosing cholangitis 24 . moreover , persistently elevated ca19 - 9 levels had a strong predictive value for a poor prognosis of hepatobiliary malignancy 2528 . our current data also supported the predictive value of ca19 - 9 in hepatic carcinoma patients . nevertheless , the sensibility and specificity in tumor diagnosis increased considerably by combination of two or more serum tumor markers 21,29 . in our study , the area under the roc curve showed that the preoperative ca19 - 9 level significantly affected the post - lt survival rate of patients . the 5-year os and rfs rates of patients with isolated increase in ca19 - 9 ( group 3 ) were significantly lower than in those with neither biomarker elevated ( group 4 ) . conceivably , a single measurement of the serum afp can not be used as an accurate predictive marker for the prognosis of liver cancer patients , whereas the combination of afp and ca19 - 9 will greatly improve the prognostic prediction . we therefore proposed selection of patients with hepatic carcinoma who exceeded the milan criteria using preoperative serum afp and ca19 - 9 levels . it should be noted that an elevated ca19 - 9 level occurred more commonly in icc or chcc - cc patients . however , when we removed the seven non - hcc patients from the study , the new results based on the 219 hcc patients were almost the same as the initial findings of the study . in addition , the conclusion was also found to be applicable to patients with icc or chcc - cc . however , being a retrospective study , we could not obtain afp or ca19 - 9 levels at different time points before and after surgery to perform a time - dependent analysis . moreover , the difference in os rates between dp and sp groups of patients failed to reach statistical significance probably because of a limited patient number of the dp group , although tmt correlated extremely well with the rfs rates after lt . in addition , in the current study , tumor size also remained an independent factor for the survival of patients on the basis of the multivariate cox analysis . using newly established lt eligibility criteria , two patients with a tumor larger than 10 cm were found among the 30 newly eligible patients for lt , one of whom with a maximum tumor size of 14 cm achieved long - term survival . most high - risk patients for tumor recurrence with large tumors had been filtered out by the proposed criteria , and thus , we did not set a limitation of tumor size and number in the criteria . as expected , the 5-year os and rfs rates were similar between patients within the milan criteria and the newly eligible patients fulfilling the proposed criteria . the prognostic relevance of other serum markers ( such as des--carboxyprothrombin , afp - l3 fraction , vascular endothelial growth factor , and angiopoietin 2 ) was also investigated 3032 , but none of these markers were recommended to survey patients for risk for developing hcc at present . further studies may evaluate their prognostic values in liver cancer patients . in conclusion , data from this study show that the combination of afp and ca19 - 9 is able to predict os and rfs of hepatic carcinoma patients after lt and that the tmt based on preoperative serum levels of afp and ca19 - 9 could be a useful tool to select hepatic carcinoma patients for lt , especially those exceeding the milan criteria . however , further prospective studies conducted on a large scale are needed to verify the new criteria and to use these two markers to predict survival of patients with hepatic carcinoma . in conclusion , data from this study show that the combination of afp and ca19 - 9 is able to predict os and rfs of hepatic carcinoma patients after lt and that the tmt based on preoperative serum levels of afp and ca19 - 9 could be a useful tool to select hepatic carcinoma patients for lt , especially those exceeding the milan criteria . however , further prospective studies conducted on a large scale are needed to verify the new criteria and to use these two markers to predict survival of patients with hepatic carcinoma . qiang xia has received grants from the training program for superb academic leaders in shanghai health system ( no . xbr2011029 ) and the special fund for building of leading talent teams in shanghai , which supported this research . for the remaining authors

objectivethe aim of this study was to assess serum levels of presurgical -fetoprotein ( afp ) and carbohydrate antigen 19 - 9 ( ca19 - 9 ) as prognostic markers in patients with hepatic carcinoma after liver transplantation ( lt).methodsa total of 226 patients were recruited for the analysis of serum afp and ca19 - 9 levels , on the basis of which the tumor marker type ( tmt ) was defined and evaluated for prognostic prediction . overall survival ( os ) and relapse - free survival ( rfs ) were analyzed using kaplan meier curves , and univariate and multivariate cox models.resultsone-year and 5-year os were 79.0 and 58.0% , respectively , whereas rfs were 70.3 and 62.2% , respectively , in this cohort of patients . there were six variables predicting both os and rfs , including tmt , tumor size , number of tumor lesions , extrahepatic or vascular invasion , and histopathological grade . among these , tmt , tumor size , and extrahepatic invasion were all independent predictors of os and rfs among these patients . further , on the basis of tmt , novel lt selection criteria for patients with hepatic carcinoma , which supplemented the milan criteria , were adopted , because the patients within the milan criteria ( n=107 ) and those exceeding milan but fulfilling the proposed criteria ( n=30 ) had similar 5-year os ( 77.8 vs. 79.3% , p=0.862 ) and rfs ( 85.5 vs. 75.1% , p=0.210 ) rates.conclusionthe data from this study showed that serum levels of preoperative afp and ca19 - 9 were able to predict survival of patients with hepatic carcinoma after lt . this study included novel criteria , adding serum afp and ca19 - 9 levels to the selection criteria for lt eligibility of patients , in addition to the milan criteria .

Introduction Patients and methods Study population and data collections Liver transplantation surgery Immunosuppressive treatment Patients follow-up and study endpoint Statistical analysis Results Patient characteristics Definition of the tumor marker type (TMT) Predictors for survival The proposed criteria for liver transplantation candidates Discussion Conclusion Conflicts of interest

to date , the milan criteria have been adopted by the united network of organ sharing ( unos ) as the standard lt selection criteria for patients with hepatocellular carcinoma ( hcc ) 2,3 . -fetoprotein ( afp ) has been widely accepted in the screening of hcc and in the identification of high - risk populations 10 , and carbohydrate antigen 19 - 9 ( ca19 - 9 ) , also called sialylated lewis ( a ) antigen , is a tumor marker for screening of different human cancers in the digestive system 11 . thus , in the current study , we assessed presurgical serum levels of afp and ca19 - 9 as prognostic markers in the prediction of overall survival ( os ) and relapse - free survival ( rfs ) for patients with hepatic carcinoma after lt . thereafter we tried to add more lt selection criteria for such patients , in addition to the milan criteria . serum levels of afp and ca19 - 9 , chest radiographs , and abdominal ultrasound scans were routinely assessed at each follow - up visit , and abdominal contrast - enhanced computed tomography was performed every 6 months during the first 2 years and annually thereafter . univariate analysis was used to analyze each factor that might have influenced the prognosis of patients with hepatic carcinoma after lt , and any variable identified as statistically significant ( p<0.05 ) in univariate analysis was subjected to multivariate cox analysis , which assessed the independent predictors for os and rfs . serum levels of afp and ca19 - 9 , chest radiographs , and abdominal ultrasound scans were routinely assessed at each follow - up visit , and abdominal contrast - enhanced computed tomography was performed every 6 months during the first 2 years and annually thereafter . univariate analysis was used to analyze each factor that might have influenced the prognosis of patients with hepatic carcinoma after lt , and any variable identified as statistically significant ( p<0.05 ) in univariate analysis was subjected to multivariate cox analysis , which assessed the independent predictors for os and rfs . further , according to the child pugh classification , 86 ( 38.1% ) patients were of child s class a , 100 ( 44.2% ) patients were of child s class b , and 40 ( 17.7% ) patients were of child s class c. the majority of the patients ( 54.9% ) had a model for end - stage liver disease score of 1019 . fifty - four ( 23.9% ) patients and 25 ( 11.1% ) patients had a maximum tumor size of 510 cm and greater than 10 cm , respectively , whereas 40 ( 17.7% ) patients were identified as having a vascular invasion and 21 ( 9.3% ) patients as having an extrahepatic invasion . one - year and 5-year os rates among these 226 patients were 79.0 and 58.0% , respectively , whereas rfs rates were 70.3 and 62.2% , respectively . histopathologic features of patients ( n=226 ) to define tmt , we assessed os and rfs of these patients with different preoperative serum levels of afp or ca19 - 9 by interaction between preoperative afp and ca19 - 9 levels and os and rfs rates . 1 , the area under the roc curve ( area=0.613 , p=0.005 ) showed that an elevated preoperative serum level of ca19 - 9 was a useful predictor for high mortality of hepatic carcinoma patients within 3 years after lt . the data showed that those patients with a preoperative ca19 - 9 level greater than 100 u / ml had a significantly worse prognosis than those with a ca19 - 9 level of 100 u / ml or lower ( 5-year os : 32.4 vs. 62.2% , p<0.001 ; 5-year rfs : 35.1 vs. 66.1% , p<0.001 ; fig . further , there were seven non - hcc patients enrolled in this study , including five patients with intrahepatic cholangiocarcinoma ( icc ) and two patients with combined hcc - cholangiocarcinoma ( chcc - cc ) . serum levels of preoperative afp of 400 ng / ml or lower versus greater than 400 ng / ml showed a significant survival benefit on both 5-year os ( 69.5 vs. 38.7% , p<0.001 ) and rfs ( 76.1 vs. 40.3% , p<0.001 ; fig . , we analyzed os and rfs of these patients according to afp and ca19 - 9 levels , which led to their segregation into four groups : group 1 patients ( n=13 ) had elevated levels of both preoperative afp ( > 400 ng / ml ) and ca19 - 9 ( > 100 u / ml ) ; group 2 ( n=72 ) had a preoperative afp level of greater than 400 ng / ml but a ca19 - 9 level of 100 u / ml or lower ; group 3 ( n=19 ) had a preoperative afp level of 400 ng / ml or lower , but a high level ( > 100 u / ml ) of preoperative ca19 - 9 ; and group 4 ( n=122 ) had low levels of both afp and ca19 - 9 ( 400 ng / ml and 100 u / ml , respectively ) . moreover , both 5-year os ( 41.2 vs. 36.8% , p=0.118 ) and rfs ( 45.3 vs. 56.1% , p=0.649 ) rates were equivalent between groups 2 and 3 . thus , on the basis of these data , tmt was defined , and hepatic carcinoma patients could be classified on the basis of tmt as follows : type negative ( n ) , 122 patients ( group 4 ) ; type single positive ( sp ) , 91 patients ( groups 2 and 3 ) ; and type double positive ( dp ) , 13 patients ( group 1 ) . the area under the roc curve ( area=0.613 , p=0.005 ) showed that an elevated preoperative ca19 - 9 level was a significant predictor for high mortality of patients within 3 years after lt . comparison of os and rfs between patients with a preoperative ca19 - 9 level 100 u / ml and those with a preoperative ca19 - 9 level > 100 u / ml . ca19 - 9 , carbohydrate antigen 19 - 9 ; os , overall survival ; rfs , relapse - free survival . comparison of os and rfs between patients with a preoperative afp level 400 ng / ml and those with a preoperative afp level > 400 ng / ml . afp , -fetoprotein ; ca19 - 9 , carbohydrate antigen 19 - 9 ; os , overall survival ; rfs , relapse - free survival . a total of 14 variables that might affect the os or rfs of patients with hepatic carcinoma were subjected to univariate analysis , including age ( 50 or > 50 years ) , sex ( male or female ) , child pugh class ( a , b , or c ) , model for end - stage liver disease score ( < 10 , 1019 , or 20 ) , primary or salvage lt , preoperative downstaging treatment ( yes or no ) , surgical technique ( living donor lt or deceased donor lt ) , tmt ( type n , sp , or dp ) , tumor pathological type ( hcc or non - hcc ) , tumor size ( 5 , 510 , or > 10 cm ) , tumor number ( single or multiple ) , extrahepatic invasion ( presence or absence ) , vascular invasion ( presence or absence ) , and histopathologic grading ( i ii or iii ) . we found that six variables were significant predictors for both os and rfs , including tmt ( type n , sp , or dp ) , tumor size ( 5 , 510 , or > 10 cm ) , tumor number ( single or multiple ) , extrahepatic invasion ( presence or absence ) , vascular invasion ( presence or absence ) , and histopathologic grading ( i ii or iii ) . the multivariate cox analysis showed that tmt , tumor size , and extrahepatic invasion were all independent predictors for os and rfs of these patients , whereas vascular invasion was an independent predictor for rfs ( table 5 ) . univariate analysis of variables that significantly affected os or rfs independent variables in the multivariate analysis for os and rfs to propose lt eligibility criteria for patients with hepatic carcinoma , in addition to the milan criteria , using our current data , we further exploited tmt as a prognostic predictor for survival in these patients and found that the n - group patients were a special subgroup with a favorable prognosis . therefore , we proposed additional criteria for lt eligibility for hepatic carcinoma patients who did not meet the milan criteria , which could consist of those within the n group , who were free from vascular invasion and extrahepatic metastasis , regardless of tumor size and number . the os rates of patients meeting the milan criteria ( n=107 ) and those exceeding the milan criteria but fulfilling the proposed criteria ( n=30 ) were 87.7 versus 96.6% for 1 year and 77.8 versus 79.3% for 5 years , respectively ( p=0.862 ) , whereas the rfs rates were 90.7 versus 86.0% for 1 year and 85.5 versus 75.1% for 5 years , respectively ( p=0.210 ) . the median tumor size in patients exceeding the milan criteria but fulfilling the proposed criteria was 5.8 cm ( range from 1.0 to 14.0 cm ) , and multiple tumor lesions occurred in 18 ( 60% ) patients . fifty - four ( 23.9% ) patients and 25 ( 11.1% ) patients had a maximum tumor size of 510 cm and greater than 10 cm , respectively , whereas 40 ( 17.7% ) patients were identified as having a vascular invasion and 21 ( 9.3% ) patients as having an extrahepatic invasion . one - year and 5-year os rates among these 226 patients were 79.0 and 58.0% , respectively , whereas rfs rates were 70.3 and 62.2% , respectively . to define tmt , we assessed os and rfs of these patients with different preoperative serum levels of afp or ca19 - 9 by interaction between preoperative afp and ca19 - 9 levels and os and rfs rates . 1 , the area under the roc curve ( area=0.613 , p=0.005 ) showed that an elevated preoperative serum level of ca19 - 9 was a useful predictor for high mortality of hepatic carcinoma patients within 3 years after lt . the data showed that those patients with a preoperative ca19 - 9 level greater than 100 u / ml had a significantly worse prognosis than those with a ca19 - 9 level of 100 u / ml or lower ( 5-year os : 32.4 vs. 62.2% , p<0.001 ; 5-year rfs : 35.1 vs. 66.1% , p<0.001 ; fig . further , there were seven non - hcc patients enrolled in this study , including five patients with intrahepatic cholangiocarcinoma ( icc ) and two patients with combined hcc - cholangiocarcinoma ( chcc - cc ) . serum levels of preoperative afp of 400 ng / ml or lower versus greater than 400 ng / ml showed a significant survival benefit on both 5-year os ( 69.5 vs. 38.7% , p<0.001 ) and rfs ( 76.1 vs. 40.3% , p<0.001 ; fig . thereafter , we analyzed os and rfs of these patients according to afp and ca19 - 9 levels , which led to their segregation into four groups : group 1 patients ( n=13 ) had elevated levels of both preoperative afp ( > 400 ng / ml ) and ca19 - 9 ( > 100 u / ml ) ; group 2 ( n=72 ) had a preoperative afp level of greater than 400 ng / ml but a ca19 - 9 level of 100 u / ml or lower ; group 3 ( n=19 ) had a preoperative afp level of 400 ng / ml or lower , but a high level ( > 100 u / ml ) of preoperative ca19 - 9 ; and group 4 ( n=122 ) had low levels of both afp and ca19 - 9 ( 400 ng / ml and 100 u / ml , respectively ) . moreover , both 5-year os ( 41.2 vs. 36.8% , p=0.118 ) and rfs ( 45.3 vs. 56.1% , p=0.649 ) rates were equivalent between groups 2 and 3 . thus , on the basis of these data , tmt was defined , and hepatic carcinoma patients could be classified on the basis of tmt as follows : type negative ( n ) , 122 patients ( group 4 ) ; type single positive ( sp ) , 91 patients ( groups 2 and 3 ) ; and type double positive ( dp ) , 13 patients ( group 1 ) . the area under the roc curve ( area=0.613 , p=0.005 ) showed that an elevated preoperative ca19 - 9 level was a significant predictor for high mortality of patients within 3 years after lt . comparison of os and rfs between patients with a preoperative ca19 - 9 level 100 u / ml and those with a preoperative ca19 - 9 level > 100 u / ml . ca19 - 9 , carbohydrate antigen 19 - 9 ; os , overall survival ; rfs , relapse - free survival . afp , -fetoprotein ; ca19 - 9 , carbohydrate antigen 19 - 9 ; os , overall survival ; rfs , relapse - free survival . a total of 14 variables that might affect the os or rfs of patients with hepatic carcinoma were subjected to univariate analysis , including age ( 50 or > 50 years ) , sex ( male or female ) , child pugh class ( a , b , or c ) , model for end - stage liver disease score ( < 10 , 1019 , or 20 ) , primary or salvage lt , preoperative downstaging treatment ( yes or no ) , surgical technique ( living donor lt or deceased donor lt ) , tmt ( type n , sp , or dp ) , tumor pathological type ( hcc or non - hcc ) , tumor size ( 5 , 510 , or > 10 cm ) , tumor number ( single or multiple ) , extrahepatic invasion ( presence or absence ) , vascular invasion ( presence or absence ) , and histopathologic grading ( i ii or iii ) . we found that six variables were significant predictors for both os and rfs , including tmt ( type n , sp , or dp ) , tumor size ( 5 , 510 , or > 10 cm ) , tumor number ( single or multiple ) , extrahepatic invasion ( presence or absence ) , vascular invasion ( presence or absence ) , and histopathologic grading ( i ii or iii ) . the multivariate cox analysis showed that tmt , tumor size , and extrahepatic invasion were all independent predictors for os and rfs of these patients , whereas vascular invasion was an independent predictor for rfs ( table 5 ) . univariate analysis of variables that significantly affected os or rfs independent variables in the multivariate analysis for os and rfs to propose lt eligibility criteria for patients with hepatic carcinoma , in addition to the milan criteria , using our current data , we further exploited tmt as a prognostic predictor for survival in these patients and found that the n - group patients were a special subgroup with a favorable prognosis . therefore , we proposed additional criteria for lt eligibility for hepatic carcinoma patients who did not meet the milan criteria , which could consist of those within the n group , who were free from vascular invasion and extrahepatic metastasis , regardless of tumor size and number . the os rates of patients meeting the milan criteria ( n=107 ) and those exceeding the milan criteria but fulfilling the proposed criteria ( n=30 ) were 87.7 versus 96.6% for 1 year and 77.8 versus 79.3% for 5 years , respectively ( p=0.862 ) , whereas the rfs rates were 90.7 versus 86.0% for 1 year and 85.5 versus 75.1% for 5 years , respectively ( p=0.210 ) . the median tumor size in patients exceeding the milan criteria but fulfilling the proposed criteria was 5.8 cm ( range from 1.0 to 14.0 cm ) , and multiple tumor lesions occurred in 18 ( 60% ) patients . single nodule 5 cm , or no more than three nodules , with each measuring 3 cm or less ) on the basis of a retrospective study of 48 patients who underwent lt for hcc , and the milan criteria have been used thereafter as a guideline for candidate selection for lt in many transplant centers worldwide . indeed , our previous study 15 verified both the expanded criteria in patients with hepatitis b - related hcc using a prospectively collected database , and the data suggested that the 1- , 3- , and 5-year recurrence rates of the newly eligible patients selected using the shanghai or hangzhou criteria were significantly higher than those among patients fulfilling the milan criteria . the 5-year os and rfs rates of patients with isolated increase in ca19 - 9 ( group 3 ) were significantly lower than in those with neither biomarker elevated ( group 4 ) . conceivably , a single measurement of the serum afp can not be used as an accurate predictive marker for the prognosis of liver cancer patients , whereas the combination of afp and ca19 - 9 will greatly improve the prognostic prediction . we therefore proposed selection of patients with hepatic carcinoma who exceeded the milan criteria using preoperative serum afp and ca19 - 9 levels . in addition , in the current study , tumor size also remained an independent factor for the survival of patients on the basis of the multivariate cox analysis . as expected , the 5-year os and rfs rates were similar between patients within the milan criteria and the newly eligible patients fulfilling the proposed criteria . in conclusion , data from this study show that the combination of afp and ca19 - 9 is able to predict os and rfs of hepatic carcinoma patients after lt and that the tmt based on preoperative serum levels of afp and ca19 - 9 could be a useful tool to select hepatic carcinoma patients for lt , especially those exceeding the milan criteria . however , further prospective studies conducted on a large scale are needed to verify the new criteria and to use these two markers to predict survival of patients with hepatic carcinoma . in conclusion , data from this study show that the combination of afp and ca19 - 9 is able to predict os and rfs of hepatic carcinoma patients after lt and that the tmt based on preoperative serum levels of afp and ca19 - 9 could be a useful tool to select hepatic carcinoma patients for lt , especially those exceeding the milan criteria . however , further prospective studies conducted on a large scale are needed to verify the new criteria and to use these two markers to predict survival of patients with hepatic carcinoma .

biddi making is a skilled job in which tobacco is rolled in a processed kendu leaf and tied with a cotton thread . the kendu leaves are moistened and then cut into pieces of required size and shape . tobacco mixed with hand and then the processed tobacco is wrapped and rolled in the prepared cut piece of kendu leaf . the biddi workers work from morning ( 6 am ) to 11 am and again from 2 pm to 10 pm . some biddi factories were there at one time but now the factories are closed . only a small group works together in some places . the airborne tobacco dust around the work places of biddi making units is the potential risk factors causing respiratory disorders to the biddi binders . the tobacco and the leaves were kept on the lap of the biddi binders and it is very much close to the nostrils and mouth . besides these , different microbes and airborne fungal spores may be added into the working atmosphere from the processed kendu leaves . these are all the probable risk factors causing respiratory disorders among the workers . keeping in mind the long - term exposure of tobacco dust exposure on lung function test of biddi binders , a follow - up study was conducted to verify the lung function changes after 2 years of exposure . in both male and female biddi binders , a validated questionnaire was administered and relevant information such as history of illness , was noted . this is a follow - up study after 2 years of the initial study . a total of 86 ( male , 41 ; female , 45 ) biddi binders were followed up . the parameters of importance were compared with the initial study findings and the results are discussed in the following section . only 86 biddi binders of initial study group had been followed up . so the present reporting compared the results of the initial study group with those of their follow - up study group . a total of 89 biddi binders ( male , 43 ; female , 46 ) were studied . vital capacity ( vc ) and forced vital capacity ( fvc ) was recorded by spirovit sp-10 ( schiller health care pvt ltd . , switzerland ) and peak expiratory flow rate by wrights peak flow meter ( clement and clarke , uk ) . forced expiratory volume in the 1 s ( fev1 ) and the 3 s ( fev3 ) , forced expiratory volume in 1 and 3 s as the percentage of fvc ( fev1 % and fev3 % ) , forced expiratory flow at fef200 ml-1200 ml , fef25%-75% , and fef75%-85% was calculated from the tracings . all measured lung volumes obtained was expressed in body temperature pressure saturated with water vapor ( btps ) . body surface area ( bsa ) the criteria followed for categorization of the severity of restrictive impairment are based on the ratios between predicted and observed values of vc and obstructive impairment based on fev1 % . in both male and female biddi binders , a validated questionnaire was administered and relevant information such as history of illness , was noted . this is a follow - up study after 2 years of the initial study . a total of 86 ( male , 41 ; female , 45 ) biddi binders were followed up . the parameters of importance were compared with the initial study findings and the results are discussed in the following section . only 86 biddi binders of initial study group had been followed up . so the present reporting compared the results of the initial study group with those of their follow - up study group . a total of 89 biddi binders ( male , 43 ; female , 46 ) were studied . vital capacity ( vc ) and forced vital capacity ( fvc ) was recorded by spirovit sp-10 ( schiller health care pvt ltd . , switzerland ) and peak expiratory flow rate by wrights peak flow meter ( clement and clarke , uk ) . forced expiratory volume in the 1 s ( fev1 ) and the 3 s ( fev3 ) , forced expiratory volume in 1 and 3 s as the percentage of fvc ( fev1 % and fev3 % ) , forced expiratory flow at fef200 ml-1200 ml , fef25%-75% , and fef75%-85% was calculated from the tracings . all measured lung volumes obtained was expressed in body temperature pressure saturated with water vapor ( btps ) . body surface area ( bsa ) the criteria followed for categorization of the severity of restrictive impairment are based on the ratios between predicted and observed values of vc and obstructive impairment based on fev1 % . the average age of the male subjects of the initial study and the follow - up study were 49.66 9.88 and 51.54 10.54 years and 36.09 9.34 and 38.34 9.67 years for females , respectively . the mean age of the total population male and female combined at the initial study is 42.04 12.03 and the follow - up study is 44.63 12.03 . their mean height and weight of the first study ( initial study ) were 155.41 7.61 cm ( male , 161.02 6.50 cm ; female , 151.29 4.41 cm ) and in the follow - up study were 155.49 7.34 ( male , 160.42 6.81 cm and female 151.00 4.34 cm ) ; similarly the weight in the initial study was 47.71 9.16 kg ( male , 50.88 9.78 kg ; female , 44.82 9.16 kg ) and in the follow - up study , the mean weight was 49.44 9.31 kg ( male , 52.93 9.94 and female 46.26 7.47 ) . it may been seen that the high prevalence rates of weakness , giddiness , chronic diarrhea , and dyspepsia were noted in both initial and follow - up study groups irrespective of gender . the symptoms of chronic diarrhea and dyspepsia in the follow - up study group showed significant increase at various levels ( p < 0.05 ; p < 0.005 ; p < 0.001 ) both for male and female . the symptom of burning sensation during passing of urine was significantly more in the follow - up study group ( both male and female ) compared with the initial study group ( p < 0.01 ; p < 0.001 ) . general symptoms of the both gender biddi binders the distribution of respiratory symptoms as reported by the subjects of both the initial and follow - up study groups is depicted in figure 2 . a high prevalence of respiratory complaints such as cough , sputum , breathlessness , and chronic bronchitis was reported in both the initial and follow - up studies . however , there was a general decline in the rates of all the complaints in the follow - up group . respiratory symptoms of the both gender biddi binders the distribution of symptoms related to musculoskeletal system in the initial and follow - up studies is summarized in figure 3 . pain in neck , joint pain ( knee and shoulder ) , and low back pain were showing very high both in the follow - up and initial studies . however , the above - reported symptoms were more in the follow - up study group . typical posture of biddi rolling might be the most probable cause of these ailments . repetitive movements of the hands and awkward postures may also be responsible for causing the above - mentioned symptoms . a substantial number of subjects in both the follow - up and initial study groups complained of peripheral neuropathy . although the prevalence rate of peripheral neuropathy ( tingling , numbness , burning sensation , and weakness of limbs ) in males in the follow - up study group ( 68.3% ) was less than that in the initial study group ( 82.9% ) , but in females the follow up study group ( 77.8% ) showed significantly higher rates ( p < 0.05 ) than in the initial study group ( 57.8% ) . musculo - skeletal system of the both gender biddi binders other important symptoms , such as burning and itching of eye , dimness of vision , loss of appetite , feeling of cramps , and others , are given in figure 4 . loss of appetite and feeling of cramps of males of follow - up study showed significantly higher rate compared with that of male subjects of initial study ( p < 0.001 , p < 0.05 ) . similarly , significantly more number of females complained loss of appetite and feeling of cramps in the follow - up study ( p < 0.001 ; p < 0.05 ) . it is a matter of concern that the overall complaints of worm infestations has increased to 26.7% from 10.5% of the initial study . as usual thread worm / pin worm , was the main type of worms that infected them and are significantly more in the follow - up study group compared with the initial study group ( p < 0.01 ) . important body symptoms of the both gender biddi binders the distribution of angular stomatitis and glossitis showed lower rates in males in the follow - up study than that of the initial study group . hypertension [ blood pressure ( bp ) > 140/90 mmhg ] was observed in 5.8% subjects in the follow - up study ( male , 4.9% ; female , 6.7% ) . only systolic hypertension ( systolic bp > 140 mmhg ) was noted in male -12.2% and female - 8.9% compared with initial study male -2.4% and female -4.4% . diastolic hypertension ( bp > 90 mmhg ) was observed only in the follow - up study . most of the signs and symptoms may be related to their work methods , handling of tobacco dusts , and likely due to nutritional deficiency . table 1 represents the physical characteristics of the biddi binders of the follow - up and initial studies . in the follow - up study , the same biddi binders ( male and female ) the mean value of age , weight , bsa , and body mass index in males was found significantly higher in the follow - up study compared with the earlier initial study results . similarly in females , age , bsa , and body mass index were significantly higher . physical parameters of male and female biddi binders in initial and follow - up studies ( meansd ) pulmonary function test values of the male and female biddi binders of both the studies presented in figures 58 show the relative significant decline of the lung volumes svc , fvc , fev1 and the flow rates fef0.2 - 1.2 l , fef25%-75% , fef75%-85% in male and female biddi binders in the follow - up study ( p < 0.05 , p < 0.01 , p < 0.001 ) . the pefr levels had no significant change in males and females between the initial and follow - up studies . lung volume of the male biddi binder lung volume of the female biddi binder lung flow rate of the male biddi binder lung flow rate of the female biddi binder the male and female biddi binders were divided into the following age groups as 2029 years , 3039 years , 4049 years , 5059 years , 6069 years , and > 70 years . all lung volumes and the flow rates gradually declined with the advancing age in both initial and follow - up studies . as the male biddi binders were categorized as nonsmoker , smoker , and exsmokers according to their smoking habit , their pft values were found to be less in the follow - up study as compared with the initial study . the nonsmoker has the higher lung volumes and flow rates compared with smokers and exsmokers . bronchodilator drugs ( bd ) were generally sympathomimatic agents that actively enlarge the lumen of bronchioles by effect on bronchodilator smooth muscles and enlarge the lumen of the bronchioles to ease obstruction . these drugs are widely and effectively used to test the reversibility of airways obstruction . the drug can be administered systemically in the form of aerosol . for each individual , the pfts were performed after 30 min of bronchodilator use . puff to get the optimum results . after the maximum expiration up to the rv , the mouthpiece of the inhaler was placed well into the mouth and lips close firmly around it . then the subjects were asked to inhale deeply through the mouth while activating the nebulizer . after holding the breath for a few seconds , the mouthpiece was removed and the patients were asked to inhale slowly , this procedure was repeated once . the bronchodilator aerosol used in the present study , which contained 100 g of drug , was used in one puff and two puffs of the drug and was administered to each subject after and before bronchodilator pft . the action of single oral dose of single inhalation lasts for about 46 h. the workers were directed not to take any bronchodilator drug at least 24 h before the test . the bronchodilator aerosol was administered to the biddi binders after the completion of the initial pft , a gap of about 30 min was given before and after bronchodilator was administered . the mean difference of lung volumes after the bronchodilatation showed significant difference in all pft parameters both in male and female biddi binders . the positive bronchodilator response was defined for fev1 and fvc as an increase of > 12% of baseline value and 200 ml according to ats criteria . the criteria of positive bronchodilatation were fulfilled by svc , fvc , fev1 , and inflow rates of male biddi binders . in female biddi binders , the positive bronchodilatation was found in all these parameters such as male biddi binders except fef0.2 - 1.2 l. the mean difference of the values between the before bronchodilator and after bronchodilator use was significant except in few of the flow rates . sulbutamol bronchodilator aerosols are widely used to judge the reversibility of the airways obstruction present or not . lung volumes of the biddi binders before and after the administration of bronchodilator aerosol ( meansd ) flow rates of the biddi binders before and after the administration of bronchodilator aerosol ( meansd ) salbutamol aerosol as bronchodilator drugs are widely and effectively used to judge the reversibility of the obstruction of the bronchial tree . the tests of reversibility of airway obstruction are commonly studied with ventilatory pulmonary function tests by spirometric method before and after the administration of bronchodilator drugs . response to bronchodilator aerosol is commonly tested by the measurement of fev1 before and after the dose of the drug . astin evidenced that the possible causes of airway resistance in these subjects are not fully known , although the possible causes might be mucous gland hypertrophy , mucosal edema , intraluminal secretion , and bronchial muscle constrictions . it has been found that a significant part of these increased airway resistance are reversible in these subjects after bronchodilator aerosol administration . in the present study , there is a significant increase in svc , fvc , fev1 , fev1% , and fef75%-85% after the administration of salbutamol bronchodilator aerosol reflects that there is some obstruction . in the present study , the airway obstruction of the biddi binders is reversible and it satisfies the criteria of positive bronchodilator response according to the criteria established by american thoracic society . a significant part of the increased airway resistance was reversed in the present subject in the bronchodilator use . the statistically significant changes of pft values in some parameters might be due to the reversal of smooth muscle tone present as evidenced [ figures 912 ] . lung volumes of the male biddi binders before and after the administrations of bronchodilator lung volumes of the female biddi binders before and after the administrations of bronchodilator lung flow rates of the male biddi binders before and after the administrations of bronchodilator lung flow rates of the female biddi binders before and after the administrations of bronchodilator the average age of the male subjects of the initial study and the follow - up study were 49.66 9.88 and 51.54 10.54 years and 36.09 9.34 and 38.34 9.67 years for females , respectively . the mean age of the total population male and female combined at the initial study is 42.04 12.03 and the follow - up study is 44.63 12.03 . their mean height and weight of the first study ( initial study ) were 155.41 7.61 cm ( male , 161.02 6.50 cm ; female , 151.29 4.41 cm ) and in the follow - up study were 155.49 7.34 ( male , 160.42 6.81 cm and female 151.00 4.34 cm ) ; similarly the weight in the initial study was 47.71 9.16 kg ( male , 50.88 9.78 kg ; female , 44.82 9.16 kg ) and in the follow - up study , the mean weight was 49.44 9.31 kg ( male , 52.93 9.94 and female 46.26 7.47 ) . it may been seen that the high prevalence rates of weakness , giddiness , chronic diarrhea , and dyspepsia were noted in both initial and follow - up study groups irrespective of gender . the symptoms of chronic diarrhea and dyspepsia in the follow - up study group showed significant increase at various levels ( p < 0.05 ; p < 0.005 ; p < 0.001 ) both for male and female . the symptom of burning sensation during passing of urine was significantly more in the follow - up study group ( both male and female ) compared with the initial study group ( p < 0.01 ; p < 0.001 ) . general symptoms of the both gender biddi binders the distribution of respiratory symptoms as reported by the subjects of both the initial and follow - up study groups is depicted in figure 2 . a high prevalence of respiratory complaints such as cough , sputum , breathlessness , and chronic bronchitis was reported in both the initial and follow - up studies . however , there was a general decline in the rates of all the complaints in the follow - up group . respiratory symptoms of the both gender biddi binders the distribution of symptoms related to musculoskeletal system in the initial and follow - up studies is summarized in figure 3 . pain in neck , joint pain ( knee and shoulder ) , and low back pain were showing very high both in the follow - up and initial studies . however , the above - reported symptoms were more in the follow - up study group . typical posture of biddi rolling might be the most probable cause of these ailments . repetitive movements of the hands and awkward postures may also be responsible for causing the above - mentioned symptoms . a substantial number of subjects in both the follow - up and initial study groups complained of peripheral neuropathy . although the prevalence rate of peripheral neuropathy ( tingling , numbness , burning sensation , and weakness of limbs ) in males in the follow - up study group ( 68.3% ) was less than that in the initial study group ( 82.9% ) , but in females the follow up study group ( 77.8% ) showed significantly higher rates ( p < 0.05 ) than in the initial study group ( 57.8% ) . musculo - skeletal system of the both gender biddi binders other important symptoms , such as burning and itching of eye , dimness of vision , loss of appetite , feeling of cramps , and others , are given in figure 4 . loss of appetite and feeling of cramps of males of follow - up study showed significantly higher rate compared with that of male subjects of initial study ( p < 0.001 , p < 0.05 ) . similarly , significantly more number of females complained loss of appetite and feeling of cramps in the follow - up study ( p < 0.001 ; p < 0.05 ) . it is a matter of concern that the overall complaints of worm infestations has increased to 26.7% from 10.5% of the initial study . as usual thread worm / pin worm , was the main type of worms that infected them and are significantly more in the follow - up study group compared with the initial study group ( p < 0.01 ) . important body symptoms of the both gender biddi binders the distribution of angular stomatitis and glossitis showed lower rates in males in the follow - up study than that of the initial study group . hypertension [ blood pressure ( bp ) > 140/90 mmhg ] was observed in 5.8% subjects in the follow - up study ( male , 4.9% ; female , 6.7% ) . only systolic hypertension ( systolic bp > 140 mmhg ) was noted in male -12.2% and female - 8.9% compared with initial study male -2.4% and female -4.4% . diastolic hypertension ( bp > 90 mmhg ) was observed only in the follow - up study . most of the signs and symptoms may be related to their work methods , handling of tobacco dusts , and likely due to nutritional deficiency . table 1 represents the physical characteristics of the biddi binders of the follow - up and initial studies . in the follow - up study , the same biddi binders ( male and female ) the mean value of age , weight , bsa , and body mass index in males was found significantly higher in the follow - up study compared with the earlier initial study results . similarly in females , age , bsa , and body mass index were significantly higher . physical parameters of male and female biddi binders in initial and follow - up studies ( meansd ) pulmonary function test values of the male and female biddi binders of both the studies presented in figures 58 show the relative significant decline of the lung volumes svc , fvc , fev1 and the flow rates fef0.2 - 1.2 l , fef25%-75% , fef75%-85% in male and female biddi binders in the follow - up study ( p < 0.05 , p < 0.01 , p < 0.001 ) . the pefr levels had no significant change in males and females between the initial and follow - up studies . lung volume of the male biddi binder lung volume of the female biddi binder lung flow rate of the male biddi binder lung flow rate of the female biddi binder the male and female biddi binders were divided into the following age groups as 2029 years , 3039 years , 4049 years , 5059 years , 6069 years , and > 70 years . all lung volumes and the flow rates gradually declined with the advancing age in both initial and follow - up studies . as the male biddi binders were categorized as nonsmoker , smoker , and exsmokers according to their smoking habit , their pft values were found to be less in the follow - up study as compared with the initial study . the nonsmoker has the higher lung volumes and flow rates compared with smokers and exsmokers . bronchodilator drugs ( bd ) were generally sympathomimatic agents that actively enlarge the lumen of bronchioles by effect on bronchodilator smooth muscles and enlarge the lumen of the bronchioles to ease obstruction . the drug can be administered systemically in the form of aerosol . for each individual , the pfts were performed after 30 min of bronchodilator use . a dose of 200 mg was delivered in the form of a puff to get the optimum results . after the maximum expiration up to the rv , the mouthpiece of the inhaler was placed well into the mouth and lips close firmly around it . then the subjects were asked to inhale deeply through the mouth while activating the nebulizer . after holding the breath for a few seconds , the mouthpiece was removed and the patients were asked to inhale slowly , this procedure was repeated once . the bronchodilator aerosol used in the present study , which contained 100 g of drug , was used in one puff and two puffs of the drug and was administered to each subject after and before bronchodilator pft . the action of single oral dose of single inhalation lasts for about 46 h. the workers were directed not to take any bronchodilator drug at least 24 h before the test . the bronchodilator aerosol was administered to the biddi binders after the completion of the initial pft , a gap of about 30 min was given before and after bronchodilator was administered . the mean difference of lung volumes after the bronchodilatation showed significant difference in all pft parameters both in male and female biddi binders . the positive bronchodilator response was defined for fev1 and fvc as an increase of > 12% of baseline value and 200 ml according to ats criteria . the criteria of positive bronchodilatation were fulfilled by svc , fvc , fev1 , and inflow rates of male biddi binders . in female biddi binders , the positive bronchodilatation was found in all these parameters such as male biddi binders except fef0.2 - 1.2 l. the mean difference of the values between the before bronchodilator and after bronchodilator use was significant except in few of the flow rates . sulbutamol bronchodilator aerosols are widely used to judge the reversibility of the airways obstruction present or not . lung volumes of the biddi binders before and after the administration of bronchodilator aerosol ( meansd ) flow rates of the biddi binders before and after the administration of bronchodilator aerosol ( meansd ) salbutamol aerosol as bronchodilator drugs are widely and effectively used to judge the reversibility of the obstruction of the bronchial tree . the tests of reversibility of airway obstruction are commonly studied with ventilatory pulmonary function tests by spirometric method before and after the administration of bronchodilator drugs . response to bronchodilator aerosol is commonly tested by the measurement of fev1 before and after the dose of the drug . astin evidenced that the possible causes of airway resistance in these subjects are not fully known , although the possible causes might be mucous gland hypertrophy , mucosal edema , intraluminal secretion , and bronchial muscle constrictions . it has been found that a significant part of these increased airway resistance are reversible in these subjects after bronchodilator aerosol administration . in the present study , there is a significant increase in svc , fvc , fev1 , fev1% , and fef75%-85% after the administration of salbutamol bronchodilator aerosol reflects that there is some obstruction . in the present study , the airway obstruction of the biddi binders is reversible and it satisfies the criteria of positive bronchodilator response according to the criteria established by american thoracic society . a significant part of the increased airway resistance was reversed in the present subject in the bronchodilator use . the statistically significant changes of pft values in some parameters might be due to the reversal of smooth muscle tone present as evidenced [ figures 912 ] . lung volumes of the male biddi binders before and after the administrations of bronchodilator lung volumes of the female biddi binders before and after the administrations of bronchodilator lung flow rates of the male biddi binders before and after the administrations of bronchodilator lung flow rates of the female biddi binders before and after the administrations of bronchodilator comparison of initial and follow - up studies provides evidence of relative increase of the prevalence of various ailments as well as pft values , irrespective of gender and age group after continuation for 2 years of exposure in the same occupational activity . administration of bronchodilator drug could inevitably bring positive bronchodilatation and easing of airway airway resistance of the biddi binders indicating the obstruction in the airways might be reversible in nature .

intorduction : the tobacco dusts get air borne during biddi making and it is inhaled by the biddi binders , which affects their health.results:in a follow - up study , 86 biddi binders ( male , 41 ; female , 45 ) were studied at a gap of 2 years . a high respiratory morbidity was observed among males than females both in the initial and follow - up study . the main complaints such as cough , sputum , and breathlessness showed high prevalence rates in the follow - up study . the high prevalence rates of weakness , giddiness , chronic diarrhea , and dyspepsia were noted in most of the cases the above complaints showed higher rates in the follow - up study . pulmonary function test ( pft ) values in the follow - up study of male and female biddi binders showed lowered compared with the initial study of same gender . in male and female biddi binders , the lung volumes svc , fvc , fev1 , and the flow rates fef0.2 - 1.2 l , fef25%-75% , fef75%-85% were significantly lowered in the follow - up study compared with the initial study . age - related decrement in pft was observed in both the studies . in nonsmokers , smokers , and ex - smokers , the pft values are lowered . the current smokers have the lowest values in both the studies . with the administration of the bronchodilator aerosol ( salbutamol ) in 63 biddi binders ( male , 27 ; female , 36 ) , the effect of bronchodilator aerosol on the pft parameters showed significant changes as all pft parameters showed positive bronchodilatation.conclusion:the pattern of bronchodilator response on pft values of the biddi binders suggests that the obstructions in the airways are reversible in nature .

INTRODUCTION MATERIALS AND METHODS Medical study RESULTS AND DISCUSSION Medical study Pulmonary function tests Bronchodilator study CONCLUSION

the tobacco and the leaves were kept on the lap of the biddi binders and it is very much close to the nostrils and mouth . keeping in mind the long - term exposure of tobacco dust exposure on lung function test of biddi binders , a follow - up study was conducted to verify the lung function changes after 2 years of exposure . in both male and female biddi binders , a validated questionnaire was administered and relevant information such as history of illness , was noted . this is a follow - up study after 2 years of the initial study . a total of 86 ( male , 41 ; female , 45 ) biddi binders were followed up . the parameters of importance were compared with the initial study findings and the results are discussed in the following section . so the present reporting compared the results of the initial study group with those of their follow - up study group . a total of 89 biddi binders ( male , 43 ; female , 46 ) were studied . forced expiratory volume in the 1 s ( fev1 ) and the 3 s ( fev3 ) , forced expiratory volume in 1 and 3 s as the percentage of fvc ( fev1 % and fev3 % ) , forced expiratory flow at fef200 ml-1200 ml , fef25%-75% , and fef75%-85% was calculated from the tracings . in both male and female biddi binders , a validated questionnaire was administered and relevant information such as history of illness , was noted . this is a follow - up study after 2 years of the initial study . a total of 86 ( male , 41 ; female , 45 ) biddi binders were followed up . the parameters of importance were compared with the initial study findings and the results are discussed in the following section . so the present reporting compared the results of the initial study group with those of their follow - up study group . a total of 89 biddi binders ( male , 43 ; female , 46 ) were studied . forced expiratory volume in the 1 s ( fev1 ) and the 3 s ( fev3 ) , forced expiratory volume in 1 and 3 s as the percentage of fvc ( fev1 % and fev3 % ) , forced expiratory flow at fef200 ml-1200 ml , fef25%-75% , and fef75%-85% was calculated from the tracings . the average age of the male subjects of the initial study and the follow - up study were 49.66 9.88 and 51.54 10.54 years and 36.09 9.34 and 38.34 9.67 years for females , respectively . the mean age of the total population male and female combined at the initial study is 42.04 12.03 and the follow - up study is 44.63 12.03 . their mean height and weight of the first study ( initial study ) were 155.41 7.61 cm ( male , 161.02 6.50 cm ; female , 151.29 4.41 cm ) and in the follow - up study were 155.49 7.34 ( male , 160.42 6.81 cm and female 151.00 4.34 cm ) ; similarly the weight in the initial study was 47.71 9.16 kg ( male , 50.88 9.78 kg ; female , 44.82 9.16 kg ) and in the follow - up study , the mean weight was 49.44 9.31 kg ( male , 52.93 9.94 and female 46.26 7.47 ) . it may been seen that the high prevalence rates of weakness , giddiness , chronic diarrhea , and dyspepsia were noted in both initial and follow - up study groups irrespective of gender . the symptoms of chronic diarrhea and dyspepsia in the follow - up study group showed significant increase at various levels ( p < 0.05 ; p < 0.005 ; p < 0.001 ) both for male and female . the symptom of burning sensation during passing of urine was significantly more in the follow - up study group ( both male and female ) compared with the initial study group ( p < 0.01 ; p < 0.001 ) . general symptoms of the both gender biddi binders the distribution of respiratory symptoms as reported by the subjects of both the initial and follow - up study groups is depicted in figure 2 . a high prevalence of respiratory complaints such as cough , sputum , breathlessness , and chronic bronchitis was reported in both the initial and follow - up studies . respiratory symptoms of the both gender biddi binders the distribution of symptoms related to musculoskeletal system in the initial and follow - up studies is summarized in figure 3 . pain in neck , joint pain ( knee and shoulder ) , and low back pain were showing very high both in the follow - up and initial studies . however , the above - reported symptoms were more in the follow - up study group . a substantial number of subjects in both the follow - up and initial study groups complained of peripheral neuropathy . although the prevalence rate of peripheral neuropathy ( tingling , numbness , burning sensation , and weakness of limbs ) in males in the follow - up study group ( 68.3% ) was less than that in the initial study group ( 82.9% ) , but in females the follow up study group ( 77.8% ) showed significantly higher rates ( p < 0.05 ) than in the initial study group ( 57.8% ) . loss of appetite and feeling of cramps of males of follow - up study showed significantly higher rate compared with that of male subjects of initial study ( p < 0.001 , p < 0.05 ) . as usual thread worm / pin worm , was the main type of worms that infected them and are significantly more in the follow - up study group compared with the initial study group ( p < 0.01 ) . important body symptoms of the both gender biddi binders the distribution of angular stomatitis and glossitis showed lower rates in males in the follow - up study than that of the initial study group . hypertension [ blood pressure ( bp ) > 140/90 mmhg ] was observed in 5.8% subjects in the follow - up study ( male , 4.9% ; female , 6.7% ) . only systolic hypertension ( systolic bp > 140 mmhg ) was noted in male -12.2% and female - 8.9% compared with initial study male -2.4% and female -4.4% . diastolic hypertension ( bp > 90 mmhg ) was observed only in the follow - up study . table 1 represents the physical characteristics of the biddi binders of the follow - up and initial studies . in the follow - up study , the same biddi binders ( male and female ) the mean value of age , weight , bsa , and body mass index in males was found significantly higher in the follow - up study compared with the earlier initial study results . physical parameters of male and female biddi binders in initial and follow - up studies ( meansd ) pulmonary function test values of the male and female biddi binders of both the studies presented in figures 58 show the relative significant decline of the lung volumes svc , fvc , fev1 and the flow rates fef0.2 - 1.2 l , fef25%-75% , fef75%-85% in male and female biddi binders in the follow - up study ( p < 0.05 , p < 0.01 , p < 0.001 ) . lung volume of the male biddi binder lung volume of the female biddi binder lung flow rate of the male biddi binder lung flow rate of the female biddi binder the male and female biddi binders were divided into the following age groups as 2029 years , 3039 years , 4049 years , 5059 years , 6069 years , and > 70 years . all lung volumes and the flow rates gradually declined with the advancing age in both initial and follow - up studies . as the male biddi binders were categorized as nonsmoker , smoker , and exsmokers according to their smoking habit , their pft values were found to be less in the follow - up study as compared with the initial study . the bronchodilator aerosol used in the present study , which contained 100 g of drug , was used in one puff and two puffs of the drug and was administered to each subject after and before bronchodilator pft . the bronchodilator aerosol was administered to the biddi binders after the completion of the initial pft , a gap of about 30 min was given before and after bronchodilator was administered . the mean difference of lung volumes after the bronchodilatation showed significant difference in all pft parameters both in male and female biddi binders . the criteria of positive bronchodilatation were fulfilled by svc , fvc , fev1 , and inflow rates of male biddi binders . in female biddi binders , the positive bronchodilatation was found in all these parameters such as male biddi binders except fef0.2 - 1.2 l. the mean difference of the values between the before bronchodilator and after bronchodilator use was significant except in few of the flow rates . lung volumes of the biddi binders before and after the administration of bronchodilator aerosol ( meansd ) flow rates of the biddi binders before and after the administration of bronchodilator aerosol ( meansd ) salbutamol aerosol as bronchodilator drugs are widely and effectively used to judge the reversibility of the obstruction of the bronchial tree . in the present study , there is a significant increase in svc , fvc , fev1 , fev1% , and fef75%-85% after the administration of salbutamol bronchodilator aerosol reflects that there is some obstruction . in the present study , the airway obstruction of the biddi binders is reversible and it satisfies the criteria of positive bronchodilator response according to the criteria established by american thoracic society . lung volumes of the male biddi binders before and after the administrations of bronchodilator lung volumes of the female biddi binders before and after the administrations of bronchodilator lung flow rates of the male biddi binders before and after the administrations of bronchodilator lung flow rates of the female biddi binders before and after the administrations of bronchodilator the average age of the male subjects of the initial study and the follow - up study were 49.66 9.88 and 51.54 10.54 years and 36.09 9.34 and 38.34 9.67 years for females , respectively . the mean age of the total population male and female combined at the initial study is 42.04 12.03 and the follow - up study is 44.63 12.03 . their mean height and weight of the first study ( initial study ) were 155.41 7.61 cm ( male , 161.02 6.50 cm ; female , 151.29 4.41 cm ) and in the follow - up study were 155.49 7.34 ( male , 160.42 6.81 cm and female 151.00 4.34 cm ) ; similarly the weight in the initial study was 47.71 9.16 kg ( male , 50.88 9.78 kg ; female , 44.82 9.16 kg ) and in the follow - up study , the mean weight was 49.44 9.31 kg ( male , 52.93 9.94 and female 46.26 7.47 ) . it may been seen that the high prevalence rates of weakness , giddiness , chronic diarrhea , and dyspepsia were noted in both initial and follow - up study groups irrespective of gender . the symptoms of chronic diarrhea and dyspepsia in the follow - up study group showed significant increase at various levels ( p < 0.05 ; p < 0.005 ; p < 0.001 ) both for male and female . the symptom of burning sensation during passing of urine was significantly more in the follow - up study group ( both male and female ) compared with the initial study group ( p < 0.01 ; p < 0.001 ) . general symptoms of the both gender biddi binders the distribution of respiratory symptoms as reported by the subjects of both the initial and follow - up study groups is depicted in figure 2 . a high prevalence of respiratory complaints such as cough , sputum , breathlessness , and chronic bronchitis was reported in both the initial and follow - up studies . respiratory symptoms of the both gender biddi binders the distribution of symptoms related to musculoskeletal system in the initial and follow - up studies is summarized in figure 3 . pain in neck , joint pain ( knee and shoulder ) , and low back pain were showing very high both in the follow - up and initial studies . however , the above - reported symptoms were more in the follow - up study group . a substantial number of subjects in both the follow - up and initial study groups complained of peripheral neuropathy . although the prevalence rate of peripheral neuropathy ( tingling , numbness , burning sensation , and weakness of limbs ) in males in the follow - up study group ( 68.3% ) was less than that in the initial study group ( 82.9% ) , but in females the follow up study group ( 77.8% ) showed significantly higher rates ( p < 0.05 ) than in the initial study group ( 57.8% ) . loss of appetite and feeling of cramps of males of follow - up study showed significantly higher rate compared with that of male subjects of initial study ( p < 0.001 , p < 0.05 ) . as usual thread worm / pin worm , was the main type of worms that infected them and are significantly more in the follow - up study group compared with the initial study group ( p < 0.01 ) . important body symptoms of the both gender biddi binders the distribution of angular stomatitis and glossitis showed lower rates in males in the follow - up study than that of the initial study group . hypertension [ blood pressure ( bp ) > 140/90 mmhg ] was observed in 5.8% subjects in the follow - up study ( male , 4.9% ; female , 6.7% ) . diastolic hypertension ( bp > 90 mmhg ) was observed only in the follow - up study . table 1 represents the physical characteristics of the biddi binders of the follow - up and initial studies . in the follow - up study , the same biddi binders ( male and female ) the mean value of age , weight , bsa , and body mass index in males was found significantly higher in the follow - up study compared with the earlier initial study results . physical parameters of male and female biddi binders in initial and follow - up studies ( meansd ) pulmonary function test values of the male and female biddi binders of both the studies presented in figures 58 show the relative significant decline of the lung volumes svc , fvc , fev1 and the flow rates fef0.2 - 1.2 l , fef25%-75% , fef75%-85% in male and female biddi binders in the follow - up study ( p < 0.05 , p < 0.01 , p < 0.001 ) . lung volume of the male biddi binder lung volume of the female biddi binder lung flow rate of the male biddi binder lung flow rate of the female biddi binder the male and female biddi binders were divided into the following age groups as 2029 years , 3039 years , 4049 years , 5059 years , 6069 years , and > 70 years . all lung volumes and the flow rates gradually declined with the advancing age in both initial and follow - up studies . as the male biddi binders were categorized as nonsmoker , smoker , and exsmokers according to their smoking habit , their pft values were found to be less in the follow - up study as compared with the initial study . the bronchodilator aerosol used in the present study , which contained 100 g of drug , was used in one puff and two puffs of the drug and was administered to each subject after and before bronchodilator pft . the bronchodilator aerosol was administered to the biddi binders after the completion of the initial pft , a gap of about 30 min was given before and after bronchodilator was administered . the mean difference of lung volumes after the bronchodilatation showed significant difference in all pft parameters both in male and female biddi binders . the criteria of positive bronchodilatation were fulfilled by svc , fvc , fev1 , and inflow rates of male biddi binders . in female biddi binders , the positive bronchodilatation was found in all these parameters such as male biddi binders except fef0.2 - 1.2 l. the mean difference of the values between the before bronchodilator and after bronchodilator use was significant except in few of the flow rates . lung volumes of the biddi binders before and after the administration of bronchodilator aerosol ( meansd ) flow rates of the biddi binders before and after the administration of bronchodilator aerosol ( meansd ) salbutamol aerosol as bronchodilator drugs are widely and effectively used to judge the reversibility of the obstruction of the bronchial tree . in the present study , there is a significant increase in svc , fvc , fev1 , fev1% , and fef75%-85% after the administration of salbutamol bronchodilator aerosol reflects that there is some obstruction . in the present study , the airway obstruction of the biddi binders is reversible and it satisfies the criteria of positive bronchodilator response according to the criteria established by american thoracic society . lung volumes of the male biddi binders before and after the administrations of bronchodilator lung volumes of the female biddi binders before and after the administrations of bronchodilator lung flow rates of the male biddi binders before and after the administrations of bronchodilator lung flow rates of the female biddi binders before and after the administrations of bronchodilator comparison of initial and follow - up studies provides evidence of relative increase of the prevalence of various ailments as well as pft values , irrespective of gender and age group after continuation for 2 years of exposure in the same occupational activity . administration of bronchodilator drug could inevitably bring positive bronchodilatation and easing of airway airway resistance of the biddi binders indicating the obstruction in the airways might be reversible in nature .

techniques have been prevalently employed for the identification and relative / absolute quantification of proteins . lc ms - based quantification approaches can be roughly divided into two main categories : ( i ) labeling techniques such as isobaric tags for relative and absolute quantification ( itraq ) , tandem mass tags ( tmts ) , stable isotope labeling by amino acids in cell culture ( silac ) , and neutron - encoded mass signatures ( neucode ) and ( ii ) label - free methods such as spectral counting and peptide ion - current - based approaches . recently , because of their simplicity , cost - effectiveness , and feasibility of multiple biological samples analyses , ion - current - based approaches have emerged as an attractive tool in quantitative proteomics . this trend has been also boosted by the dramatically increasing availability of high - resolution ms instrumentations in the past few years . besides the well - controlled sample preparation and lc / ms procedures , an appropriate method for data analysis is also essential to achieve confident and accurate ion - current - based quantification . for instance , normalization is often applied in label - free quantitative proteomics to reduce the effect of the complicated analytical variability and systematic bias . many normalization methods such as central tendency , lowess regression , and quantile normalization were first used in the analysis of microarray data and have been recently adapted for analyzing proteomics data . the evaluation of different normalization approaches has been widely performed based on high - abundance peptides ( common to all or the majority of lc kultima et al . demonstrated that the regrrun ( linear regression followed by analysis order normalization ) effectively decreased the median sd by 43% on average compared with raw data in peaks that successfully matched across more than 50% lc ms analyses . in addition , many factors involved in the normalization procedure such as imputation ( for missing values ) , retention time , precursor m / z , and prefractionation of sample also have been studied in label - free quantification . another important issue for data analysis is choice of methods to compute protein ratios based on peptide quantitative information , which has been widely studied for labeling techniques . it has been demonstrated that a simple sum - of - intensities algorithm achieved superior performance over other algorithms such as average of the ratios , libra ratio , linear regression , and total least - squares for estimation of true protein ratios . a systematic evaluation in this regard has not been conducted for ion - current - based label - free method , although various methods were applied in popular packages and procedures . the sum or average intensity method has been employed in packages such as the intensity - based absolute quantification ( ibaq , though the intensity is divided by the number of theoretically observable peptides ) , progenesis lc ms software ( nonlinear dynamics limited , newcastle upon tyne , u.k . ) , and the ion - current - based method we developed previously . packages such as census and sieve ( thermo fisher scientific , san jose , ca ) have applied a variance - weighted method ( based on standard deviation or coefficient variation of peaks / peptides ) to calculate protein quantitation ratios . other protein ratio estimation methods such as top3 ( using the sum intensities of the top - three unique peptides ) and average ratios are also employed in quantitative proteomics . in this study , we developed and optimized a new label - free quantitative procedure for ion - current - based quantification , ican ( ion - current - based analysis ) , and evaluated its capacity for proteomic quantification and the discovery of significantly different proteins , even for these with small - fold changes ( 1.5-fold ) . key quantitative features such as frame filtering , normalization , protein ratio determination , and statistical analysis were comprehensively evaluated and optimized . with these optimizations , ican significantly improved the quantitative accuracy and sensitivity and performance in discovering altered proteins over existing methods . the pc3-ln4 cells and e. coli cells were from kinex pharmaceuticals ( buffalo , ny ) . the rat brain samples were from buffalo general medical center ( buffalo , ny ) . cell or tissue samples were homogenized in an ice - cold lysis buffer ( 50 mm tris - formic acid , 150 mm nacl , 0.5% sodium deoxycholate , 2% sds , 2% np-40 , ph 8.0 ) using a polytron homogenizer ( kinematica ag , switzerland ) . after homogenization performed for a 510s burst at 15 000 rpm for 10 times , the mixture was then sonicated in a cold room for 10 min with a low - power sonicator until the solution was clear . lysates were centrifuged at 140 000 g for 1 h at 4 c . the supernatant was collected and stored at 80 c until analysis . for preparation of moderate - abundance proteins ( maps ) , the plasma sample ( 200 ul ) from a healthy young woman was fractionated with igy14-supermix tandem column ( sigma - aldrich ) , as previously reported . three buffers ( dilution / washing buffer : 10 mm tris - hcl , 150 mm nacl , ph7.4 ( tbs ) ; stripping buffer : 100 mm glycine , ph2.5 ; neutralization buffer : 100 mm tris - hcl , ph8.0 ) were , respectively , used for loading / washing , eluting , and neutralization . the resulting flow - through fraction ( low - abundance proteins ) and the bound / eluted fractions from igy-14 ( high - abundance proteins ) and from supermix ( maps ) were collected separately . all fractions were then individually concentrated in amicon centrifugal filter with 3-kda molecular mass cutoff ( emd millipore ) , followed by buffer exchange to 50 mm nh4hco3 according to the manufacturer s instruction . protein concentration was measured using bca protein assay ( pierce , rockford , il ) . the amounts of 100 and 90 g e. coli extracts were , respectively , spiked with bovine serum albumin ( bsa ) at four different levels ( 0.025 , 0.05 , 0.075 , and 0.1% of total proteins ) and maps at two different levels ( 5 g and 7.5 g ) . all samples ( each containing 100 g of total protein ) were reduced with tcep ( 3 mm ) for 10 min and then alkylated with 20 mm iam for 30 min in darkness . a precipitation / on - pellet - digestion procedure was applied to performed precipitation and tryptic digestion as previously described . peptide samples were analyzed using an ultrahigh pressure eksigent ( dublin , ca ) nano-2d ultracapillary / nano - lc system coupled to a ltq / orbitrap xl hybrid mass spectrometer ( thermo fisher scientific , san jose , ca ) . the mobile phase consisted of 0.1% formic acid in 2% acetonitrile ( a ) and 0.1% formic acid in 88% acetonitrile ( b ) . samples were loaded onto a reversed - phase trap ( 300 m i d 1 cm ) , with 1% mobile phase b at a flow rate of 10 l / min , and the trap was washed for 3 min . a series of nanoflow gradients ( flow rate , 250 nl / min ) was used to back - flush the trapped samples onto the nano - lc column ( 75 m i d 75 cm , packed with 3 m particles ) for separation . the nano - lc column was heated to 52 c to greatly improve both chromatographic resolution and reproducibility . to stabilize ionization efficiency , the spray tip was cleaned by dripping 50% methanol by gravity after every three runs . the parameters for ms were demonstrated in our previous publications . in this study , for the spiked - in bsa experiment , each group at different bsa concentration was analyzed four times ; for the spiked - in map experiments , two groups at different maps concentration were alternatively analyzed five times . five consecutive runs of the rat brain sample and six runs with different load amount of pc3-ln4 cell ( 1 and 2 g , three replicates per group ) were further analyzed to assess different normalization methods . in addition , to assess the performance of biomarker discovery by ican and ibaq , we employed the study 6 ltq orbitrap xl @p65 data set generated by the program of clinical proteomic technology assessment for cancer ( cptac ) . according to the publicly available documentation associated with this study , the universal proteomics standard set 1 ( ups1 , a 48-protein equimolar standard ) was spiked at amounts of 0.25 and 0.74 fmol/l into yeast lysate for sets a and b , the subset of studies investigated in the current work . proteome discoverer version 1.4.1.14 ( thermo - scientific ) was used to perform database searching against swiss - prot protein database ( version 06/13/2012 ) for the bsa spiked - in experiment , five consecutive lc ms runs experiment , and six lc ms runs with different load amount experiment . maxquant v1.4.1.2 , incorporated with the andromeda search engine , was used for the map spiked - in experiment and cptac study 6 data . a total of 7766 protein entries , 20 238 entries , 4431 entries , and 7801 entries were presented in respective rat , human , e. coli , and yeast database . the databases were augmented with sequence of bsa , the ups1 48 proteins ( sigma - aldrich ) , and 118 maps ( achieved from three replicate lc the search parameters used were as follows : 10 ppm tolerance for precursor ion masses and 1.0 da for fragment ion masses . carbamidomethylation of cysteines was set as a fixed modification , and a variable modification of methionine oxidation was allowed . the false discovery rate ( fdr ) was determined by using a target - decoy search strategy . the sequence database contains each sequence in both forward and reversed orientations , enabling fdr estimation . for resulted files from proteome discoverer , scaffold v4.2.0 ( proteome software , portland , or ) was used to validate ms2-based peptide and protein identification based on cutoffs of cross - correlation ( xcorr ) and delta cn values . the fdr was set to 0.01 and 0.05 , respectively , for peptide and protein identifications . for maxquant , the fdr was set to 0.01 for peptide and protein identifications , respectively . the identifications from the reverse database and common contaminants were eliminated . the protein quantitative values based on ms2-tic , nasf and empai for each data set were obtained using scaffold v4.2.0 under the same peptide / protein identification criteria . the ibaq intensities , the sum of intensities of all peptides divided by the number of theoretically observable peptides , were achieved from the maxquant using standard settings with the option the ibaq values for each protein are normalized against sum of quantitative values in individual runs . the quantitative analysis by ican was performed as shown in the pipeline ( figure 1 ) . the peak detection and chromatographic alignment based on retention time , m / z , and charge states were analyzed by sieve v2.1 ( thermo scientific , san jose , ca ) . quantitative frames / features were defined based on m / z ( width : 10 ppm ) and retention time ( width : 2.5 min ) of peptide precursors in the aligned runs . subsequently , using tools in - house the ms2 fragmentation scans associated with each frame were assigned to the peptide / protein identifications from proteome discoverer or maxquant as previously described . the loess normalization was performed to reduce the systematic bias . in the case of missing data , a value of 1000 as the baseline quantitative value was assigned . after further excluding frames shared with multiple proteins , intensities for frames with the same sequence were combined to be the unique peptide intensity and then intensities for unique peptides of the same protein were further combined to be the protein intensity with grubbs test analysis in both steps . grubbs test was performed by the listpor ( v version 2.2.2104 ) program ( panomics.pnnl.gov ) . minimum data set presence 3 and 2 , p value cutoff of 0.01 and 0.05 , were , respectively , set at frame level and unique peptide level . the relative protein ratio was calculated by comparing the summed abundance values of the protein in each group . student s t - test statistics was applied to analyze log - transformed values of protein intensities for all of these methods . abundance change 1.3-fold and p value 0.05 were used as the thresholds to define altered proteins . the p - value adjustments for multiple testing were evaluated according to sequential bonferroni correction ( sb ) , benjamini hochberg fdr control ( bh ) , and sequential fisher s combined probability test ( sfisher ) . the ican supports identification results from proteome discoverer , maxquant , and mascot . for label - free proteomic quantification , accurate and precise quantification of low - abundance proteins remains challenging . as demonstrated by various laboratories including ours , spectral count - based approaches resulted in suboptimal quantification of low - abundance proteins due to the inherent biases and variations in data - dependent sampling of fragment ions ( ms2 ) . by comparison , ion - current - based approaches have been shown to afford markedly improved quantification for low - abundance proteins when efficient and reproducible liquid - chromatography ( lc ) separation and high - resolution ms are employed . to date , owing to the prevalent use of high - resolution ms , ion - current - based methods have become the most promising label - free approaches . however , comprehensive evaluation and optimization of data analysis approaches for ion - current - based quantification have not been adequately reported . here , based on extensive evaluation and optimization , we developed an optimal ion - current - based procedure ( figure 1 ) termed ican ( ion - current - based analysis ) and assessed its capacity for proteomic quantification and discovery of significantly altered proteins , even for these with small - fold changes ( 1.5-fold ) . the ican is designed for data generated from high - resolution ms ; in the current work , we chose to interface sieve ( thermo scientific , san jose , ca ) with this pipeline , which performs peak detection and chromatographic alignment based on retention time , m / z , and charge states . each aligned quantitative feature ( i.e. , a frame , the set of peak areas of a specific peptide ) was correlated with the peptide / protein i d information from popular software such as proteome discoverer , maxquant , and mascot with scripts developed in - house . streamlined processes for frame filtering , loess normalization , and outlier detection by grubbs test on both frame and peptide levels were integrated in ican . these processes were comprehensively optimized and proved to significantly improve the quantitative accuracy and sensitivity and performance in discovering altered proteins over existing strategies . in this study , the frame identification is derived from the spectrum identification results from popular database search algorithms such as proteome discoverer and maxquant . we used in - house scripts to assign these peptide identifications to the distinguished frames . on the basis of our previous studies , it was observed that some frames contained multiple unique peptides and thus may lead to unreliable quantification . here we examined the shared frame issue using the analysis of a series of e. coli extracts spiked with bsa at four different levels ( 0.025 , 0.05 , 0.075 , and 0.1% of total proteins ; four replicates per group ) . a total of 818 proteins including bsa were identified with a peptide fdr of 0.1% ( supplemental table 1 in the supporting information ) . among the total of 13 801 quantitative frames ( supplemental table 2 in the supporting information ) , 654 ( 4.7% ) assigned to multiple peptide ids were observed . of these shared frames , 617 ( 94.3% ) frames only have two unique peptides ( supplemental figure 1 in the supporting information ) . the peptides with shared frames likely have indistinguishable m / z and retention time , or some of them were derived from misassigned peptide during database search . we evaluated different cutoff thresholds for peptide / protein identification and found that more stringent cutoffs for identification ( e.g. , lower identification fdr threshold ) reduced of the percentage of shared frames , and thus stringent criteria for identification is advisable . some representative data are shown in table 1 . moreover , the peptide fdr in shared frame - associated spectra is much higher ( 9-fold ) than the determined global peptide fdr ( table 1 ) , indicating increased incorrect identifications in shared frames . thus , in this study , those share frames containing multiple unique peptides were eliminated . spiked - in bsa experiment , 5 replicates of yeast and 20 replicates of rat brain were analyzed in this study . an optimal normalization method is indispensable to reduce systematic biases and variations and thus to ensure the accuracy and precision of relative quantification in multiple samples . previously , many normalization approaches have been evaluated for label - free quantification on relatively high - abundance peptides that are commonly identified in all or the majority of lc here we evaluated all of the identified peptides with a wide range of abundance levels by six different normalization methods , including loess , quantile , upper - quantile , maximum intensity , median intensity , and total intensity normalization ( supporting information ) . the loess and quantile normalization achieved best performances in the spiked - in bsa experiment , which decreased the median coefficient variations ( cvs ) of e. coli peptide intensities by an average of 29% compared with the original data ( figure 2 ) . ms runs of the same rat brain digest and six runs of the same digest with different load amounts , respectively , representing data sets with minimal and substantial variations of sample preparation and loading . the loess approach showed the most effective normalization ( supplemental figure 2 in the supporting information ) and thus was employed in ican and subsequent studies . bsa was spiked into e. coli extracts at four different levels ( 0.025 , 0.05 , 0.075 , and 0.1% of total proteins ; four replicates / group ) . box and whiskers ( 199 percentile ) plot was used to analyze the coefficient variations ( cvs ) of e. coli peptide intensities among these 16 lc after normalization , the level of missing data and reproducibility of the quantitative features by ican was further evaluated based on replicate lc ms runs . one of the most prominent advantages of ion - current - based approach over spectral counting or fragment - ion intensities ( ms2-tic ) is the reliable quantification of low - abundance peptides , even though a peptide was only identified for once in the entire sample set , thus substantially reducing the frequency of missing values and improving the analytical reproducibility . as shown in supplemental figure 3 in the supporting information , although several methods were employed to improve the reproducibility of lc ms analysis as previously described , only 655 ( 80.1% of all identified ) proteins were identified in all 16 lc ms runs and thereby quantifiable by spectral counting or ms2-tic without missing data . per contra , the ican was able to quantify 816 ( 99.8% ) proteins without any missing value across the 16 lc were filtered out because all frames assigned to these two proteins were shared frames . we evaluated the quantitative reproducibility of ican by correlating the protein intensities between any two of the four lc ms analyses in the spiked - in bsa ( 0.075% ) group . here the protein intensity was obtained by summing the areas of all peptide peaks assigned to the specific protein . linear regression of the correlation between two replicate runs was performed , and the r - squared values for paired correlations are all above 0.99 , indicating the excellent quantitative reproducibility ( figure 3 ) . moreover , a high quantitative reproducibility was also achieved for both high- ( the upper segment of each line ) and low - abundance proteins ( the lower segment of each line ) . the reproducibility of spectral counting or ms2-tic methods was far inferior , which is particularly sound for low - abundance proteins ( supplemental figure 4 in the supporting information ) . the excellent correlation of protein intensities between different replicates of a spiked - in bsa ( 0.075% ) group was observed . the two axes represent the quantitative abundance values of the same proteins , respectively , by the two duplicate runs . the preliminary assessment of quantitative accuracy and precision by ican was performed using the bsa spiked - in e. coli data . the expected ratio of the reference proteins ( e. coli ) was 1.00 , and the five possible changes of bsa have expected ratios of 1.33 ( 0.1%/0.075% bsa in e. coli ) , 1.50 ( 0.075%/0.05% ) , 2.00 ( 0.1%/0.05% ) , 3.00 ( 0.075%/0.025% ) , and 4.00 ( 0.1%/0.025% ) , respectively . as shown in figure 4 , ican quantified nearly all identified proteins without missing data ( as previously described ) , and the measured bsa ratios agreed very well with the expected values with small relative deviations ( 0.39.5% ) . excellent linearity between the nominal and observed ratios was achieved ( supplemental figure 5 in the supporting information ) . the ratios of reference proteins determined by ican were tightly centered around the theoretical value . the means and standard deviations of the ratios of reference proteins , were , respectively , 0.99 0.06 , 1.01 0.07 , 1.00 0.05 , 1.00 0.08 , and 0.98 0.05 for the five comparisons previously mentioned ( n = 4/group ) , reflecting the high accuracy and precision achieved by ican in calculating protein expression ratios . the popular ms2-based methods such as ms2-tic , the normalized spectral abundance factor ( nasf ) , and exponentially modified protein abundance index ( empai ) were also evaluated ( figure 4b d ) . to achieve optimal analysis for these methods , we employed only the 655 proteins that had no missing data in any of the replicates when performing quantification with these approaches . even ican calculated the lowest 20% proteins in abundance while other ms2-based methods did not , it still performed significantly better in terms of quantitative accuracy and precision , as shown in figure 4 . on the basis of these results , the following sections are focused on the comparison of ion - current - based strategies . evaluation of accuracy and precision of relative quantification analysis by ican , ms2-tic , nasf , and empai using the spiked - in bsa data . bsa was spiked into e. coli extracts at four different levels ( 0.025 , 0.05 , 0.075 , and 0.1% of total proteins ) . the expected ratio of 1 for reference proteins and five theoretical fold changes of 1.33 ( 0.1/0.075 ) , 1.50 ( 0.075/0.05 ) , 2.00 ( 0.1/0.05 ) , 3.00 ( 0.075/0.025 ) , and 4.00 ( 0.1/0.025 ) for bsa were investigated . for proteomics quantification , one of the major aims is to completely discover the true altered proteins to the extent possible , while minimizing false - positives that can otherwise lead to misleading biological clues and waste of resources in informatics analysis and validation . to evaluate the sensitivity and false positive rate ( fpr ) of biomarker discovery by ion - current - based quantification methods , we spiked a mixture of maps obtained from human plasma into e. coli extracts at two different levels ( map - a : 90 g of e. coli and 5 g of map ; map - b : 90 g of e. coli and 7.5 g of map ) . in this set , the expected ratio of reference proteins ( e. coli ) and maps were 1.00 and 1.50 ( map - b / map - a ) , respectively . a total of 775 proteins including 49 maps were identified with a peptide and protein fdr of 1% , respectively , using maxquant ( version 1.4.1.2 ) . the list of peptide and protein identifications was shown in supplemental table 3 in the supporting information . when using 1.3-fold change ( the lowest quantifiable fold - change by our ion - current - based quantitative method based on our previous investigation ) and p 0.05 ( t test ) as the cutoff thresholds , 45 of 49 ( 91.8% ) maps and none of reference proteins were determined as altered proteins ( fpr = 0% ) by ican , as shown in figure 5a ( details in supplemental table 4 in the supporting information ) . the reference proteins and map were , respectively , indicated by blue and red dots . the mean and standard deviation of the ratios of 45 altered maps quantified by ican was 1.71 0.13 , demonstrating excellent sensitivity and accuracy in discovery of changed proteins . the outstanding ability of ican for identifying altered proteins was further proved by the area under curve value of 0.97 using receiver - operating characteristic ( roc ) analysis ( supplemental figure 6 in the supporting information ) . in this study , it was showed that ican could quantify nearly all identified proteins and assign significance with high sensitivity and low fpr to small changes of 1.5-fold , providing a competitive ability in the field of quantitative proteomics . relative ratios obtained by ( a ) ican and ( b ) ibaq for a quantitative experiment of e. coli extracts spiked with human plasma moderate - abundance proteins ( maps ) ( n = 5/group ) . in total , 49 map proteins ( red dots , expected ratio is 1.5 between two groups ) and 726 e. coli proteins ( blue dots , expected ratio of 1.0 ) were quantified . the ibaq method , which divides the sum of intensities of all peptides by the number of theoretically observable peptides , was shown to be the most accurate among different absolute quantification methods in a previous work . the intensities or ibaq values for proteins were also achieved from the maxquant using standard settings with the option of match between runs selected . thus , here the same list of peptide / protein identifications from maxquant was shared and analyzed by ican and ibaq . we calculated the relative ratios of proteins by the ibaq values ( supplemental table 4 in the supporting information ) against ican . using the same threshold , 42 of 49 ( 85.7% ) maps and 7 reference proteins ( figure 5b ) were determined as altered proteins ( fpr = 14.3% ) by ibaq , with a significantly lower sensitivity and higher fpr than ican . the mean and standard deviation of the ratios of 42 altered maps quantified by ibaq was 1.8 0.2 , also indicating good accuracy and precision in discovery of changed proteins . a third - party , publicly available data set , the clinical proteomic technology assessment for cancer ( cptac ) study 6 data , was employed for further investigation of these two methods on the relative quantification . here study 6b ( 0.74 fmol/l ups1 spiked into yeast lysate ) versus 6a ( 0.25 fmol/l ups1 spiked into yeast lysate a ) samples , which contain relatively low abundance of ups proteins , were selected to analyze . the expected ratios for yeast proteins and ups were , respectively , 1.0 and 3.0 . after database searching by maxquant , a total of 777 proteins including 15 ups proteins were identified in this study . using the same threshold ( 1.3-fold and p 0.05 ) , all ( 100% ) ups with a median ratio of 3.25 and 1 yeast protein were determined by ican as significantly altered proteins , while 12 ( 80% ) ups with a median ratio of 4.78 and 5 yeast proteins were determined by ibaq ( supplemental figure 7 and supplemental table 5 in the supporting information ) . again , ican was demonstrated to be superior in that it identified more true - positives ( ups proteins ) with higher quantitative accuracy and lower fpr than ibaq method . wrong peptide identification or incorrect assignment of peptide i d to quantitative frames may severely compromise the quantification of the affected proteins ; in quantitative analysis , these incorrectly identified / assigned peptides often take the form of outliers , which must be removed to ensure reliable quantification . here we used grubbs test to identify and then eliminate outliers arising from wrong peptide assignment or large biological / technical variations before the calculation of the quantitative values of unique peptides and proteins . in this study , we further evaluated the protein ratio determination method by comparing a sum - of - intensities method with outlier removal versus other popular approaches . using the abundance values obtained by ican , approaches for aggregating quantitative data from peptide - level to protein levels such as top3 , sum - of - intensity , average ratios , variance - weighted ( on coefficient variation of peptide ) , and linear regression ( supporting information ) were evaluated versus ican . as previously described , these approaches have been widely used in quantitative proteomics . as shown in figure 6a , similar sensitivity for biomarker discovery was achieved by ican , variance - weighted , average ratio , and sum - of - intensity approaches using the spiked - in map data . the ican and variance - weighted approaches showed the lowest and second lowest fpr for identifying altered protein . without outlier analysis , variance - weighted approach achieved the comparable sensitivity with ican in discovering altered proteins in this study , while the sensitivity of other approaches are inferior ( figure 6a ) . in addition , it is clear that grubbs test outlier analysis greatly reduced the false - positives ( ican vs sum - of - intensity ) ( figure 6a ) . for instance , e. coli protein glutamine - fructose-6-phosphate transaminase ( glms , expected ratio is 1.0 ) , determined as an altered protein ( 1.56-fold and p value = 0.02 ) by sum - of - intensity method , was quantified by 11 unique peptides , but 10 ( 90.9% ) of them have ratios around the expected ones , as shown in supplemental figure 8 in the supporting information . the ican analysis ( sum - of - intensity with rejection ) removes the outlier ( red spots in supplemental figure 8 in the supporting information ) and gives the protein ratio ( 0.97-fold and p value = 0.43 ) that agrees well with most of the peptide ratio . therefore , here we utilized the sum - of - intensity with rejection for protein ratio estimation in the ion - current - based quantification procedure to replace the sum - of - intensity approach we described in previous studies . evaluation of ( a ) different methods for aggregating quantitative data from peptide - level to protein levels and ( b ) multiple testing approaches for ican - based quantification . the false - positive rate ( fpr ) and sensitivity for discovering altered proteins were investigated with the combination of statistical analysis and a fold - change filter ( 1.3-fold ) . a p value of 0.05 was adopted . for investigation of multiple testing , critical significance levels of both 0.05 and 0.10 sb , sequential bonferroni test ; bh , benjamini and hochberg test ; sfisher , sequential fisher combined probability test . we also evaluated multiple hypothesis testing such as sequential bonferroni correction ( sb ) , benjamini - hochberg fdr control ( bh ) , and sequential fisher s combined probability test ( sfisher ) to adjust the p value of t test ( supporting information ) . for investigation of multiple testing , we used 0.05 and 0.10 , respectively , as the critical significance level . with the combination of fold - change ( 1.3-fold threshold ) and statistical testing ( 0.05 or 0.10 ) , the superior performance of biomarker discovery was observed by sfisher compared with the other two methods ( figure 6b and supplemental table 6 in the supporting information ) in ion - current - based quantification . forty - three ( 87.8% ) maps and none of reference proteins were identified as altered proteins with the thresholds of 1.3-fold and p 0.05 by sfisher . the cptac data described above were also tested using multiple testing , and a similar result was shown in supplemental figure 9 and supplemental table 7 in the supporting information , indicating the superior of sfisher . ion - current - based quantitative approach has emerged as an attractive alternative to both spectral counting and labeling methods , which can analyze many biological samples for large - scale studies such as clinical and pharmaceutical investigations . recently , the wide prevalence of high - resolution ms has greatly boosted the quality of ion - current - based analysis . moreover , the substantial advancements in ms instrumentation ( e.g. , analysis of 4000 unique yeast proteins in 1 h of lc ms / ms run using a hybrid oribtrap ms instrument ) , will markedly enhance the coverage of ion - current - based analysis . for ion - current - based strategy , a data - processing procedure enabling accurate , precise , and sensitive quantification is critical . here we demonstrated that the ican procedure is optimal for ion - current - based quantitative analysis , which provides superior quantitative accuracy and higher sensitivity for biomarker discovery with a lower fdr than these popular methods we ve tested . furthermore , the comparative investigations of various quantitative features in this study provide highly valuable information for the development and evaluation of algorithms for both labeling and label - free methods .

the rapidly expanding availability of high - resolution mass spectrometry has substantially enhanced the ion - current - based relative quantification techniques . despite the increasing interest in ion - current - based methods , quantitative sensitivity , accuracy , and false discovery rate remain the major concerns ; consequently , comprehensive evaluation and development in these regards are urgently needed . here we describe an integrated , new procedure for data normalization and protein ratio estimation , termed ican , for improved ion - current - based analysis of data generated by high - resolution mass spectrometry ( ms ) . ican achieved significantly better accuracy and precision , and lower false - positive rate for discovering altered proteins , over current popular pipelines . a spiked - in experiment was used to evaluate the performance of ican to detect small changes . in this study e. coli extracts were spiked with moderate - abundance proteins from human plasma ( map , enriched by igy14-supermix procedure ) at two different levels to set a small change of 1.5-fold . forty - five ( 92% , with an average ratio of 1.71 0.13 ) of 49 identified map protein ( i.e. , the true positives ) and none of the reference proteins ( 1.0-fold ) were determined as significantly altered proteins , with cutoff thresholds of 1.3-fold change and p 0.05 . this is the first study to evaluate and prove competitive performance of the ion - current - based approach for assigning significance to proteins with small changes . by comparison , other methods showed remarkably inferior performance . ican can be broadly applicable to reliable and sensitive proteomic survey of multiple biological samples with the use of high - resolution ms . moreover , many key features evaluated and optimized here such as normalization , protein ratio determination , and statistical analyses are also valuable for data analysis by isotope - labeling methods .

Introduction Materials and Methods Results and Discussion Conclusions Data Sharing

lc ms - based quantification approaches can be roughly divided into two main categories : ( i ) labeling techniques such as isobaric tags for relative and absolute quantification ( itraq ) , tandem mass tags ( tmts ) , stable isotope labeling by amino acids in cell culture ( silac ) , and neutron - encoded mass signatures ( neucode ) and ( ii ) label - free methods such as spectral counting and peptide ion - current - based approaches . recently , because of their simplicity , cost - effectiveness , and feasibility of multiple biological samples analyses , ion - current - based approaches have emerged as an attractive tool in quantitative proteomics . this trend has been also boosted by the dramatically increasing availability of high - resolution ms instrumentations in the past few years . besides the well - controlled sample preparation and lc / ms procedures , an appropriate method for data analysis is also essential to achieve confident and accurate ion - current - based quantification . many normalization methods such as central tendency , lowess regression , and quantile normalization were first used in the analysis of microarray data and have been recently adapted for analyzing proteomics data . in addition , many factors involved in the normalization procedure such as imputation ( for missing values ) , retention time , precursor m / z , and prefractionation of sample also have been studied in label - free quantification . a systematic evaluation in this regard has not been conducted for ion - current - based label - free method , although various methods were applied in popular packages and procedures . , and the ion - current - based method we developed previously . other protein ratio estimation methods such as top3 ( using the sum intensities of the top - three unique peptides ) and average ratios are also employed in quantitative proteomics . in this study , we developed and optimized a new label - free quantitative procedure for ion - current - based quantification , ican ( ion - current - based analysis ) , and evaluated its capacity for proteomic quantification and the discovery of significantly different proteins , even for these with small - fold changes ( 1.5-fold ) . key quantitative features such as frame filtering , normalization , protein ratio determination , and statistical analysis were comprehensively evaluated and optimized . for preparation of moderate - abundance proteins ( maps ) , the plasma sample ( 200 ul ) from a healthy young woman was fractionated with igy14-supermix tandem column ( sigma - aldrich ) , as previously reported . the resulting flow - through fraction ( low - abundance proteins ) and the bound / eluted fractions from igy-14 ( high - abundance proteins ) and from supermix ( maps ) were collected separately . the amounts of 100 and 90 g e. coli extracts were , respectively , spiked with bovine serum albumin ( bsa ) at four different levels ( 0.025 , 0.05 , 0.075 , and 0.1% of total proteins ) and maps at two different levels ( 5 g and 7.5 g ) . in this study , for the spiked - in bsa experiment , each group at different bsa concentration was analyzed four times ; for the spiked - in map experiments , two groups at different maps concentration were alternatively analyzed five times . five consecutive runs of the rat brain sample and six runs with different load amount of pc3-ln4 cell ( 1 and 2 g , three replicates per group ) were further analyzed to assess different normalization methods . in addition , to assess the performance of biomarker discovery by ican and ibaq , we employed the study 6 ltq orbitrap xl @p65 data set generated by the program of clinical proteomic technology assessment for cancer ( cptac ) . proteome discoverer version 1.4.1.14 ( thermo - scientific ) was used to perform database searching against swiss - prot protein database ( version 06/13/2012 ) for the bsa spiked - in experiment , five consecutive lc ms runs experiment , and six lc ms runs with different load amount experiment . maxquant v1.4.1.2 , incorporated with the andromeda search engine , was used for the map spiked - in experiment and cptac study 6 data . for resulted files from proteome discoverer , scaffold v4.2.0 ( proteome software , portland , or ) was used to validate ms2-based peptide and protein identification based on cutoffs of cross - correlation ( xcorr ) and delta cn values . the fdr was set to 0.01 and 0.05 , respectively , for peptide and protein identifications . abundance change 1.3-fold and p value 0.05 were used as the thresholds to define altered proteins . by comparison , ion - current - based approaches have been shown to afford markedly improved quantification for low - abundance proteins when efficient and reproducible liquid - chromatography ( lc ) separation and high - resolution ms are employed . to date , owing to the prevalent use of high - resolution ms , ion - current - based methods have become the most promising label - free approaches . however , comprehensive evaluation and optimization of data analysis approaches for ion - current - based quantification have not been adequately reported . here , based on extensive evaluation and optimization , we developed an optimal ion - current - based procedure ( figure 1 ) termed ican ( ion - current - based analysis ) and assessed its capacity for proteomic quantification and discovery of significantly altered proteins , even for these with small - fold changes ( 1.5-fold ) . the ican is designed for data generated from high - resolution ms ; in the current work , we chose to interface sieve ( thermo scientific , san jose , ca ) with this pipeline , which performs peak detection and chromatographic alignment based on retention time , m / z , and charge states . , a frame , the set of peak areas of a specific peptide ) was correlated with the peptide / protein i d information from popular software such as proteome discoverer , maxquant , and mascot with scripts developed in - house . these processes were comprehensively optimized and proved to significantly improve the quantitative accuracy and sensitivity and performance in discovering altered proteins over existing strategies . in this study , the frame identification is derived from the spectrum identification results from popular database search algorithms such as proteome discoverer and maxquant . here we examined the shared frame issue using the analysis of a series of e. coli extracts spiked with bsa at four different levels ( 0.025 , 0.05 , 0.075 , and 0.1% of total proteins ; four replicates per group ) . spiked - in bsa experiment , 5 replicates of yeast and 20 replicates of rat brain were analyzed in this study . an optimal normalization method is indispensable to reduce systematic biases and variations and thus to ensure the accuracy and precision of relative quantification in multiple samples . previously , many normalization approaches have been evaluated for label - free quantification on relatively high - abundance peptides that are commonly identified in all or the majority of lc here we evaluated all of the identified peptides with a wide range of abundance levels by six different normalization methods , including loess , quantile , upper - quantile , maximum intensity , median intensity , and total intensity normalization ( supporting information ) . the loess and quantile normalization achieved best performances in the spiked - in bsa experiment , which decreased the median coefficient variations ( cvs ) of e. coli peptide intensities by an average of 29% compared with the original data ( figure 2 ) . bsa was spiked into e. coli extracts at four different levels ( 0.025 , 0.05 , 0.075 , and 0.1% of total proteins ; four replicates / group ) . box and whiskers ( 199 percentile ) plot was used to analyze the coefficient variations ( cvs ) of e. coli peptide intensities among these 16 lc after normalization , the level of missing data and reproducibility of the quantitative features by ican was further evaluated based on replicate lc ms runs . one of the most prominent advantages of ion - current - based approach over spectral counting or fragment - ion intensities ( ms2-tic ) is the reliable quantification of low - abundance peptides , even though a peptide was only identified for once in the entire sample set , thus substantially reducing the frequency of missing values and improving the analytical reproducibility . we evaluated the quantitative reproducibility of ican by correlating the protein intensities between any two of the four lc ms analyses in the spiked - in bsa ( 0.075% ) group . moreover , a high quantitative reproducibility was also achieved for both high- ( the upper segment of each line ) and low - abundance proteins ( the lower segment of each line ) . the reproducibility of spectral counting or ms2-tic methods was far inferior , which is particularly sound for low - abundance proteins ( supplemental figure 4 in the supporting information ) . the preliminary assessment of quantitative accuracy and precision by ican was performed using the bsa spiked - in e. coli data . the expected ratio of the reference proteins ( e. coli ) was 1.00 , and the five possible changes of bsa have expected ratios of 1.33 ( 0.1%/0.075% bsa in e. coli ) , 1.50 ( 0.075%/0.05% ) , 2.00 ( 0.1%/0.05% ) , 3.00 ( 0.075%/0.025% ) , and 4.00 ( 0.1%/0.025% ) , respectively . the means and standard deviations of the ratios of reference proteins , were , respectively , 0.99 0.06 , 1.01 0.07 , 1.00 0.05 , 1.00 0.08 , and 0.98 0.05 for the five comparisons previously mentioned ( n = 4/group ) , reflecting the high accuracy and precision achieved by ican in calculating protein expression ratios . the popular ms2-based methods such as ms2-tic , the normalized spectral abundance factor ( nasf ) , and exponentially modified protein abundance index ( empai ) were also evaluated ( figure 4b d ) . even ican calculated the lowest 20% proteins in abundance while other ms2-based methods did not , it still performed significantly better in terms of quantitative accuracy and precision , as shown in figure 4 . on the basis of these results , the following sections are focused on the comparison of ion - current - based strategies . evaluation of accuracy and precision of relative quantification analysis by ican , ms2-tic , nasf , and empai using the spiked - in bsa data . bsa was spiked into e. coli extracts at four different levels ( 0.025 , 0.05 , 0.075 , and 0.1% of total proteins ) . the expected ratio of 1 for reference proteins and five theoretical fold changes of 1.33 ( 0.1/0.075 ) , 1.50 ( 0.075/0.05 ) , 2.00 ( 0.1/0.05 ) , 3.00 ( 0.075/0.025 ) , and 4.00 ( 0.1/0.025 ) for bsa were investigated . for proteomics quantification , one of the major aims is to completely discover the true altered proteins to the extent possible , while minimizing false - positives that can otherwise lead to misleading biological clues and waste of resources in informatics analysis and validation . to evaluate the sensitivity and false positive rate ( fpr ) of biomarker discovery by ion - current - based quantification methods , we spiked a mixture of maps obtained from human plasma into e. coli extracts at two different levels ( map - a : 90 g of e. coli and 5 g of map ; map - b : 90 g of e. coli and 7.5 g of map ) . in this set , the expected ratio of reference proteins ( e. coli ) and maps were 1.00 and 1.50 ( map - b / map - a ) , respectively . when using 1.3-fold change ( the lowest quantifiable fold - change by our ion - current - based quantitative method based on our previous investigation ) and p 0.05 ( t test ) as the cutoff thresholds , 45 of 49 ( 91.8% ) maps and none of reference proteins were determined as altered proteins ( fpr = 0% ) by ican , as shown in figure 5a ( details in supplemental table 4 in the supporting information ) . the mean and standard deviation of the ratios of 45 altered maps quantified by ican was 1.71 0.13 , demonstrating excellent sensitivity and accuracy in discovery of changed proteins . in this study , it was showed that ican could quantify nearly all identified proteins and assign significance with high sensitivity and low fpr to small changes of 1.5-fold , providing a competitive ability in the field of quantitative proteomics . relative ratios obtained by ( a ) ican and ( b ) ibaq for a quantitative experiment of e. coli extracts spiked with human plasma moderate - abundance proteins ( maps ) ( n = 5/group ) . in total , 49 map proteins ( red dots , expected ratio is 1.5 between two groups ) and 726 e. coli proteins ( blue dots , expected ratio of 1.0 ) were quantified . using the same threshold , 42 of 49 ( 85.7% ) maps and 7 reference proteins ( figure 5b ) were determined as altered proteins ( fpr = 14.3% ) by ibaq , with a significantly lower sensitivity and higher fpr than ican . the mean and standard deviation of the ratios of 42 altered maps quantified by ibaq was 1.8 0.2 , also indicating good accuracy and precision in discovery of changed proteins . using the same threshold ( 1.3-fold and p 0.05 ) , all ( 100% ) ups with a median ratio of 3.25 and 1 yeast protein were determined by ican as significantly altered proteins , while 12 ( 80% ) ups with a median ratio of 4.78 and 5 yeast proteins were determined by ibaq ( supplemental figure 7 and supplemental table 5 in the supporting information ) . in this study , we further evaluated the protein ratio determination method by comparing a sum - of - intensities method with outlier removal versus other popular approaches . using the abundance values obtained by ican , approaches for aggregating quantitative data from peptide - level to protein levels such as top3 , sum - of - intensity , average ratios , variance - weighted ( on coefficient variation of peptide ) , and linear regression ( supporting information ) were evaluated versus ican . as shown in figure 6a , similar sensitivity for biomarker discovery was achieved by ican , variance - weighted , average ratio , and sum - of - intensity approaches using the spiked - in map data . without outlier analysis , variance - weighted approach achieved the comparable sensitivity with ican in discovering altered proteins in this study , while the sensitivity of other approaches are inferior ( figure 6a ) . for instance , e. coli protein glutamine - fructose-6-phosphate transaminase ( glms , expected ratio is 1.0 ) , determined as an altered protein ( 1.56-fold and p value = 0.02 ) by sum - of - intensity method , was quantified by 11 unique peptides , but 10 ( 90.9% ) of them have ratios around the expected ones , as shown in supplemental figure 8 in the supporting information . the ican analysis ( sum - of - intensity with rejection ) removes the outlier ( red spots in supplemental figure 8 in the supporting information ) and gives the protein ratio ( 0.97-fold and p value = 0.43 ) that agrees well with most of the peptide ratio . therefore , here we utilized the sum - of - intensity with rejection for protein ratio estimation in the ion - current - based quantification procedure to replace the sum - of - intensity approach we described in previous studies . the false - positive rate ( fpr ) and sensitivity for discovering altered proteins were investigated with the combination of statistical analysis and a fold - change filter ( 1.3-fold ) . with the combination of fold - change ( 1.3-fold threshold ) and statistical testing ( 0.05 or 0.10 ) , the superior performance of biomarker discovery was observed by sfisher compared with the other two methods ( figure 6b and supplemental table 6 in the supporting information ) in ion - current - based quantification . forty - three ( 87.8% ) maps and none of reference proteins were identified as altered proteins with the thresholds of 1.3-fold and p 0.05 by sfisher . ion - current - based quantitative approach has emerged as an attractive alternative to both spectral counting and labeling methods , which can analyze many biological samples for large - scale studies such as clinical and pharmaceutical investigations . recently , the wide prevalence of high - resolution ms has greatly boosted the quality of ion - current - based analysis . , analysis of 4000 unique yeast proteins in 1 h of lc ms / ms run using a hybrid oribtrap ms instrument ) , will markedly enhance the coverage of ion - current - based analysis . for ion - current - based strategy , a data - processing procedure enabling accurate , precise , and sensitive quantification is critical . here we demonstrated that the ican procedure is optimal for ion - current - based quantitative analysis , which provides superior quantitative accuracy and higher sensitivity for biomarker discovery with a lower fdr than these popular methods we ve tested . furthermore , the comparative investigations of various quantitative features in this study provide highly valuable information for the development and evaluation of algorithms for both labeling and label - free methods .

inflammatory bowel disease ( ibd ) is a chronic inflammation of the gastrointestinal tract thought to be a result of dysregulated or aberrant immune response to intestinal flora and multiple environmental factors with regard to genetic predisposition . exposure of intestinal epithelial cells ( iec ) to bacterial components and products can potentially initiate intestinal inflammation by their release of cytokines chemokines and recruitment of inflammatory cells . iec can also respond to a broad array of cytokines with altered gene expression and growth characteristics . cytokine that plays a crucial role in the inflammatory diseases such as ibd is il-1. enhanced level of this cytokine has been determined in mucosal tissues infected with enteropathogenic bacteria , as well as in mucosal biopsies with active ibd . il-1 activates intracellular signaling cascades in iec leading to the increase of expression and secretion of proinflammatory cytokines and chemokines , uncontrolled intestinal inflammation , and disruption of epithelial function [ 3 , 4 ] . lipopolysaccharide ( lps ) or endotoxin , the key component of the cell wall of gram - negative bacteria , stimulates activation of transcription factors and production of proinflammatory cytokines . the expression of lps specific toll - like receptor 4 ( tlr-4 ) in human colorectal cancer cells highlighted a key function of tlr system in the development of colitis - associated tumors , suggesting a role of this receptor in colorectal cancer development and progression . cytokines with anti - inflammatory properties have been implicated in the prevention of inappropriate immune activation by intestinal flora . transforming growth factor is a strong anti - inflammatory cytokine with multipotent mechanism of action . in mammals three isoforms ( tgf-1 , -2 , and -3 ) have been described , which share 75% amino acid sequence homology but are encoded by different genes . tgf-s have strong impact on the inflammatory responses and tumor microenvironment including fibroblasts , endothelial cells and immune cells . tgf-s suppress cytotoxic t - cell differentiation and inhibit nk cell and neutrophil effector functions . also , they have been shown to suppress mhc i and mhc ii expression . on the other hand , overexpression of tgf- could induce the secretion of proinflammatory cytokines , for example , tnf- , il-1 , il-6 , and il-8 . tgf- signalling is mediated by three specific types of cell surface proteins : tgf- receptor i ( tri ) , ii ( trii ) , and iii ( triii ) . tgf- initiates its signaling by binding to trii , which has intrinsic serine - threonine kinase activity . then , trii recruits and phosphorylates tri , establishing heterotetrameric complex consisting of two trii and two tri . tri initiates phosphorylation of the adaptor proteins smad2 and smad3 which is followed by the formation of complex with smad4 . nuclear smad complexes bind to smad - binding elements on dna , affecting transcriptional activity which is dependent on their interaction with coactivators . smad7 differs structurally from other members of smad family and functions as a negative regulator of tgf- signaling . its gene expression is induced by tgf- ; thus smad7 represents negative feedback loop , restraining tgf- activity . it recruits the gadd34 complex to the tri , thus preventing smad2/smad3 phosphorylation and tgf- signal transduction . smad7 also contributes to tri dephosphorylation and ubiquitination and proteasomal degradation of the tgf- receptor complex . transcription factors , histone readers , modifiers , and chromatin remodelers that bind to activated smad determine what genes and how they will be affected by signal transduction complexes . tgf- can also activate other non - smad signalling pathways , including pi3k , mapk , traf6 , and mtorc . some of the important downstream targets of tgf- signaling include cell cycle checkpoint genes , the activation of which leads to growth arrest . yet , tgf- signaling can also directly stimulate the production of several mitogenic growth factors which can drive the carcinogenic process . a number of inflammatory diseases including inflammatory bowel disease and cancer are associated with abnormal tgf-s regulation [ 5 , 17 , 18 ] . moreover it was associated with a reduction in smad3 phosphorylation , which is crucial for anti - inflammatory action of tgf-. proinflammatory stimuli , such as tnf- , il-1 , and inf- , also induce smad7 expression . chronic inflammation has been recognized to be associated with a high cancer risk and may be involved in all stages of tumor development , that is , initiation , promotion , and progression . colorectal cancer is one of the most common cancers , accounting for 8% of all cancer deaths , making it the fourth cause of cancer deaths . due to high mortality and extensive anticancer drugs toxicity there has been growing interest in substances that may have chemopreventive action , that is , can prevent or delay the development of cancer [ 24 , 25 ] . diet has been proved to play a significant role in the aetiology of colorectal cancer . consumption of dietary components with anti - inflammatory activity has been associated with reduced risk of developing colorectal cancer . one of the essential components of high fiber diet is inositol hexaphosphate ( ip6 ) . it is a naturally occurring hexaphosphorylated carbohydrate , found in both plant and mammalian cells . with intracellular concentration of about 100 m , ip6 participates in a variety of cellular functions such as signal transduction , regulation of cell proliferation , and differentiation . ip6 has been shown in in vitro studies to inhibit growth of human breast , colon , prostate , and liver cancer cells . its anticancer properties have been documented to result from its antiproliferative , proapoptotic , and antiangiogenic effects . ip6 is also known for its antioxidant properties , prevention against formation of kidney stones , high blood cholesterol level and heart and liver diseases . its antioxidant action was recognized in experimental models of myocardial reperfusion injury , pulmonary inflammation , and peptic ulcer induction . therefore , ip6 is believed to have potential to serve as preventive agent for chronic inflammation and carcinogenesis . recently , it has been revealed that ip6 has strong impact on transcriptional activity of tgf-s and their receptor genes in colon cancer cells . the aim of the present study was to examine the potential of ip6 to affect proinflammatory agents - influenced changes in transcriptional activity of the genes encoding tgf-1 , tgf-2 , and tgf-3 and their receptors tri , trii , and triii in human colon caco-2 cells . the caco-2 human intestinal epithelial cells ( dsmz , braunschweig , germany ) were routinely cultured in rpmi 1640 medium ( sigma aldrich ) supplemented with 10% fetal bovine serum ( gibcobrl ) , 100 u / ml penicillin and 100 g / ml streptomycin ( both from sigma aldrich ) and 10 mm hepes ( gibcobrl ) . cells were seeded into six - well plates ( nunc international ) at a density of 4.5 10 per well and allowed to grow to 80% confluency in 3 ml of medium . after three days the culture media were changed to media with 2% fbs and cells were then cultured for 2 days . they were then stimulated with 100 g / ml lps ( escherichia coli serotype 055:b5 , salmonella enterica serotype typhimurium ; both from sigma aldrich ) , or 1 ng / ml il-1 ( sigma aldrich ) for 30 min . afterwards cells were treated with 2.5 mm ip6 as dipotassium salt ( distilled water dissolved and ph 7.4 adjusted ) ( sigma aldrich ) for 3 and 12 h. in separate cultures , cells were incubated with lps or il-1 at the indicated concentrations and for the indicated times . total rna was extracted from cells using trizol reagent ( invitrogen ) according to the manufacturer 's specifications . integrity of the rna extracts was qualitatively checked by electrophoresis in 1.0% agarose gel stained with ethidium bromide . rna concentration was determined spectrophotometrically on the basis of absorbance values at a wavelength of 260 nm using a genequant pro ( amersham biosciences ) . detection of the expression of genes encoding tgf- isoforms and their receptors was carried out using a qrt - pcr technique with a sybr green chemistry ( sybr green quantitect rt - pcr kit , qiagen ) and opticon dna engine continuous fluorescence detector ( mj research ) as described previously . oligonucleotide primers specific for tgf-1 , tgf-2 , tgf-3 , tri , trii , and triii mrnas were designed using primer express 2.0 software ( pe applied biosystems , usa ) ( table 1 ) . the thermal profile for one - step rt - pcr was as follows : 50c for 30 min for reverse transcription and 95c for 15 min followed by 45 cycles at 94c for 15 s , 55c for 30 s , and 72c for 45 s for amplification . following rt - pcr , the samples were subjected to temperature ramp from 60c to 95c at the rate of 0.2c / s with continuous fluorescence monitoring for melting curve analysis . . a commercially available standard of -actin ( taqman dna template reagent kit , applied biosystems ) was used to estimate the mrna copy numbers of examined genes . the expression level of examined genes in cultured cells was expressed as the fold change relative to the control . the value of fold change > 1 reflects increased expression of the target gene , and a value of fold change < 1 points to a decrease in the gene expression . finally , specificity of rt - pcr reaction was confirmed by determining the characteristic temperature of melting for each amplimer and by 6% polyacrylamide gel ( paa ) electrophoresis of rt - pcr products with their visualization using silver staining . the colon cancer cells caco-2 showed constitutive expression of genes encoding all three tgf- isoforms and their receptors . in the time course of the experiment , differential tgf-1 expression after exposure of caco-2 to e. coli lps was observed . at 3 h , it decreased in comparison to control ( p = 0.025 ) and ip6 up regulated lps - evoked effect ( p = 0.032 ) . a significantly higher tgf-1 mrna level was determined following cell treatment with lps for 12 h than in unstimulated cells ( p = 0.047 ) . lps - stimulated transcription of this gene was remarkably down - regulated by ip6 at that time ( p = 0.01 ) ( figures 1(a ) and 1(b ) ) . endotoxin of s. typhimurium had no influence on tgf-1 mrna level after 3 h treatment ( p = 0.487 ) but longer exposure of cells to it ( 12 h ) caused significant decrease in transcription of the gene ( p < 0.001 ) ( figure 1(a ) ) . treated with both lps and ip6 revealed no statistically significant differences after 3 and 12 h ( p > 0.05 ) ( figure 1(b ) ) . incubation of caco-2 with il-1 for both 3 and 12 h up - regulated tgf-1 gene as compared with untreated cells ( p < 0.05 ) ( figure 1(a ) ) . after 3 h , 2.5 mm ip6 did not change tgf-1 expression stimulated by il-1 ( p = 0.268 ) . furthermore , significant decrease in the expression of this gene was revealed in cells exposed to il-1 and ip6 for 12 h ( p = 0.047 ) in comparison to the cultures treated with il-1 only ( figure 1(b ) ) . lps of e. coli gradually down - regulated tgf-2 expression within 312 h ( p < 0.05 ) ( figure 2(a ) ) and ip6 was able to enhance it markedly at both time points in comparison to lps effects only ( p < 0.05 ) ( figure 2(b ) ) . in response to lps of salmonella caco-2 exhibited significantly higher transcription of this gene than control after 3 h ( p = 0.001 ) . however , the prolongation of time to 12 h led to insignificantly reduced tgf-2 expression ( p = 0.130 ) ( figure 2(a ) ) . ip6 enhanced lps - stimulated transcription of this gene after 3 h ( p < 0.001 ) . subsequently ( 12 h ) , the combination of ip6 and lps gave rise to 2-fold increase in tgf-2 mrna level ( p = 0.002 ) compared to lps - treated cells ( figure 2(b ) ) . the tgf-2 transcript was over 2-fold higher by the treatment with il-1 for 3 h ( p = 0.007 ) and 12 h ( p = 0.001 ) as compared to control ( figure 2(a ) ) . when ip6 was added to il-1-prestimulated cultures , the level of tgf-2 mrna markedly raised at 3 h in comparison to those treated with il-1 alone ( p < 0.0001 ) ( figure 2(b ) ) . e. coli lps diminished transcriptional activity of tgf-3 gene in caco-2 cells in a time - dependent manner . the decrease of the tgf-3 mrna level was statistically significant compared to control at 12 h ( p < 0.0001 ) ( figure 3(a ) ) . ip6 enhanced the expression of this gene after 3 h ( p < 0.0001 ) and 12 h ( p < 0.0001 ) with reference to lps - stimulated cells ( figure 3(b ) ) . cell cultures treated with lps of s. typhi manifested above 2-fold increase in tgf-3 mrna expression compared to control culture at 3 h ( p = 0.016 ) and ip6 markedly enhanced lps - induced expression of this isoform ( p = 0.008 ) . incubation of cells with s. typhi lps for 12 h did not change mrna level of tgf-3 compared to control ( p = 0.286 ) . in cells exposed to lps and ip6 statistically significant increase in mrna for tgf-3 in relation to cells treated with s. typhi lps only ( p = 0.005 ) il-1 induced transcriptional activity of this gene by 2-fold after 3 h ( p = 0.009 ) and about 4-fold after 12 h ( p < ip6 modified il-1 effects by significant increasing of tgf-3 mrna expression at 3 h ( p < 0.001 ) ( figure 3(b ) ) . lipopolysaccharide of e. coli downregulated transcriptional activity of the gene encoding type i tgf- receptor in caco-2 at 3 h ( p < 0.001 ) ( figure 4(a ) ) while ip6 strongly induced it ( p < 0.0001 ) ( figure 4(b ) ) . at 12 h , the expression of the gene in control and lps - stimulated cultures was comparable ( p = 0.075 ) , and there were no changes in tri mrna amount in the cells treated with lps and ip6 ( p = 0.479 ) ( figures 4(a ) and 4(b ) ) . caco-2 exposed to lps of salmonella typhi for 3 h produced significantly higher quantity of tri transcript than control ( p < 0.001 ) . the amount of tri mrna in cells treated with lps / ip6 and lps - stimulated cells was similar ( p > 0.05 ) . treatment of caco-2 cells with salmonella typhi lps for 12 h resulted in statistically significant decrease in tri gene transcription ( p = 0.016 ) which was remarkably up - regulated by ip6 ( p < 0.004 ) ( figures 4(a ) and 4(b ) ) . by comparison , il-1 induced transcriptional activity of tri gene in caco-2 cells . the extent of stimulation by this cytokine was 3.9- and 2.6-fold after 3 h and 12 h , respectively , compared to the control ( p < 0.05 ) ( figure 4(a ) ) . nevertheless , 2.5 mm ip6 did not significantly change mrna tri expression in il-1-treated cultures throughout the time period of the experiment ( p > 0.05 ) ( figure 4(b ) ) . over the period of the experiment , a statistically significant decrease in the expression of trii in cultures treated with lps of e. coli in relation to control ( p < 0.05 ) was detected . by comparison , ip6 stimulated lps - decreased transcription of trii for both 3 h ( p = 0.017 ) and 12 h ( p = 0.003 ) ( figures 5(a ) and 5(b ) ) . the transcription of trii did not differ in the control cells and the cells stimulated with lps of salmonella for 3 h ( p = 0.129 ) . moreover , cultures treated with lps and lps / ip6 revealed similar level of trii transcript at this time point ( p = 0.468 ) . cell culturing with salmonella tyhpi lps for 12 h decreased trii mrna level as compared to the control ( p < 0.001 ) . transcriptional activity of this gene was upregulated in response to 2.5 mm ip6 ( p = 0.001 ) ( figures 5(a ) and 5(b ) ) . exposure of caco-2 to il-1 for both 3 h and 12 h resulted in above 3-fold up - regulation of trii gene as compared with untreated cells ( p < 0.05 ) . at 3 h , trii gene was found to be expressed at the same level in il-1-stimulated cells and cells treated with both il-1 and ip6 ( p = 0.312 ) . in longer - lasting cultures , transcriptional activity of this gene was significantly suppressed by ip6 in cells treated with il-1/ip6 in comparison to those challenged with il-1 alone ( p = 0.023 ) ( figures 5(a ) and 5(b ) ) . in 3 h and 12 h lasting cultures , the expression of triii was significantly lowered by lps of e. coli compared to control cells ( p < 0.05 ) ( figure 6(a ) ) . lps - treated cells exposed to ip6 presented an increase in transcriptional activity of the gene in comparison to cells incubated with lps only ( p < 0.001 ) . the considerable , that is , 3.5-fold and 2.6-fold enhancement of triii mrna expression was observed after 3 h and 12 h , respectively , ( p < 0.05 ) ( figures 6(a ) and 6(b ) ) . cells exposed to lps of s. typhi for 3 h produced significantly higher quantity of triii transcript ( p < 0.001 ) which has not been changed by ip6 ( p > 0.05 ) . furthermore , after 12 h , endotoxin of s. typhi reduced transcription of the gene encoding type iii receptor ( p = 0.043 ) , which was markedly up - regulated by ip6 ( p = 0.041 ) . treatment of cells with il-1 for both 3 h and 12 h showed a significant ( p < 0.0001 ) increase in the expression of triii in comparison with control . however , no statistically significant change in its mrna level in cultures with il-/ip6 and il-1 only was detected after 3 h ( p = 0.130 ) . after 12 h , a marked decrease in il-1-enhanced triii transcript level was observed in response to ip6 ( p < 0.001 ) ( figures 6(a ) and 6(b ) ) . the researches over the past few years have shown unique and essential roles for tgf- in regulating inflammatory and adaptive immune responses . in particular , tgf- antagonizes the activation of the key proinflammatory cytokines including il-1 and tnf- [ 13 , 32 ] . the anti - inflammatory treatment strategies can rely on inhibition of proinflammatory cytokine production , receptor binding , signaling , or induction / up - regulation of anti - inflammatory and immunoregulatory cytokines . studies conducted in vitro and in vivo have demonstrated that phytochemicals , such as curcumin , resveratrol , and genistein , exert chemopreventive effect by targeting the constituents of inflammatory signal pathways [ 32 , 33 ] . the studies of cherng et al . showed that ip6 significantly suppressed the secretion of il-10 and augmented ifn- production in human peripheral blood mononuclear cells . this compound can modulate the inflammatory response of iec by regulating their expression and secretion of cytokines and chemokines . our previous studies revealed that ip6 down - regulated both the il-1-stimulated increase of il-8 release from enterocytes and the cellular response to bacterial lps . moreover , it appeared to influence the expression of tnf- , proinflammatory cytokine , and its receptors tnfri and tnfrii in colon cancer cells . ip6 could also inhibit il-1-stimulated expression of il-6 and il-8 at the transcriptional level in iec . in the present study , we evaluated the influence of ip6 on the expression of tgf-1 , -2 , and -3 and their receptors tri , trii , and triii in human intestinal cells under inflammatory conditions . the intestinal epithelium plays important roles in maintaining immune homeostasis in the gut and participates in maintenance of tolerance toward the microflora and food antigens . the cells of intestinal epithelium are capable of producing and releasing il-1 , il-6 , tnf- , and tgf- , either spontaneously or during the course of intestinal mucosa inflammation [ 35 , 3840 ] . therefore , we used lps derived from e. coli and s. typhi , as well as il-1 as a relevant in vitro model , to study the regulation of tgf- isoforms and their receptors expression in colon epithelial cells . the cell line caco-2 ( enterocyte - like ) utilized in the present experiment is a well - established and widely used model of human intestinal barrier . lipopolysaccharide released from gram - negative bacteria cell surface is one of the most potent innate immune - activating stimuli known . over the last years , the effects of enteropathogenic and enteroinvasive bacteria and members of the normal intestinal microflora on the expression of tgf-s were examined . however , these studies revealed that commensal and pathogenic species induced fundamentally different cytokine responses in human intestinal epithelial cell lines . the results of zeuthen et al . showed that the presence and composition of enteric bacteria affects the production of iec - derived tgf- and that the modulatory effect of this cytokine is highly dependent on the bacterial stimulus . the authors indicated relatively high production of tgf-1 in nonstimulated caco-2 cells and its further increase by stimulation with both g - positive and g - negative commensals . studies demonstrated that intestinal epithelial dld1 cells increased tgf-1 and lovo cells increased tgf-2 secretion , at 12 h in response to e. coli lps . however , no significant changes in tgf-s production in hepatocellular carcinoma and myelomonocytic cell lines were observed following stimulation with lps . considerable upexpression of mrnas for tgf-1 , tgf-2 , and tgf-3 was detected in human intestinal line ht-29 after 3 h coculture with enterotoxigenic e. coli . furthermore , the infection the cells with both enteropathogenic e. coli and s. typhimurium led to the high induction of tgf-3 mrna only . there were insignificant differences in tgf-1 and tgf-2 mrnas in control and cells exposed to both pathogens . additionally , the authors indicated significantly reduced expression of all tgf- isoforms in ht-29 incubated with commensal bacteria . also , a significant increase in tgf-2 and decrease in tgf-3 mrnas in these cells co - cultured with commensal e. coli were determined . in our study , lps derived from e. coli down - regulated mrna levels of tgf-2 and tgf-3 and all types of receptors in caco-2 cells over the course of the experiment . in the case of tgf-1 , its reduced transcriptional activity was seen after 3 h of incubation . however , a longer stimulation of cells with lps caused up - expression of this isoform . ip6 elicited the opposed to e. coli lps effect by increasing downregulated transcription of the examined genes and by suppressing the expression of tgf-1 at 12 h. then , endotoxin of s. typhi promoted differential expression profile of tgf-s and their receptors in a time - dependent manner . lps to the cell cultures was manifested by higher transcriptional activity of tgf-2 , -3 , tri , and trii at 3 h , but longer stimulation of caco-2 resulted in up - regulation of tgf-1 and all receptors . ip6 had no effect on lps - altered expression of tgf-1 . however , it enhanced lps - increased mrna expression of tgf-2 and -3 . likewise , this agent was capable of activating lps - downregulated transcription of tri , ii and iii after 12 h. according to the published data , the proinflammatory cytokines like il-1 , tnf- , and ifn- increased the production of tgf- isoforms . the main source of il-1 in ibd patients is the monocyte / macrophage system and active il-1 is released into the colonic mucosa . low concentrations of il-1 have been shown to induce local inflammatory response followed by the activation of protective immune response . as shown in this study , il-1 stimulation of the epithelial cells up - regulated mrna levels of all tgf- isoforms and their receptors at both 3 h and 12 h. ip6 counteracted the stimulatory effect of il-1 on tgf-1 , trii , and triii genes expression in caco-2 cells by decreasing their mrna levels in 12 h lasting cultures . furthermore , ip6 acted synergistically with il-1 by enhancing the transcription of tgf-2 and -3 isoforms at 3 h. tgb-s exert their effects via activation of heteromeric receptor complexes of tri and trii this receptor acts as an enhancer of tgf-s activities by promoting their access to the signaling receptors , especially that of tgf-2 isoform which has a low affinity for the type ii receptor . the differential impact of ip6 on the expression of mrna tgf-s and their receptors in colon epithelium under inflammatory conditions may be related to the role which these isoforms play . the three isoforms of tgf- are distributed in specific spatial and temporal patterns in the tissues and demonstrate distinct biological activities . the tgf-2 and -3 isoforms , tgf-2 suppresses ifn- and il-1 at the transcriptional level and plays a critical role in the development of tolerance and the prevention of autoimmunity and anti - inflammatory responses . also , the increased expression of tgf-1 mrna in caco-2 cells treated with ip6 for 3 h following e. coli endotoxin pretreatment may indicate its anti - inflammatory activity in relation to this lps . a variety of pathogenic and proinflammatory stimuli upregulate smad7 mrna expression , which in turn suppresses the tgf- pathway , through activation of nf-b . show that p65/rela subunit of nf-b is required for transcriptional activation of smad7 by bacterial lps and the proinflammatory cytokines ( il-1 , tnf- ) . inositol hexaphosphate exerts influence on cells via phosphatidylinositol-3 kinase ( pi3k ) , mapk , pkc , ap-1 , and nf-b [ 28 , 29 ] . our previously published data demonstrated that ip6 modulated the expression of p65 subunit of nuclear factor b and its ib inhibitor in the intestinal epithelial cells . diseases characterized by chronic inflammation frequently result in irreversible organ dysfunction due to extensive tissue fibrosis . tgf- , in particular the tgf-1 isoform , is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix - degrading metalloproteinases ( mmps ) and their inhibitors ( timps ) . according to hong et al . , natural or synthesized agents that suppress and blockade tgf- signaling generally demonstrate anti - inflammatory and anti - fibrotic activities . underline that , there are no ibd therapies that have been shown to specifically decrease fibrosis . they investigated the ability of resveratrol , a naturally occurring phytochemical , to decrease inflammation and fibrosis in an animal model of cd and showed the reduction of inflammatory cytokines as a promising trend in decreasing tissue fibrosis . the results of the present study demonstrate that ip6 is able to significantly downregulate tgf-1 , trii , and triii activities in colon epithelial cells stimulated with proinflammatory agents il-1 and e. coli lps . moreover , in the recently published studies , we reported that ip6 influenced constitutive expression of both mmp and timp genes and downregulated il-1-stimulated transcription of some of these genes in the intestinal epithelial cells . taken together our results were consistent with the report of kamp et al . , who concluded that ip6 reduced pulmonary inflammation and fibrosis in the respiratory bronchioles of rats . in summary , the present findings suggest that ip6 can suppress the inflammation and exert chemopreventive activity through the modulation of expression of genes encoding tgf-s and their receptors . the current data confirm our previous conclusions that ip6 present in the intestinal milieu may exert immunoregulatory effects on colonic epithelium under inflammatory conditions or during microbe - induced infection / inflammation in order to maintain the colonic mucosa in a noninflammatory state or to counteract infection [ 35 , 37 ] . inositol hexaphosphate with its anti - inflammatory and antifibrotic properties seems to be an ideal drug candidate to adjunct therapy of ibd and inflammation - associated colon cancer . therefore , it is tempting to hypothesize that supplementing the diet of ip6 could be beneficial for preventing or reducing the inflammatory reactions and fibrosis in the intestine .

transforming growth factor ( tgf- ) is a multifunctional cytokine recognized as an important regulator of inflammatory responses . the effect of inositol hexaphosphate ( ip6 ) , a naturally occurring phytochemical , on the mrna expression of tgf-1 , tgf-2 , tgf-3 and tri , trii , and triii receptors stimulated with bacterial lipopolysaccharides ( escherichia coli and salmonella typhimurium ) and il-1 in intestinal cells caco-2 for 3 and 12 h was investigated . real - time qrt - pcr was used to validate mrnas level of examined genes . bacterial endotoxin promoted differential expression of tgf-s and their receptors in a time - dependent manner . il-1 upregulated mrna levels of all tgf-s and receptors at both 3 h and 12 h. ip6 elicited the opposed to lps effect by increasing downregulated transcription of the examined genes and suppressing the expression of tgf-1 at 12 h. ip6 counteracted the stimulatory effect of il-1 on tgf-1 and receptors expression by decreasing their mrna levels . ip6 enhanced lps- and il-1-stimulated mrna expression of tgf-2 and -3 . based on these studies it may be concluded that ip6 present in the intestinal milieu may exert immunoregulatory effects and chemopreventive activity on colonic epithelium under inflammatory conditions or during microbe - induced infection / inflammation by modulating the expression of genes encoding tgf-s and their receptors at transcriptional level .

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

inflammatory bowel disease ( ibd ) is a chronic inflammation of the gastrointestinal tract thought to be a result of dysregulated or aberrant immune response to intestinal flora and multiple environmental factors with regard to genetic predisposition . the expression of lps specific toll - like receptor 4 ( tlr-4 ) in human colorectal cancer cells highlighted a key function of tlr system in the development of colitis - associated tumors , suggesting a role of this receptor in colorectal cancer development and progression . transforming growth factor is a strong anti - inflammatory cytokine with multipotent mechanism of action . in mammals three isoforms ( tgf-1 , -2 , and -3 ) have been described , which share 75% amino acid sequence homology but are encoded by different genes . tgf-s have strong impact on the inflammatory responses and tumor microenvironment including fibroblasts , endothelial cells and immune cells . on the other hand , overexpression of tgf- could induce the secretion of proinflammatory cytokines , for example , tnf- , il-1 , il-6 , and il-8 . tgf- signalling is mediated by three specific types of cell surface proteins : tgf- receptor i ( tri ) , ii ( trii ) , and iii ( triii ) . then , trii recruits and phosphorylates tri , establishing heterotetrameric complex consisting of two trii and two tri . transcription factors , histone readers , modifiers , and chromatin remodelers that bind to activated smad determine what genes and how they will be affected by signal transduction complexes . chronic inflammation has been recognized to be associated with a high cancer risk and may be involved in all stages of tumor development , that is , initiation , promotion , and progression . colorectal cancer is one of the most common cancers , accounting for 8% of all cancer deaths , making it the fourth cause of cancer deaths . one of the essential components of high fiber diet is inositol hexaphosphate ( ip6 ) . it is a naturally occurring hexaphosphorylated carbohydrate , found in both plant and mammalian cells . with intracellular concentration of about 100 m , ip6 participates in a variety of cellular functions such as signal transduction , regulation of cell proliferation , and differentiation . recently , it has been revealed that ip6 has strong impact on transcriptional activity of tgf-s and their receptor genes in colon cancer cells . the aim of the present study was to examine the potential of ip6 to affect proinflammatory agents - influenced changes in transcriptional activity of the genes encoding tgf-1 , tgf-2 , and tgf-3 and their receptors tri , trii , and triii in human colon caco-2 cells . they were then stimulated with 100 g / ml lps ( escherichia coli serotype 055:b5 , salmonella enterica serotype typhimurium ; both from sigma aldrich ) , or 1 ng / ml il-1 ( sigma aldrich ) for 30 min . afterwards cells were treated with 2.5 mm ip6 as dipotassium salt ( distilled water dissolved and ph 7.4 adjusted ) ( sigma aldrich ) for 3 and 12 h. in separate cultures , cells were incubated with lps or il-1 at the indicated concentrations and for the indicated times . detection of the expression of genes encoding tgf- isoforms and their receptors was carried out using a qrt - pcr technique with a sybr green chemistry ( sybr green quantitect rt - pcr kit , qiagen ) and opticon dna engine continuous fluorescence detector ( mj research ) as described previously . oligonucleotide primers specific for tgf-1 , tgf-2 , tgf-3 , tri , trii , and triii mrnas were designed using primer express 2.0 software ( pe applied biosystems , usa ) ( table 1 ) . the thermal profile for one - step rt - pcr was as follows : 50c for 30 min for reverse transcription and 95c for 15 min followed by 45 cycles at 94c for 15 s , 55c for 30 s , and 72c for 45 s for amplification . a commercially available standard of -actin ( taqman dna template reagent kit , applied biosystems ) was used to estimate the mrna copy numbers of examined genes . the expression level of examined genes in cultured cells was expressed as the fold change relative to the control . the value of fold change > 1 reflects increased expression of the target gene , and a value of fold change < 1 points to a decrease in the gene expression . the colon cancer cells caco-2 showed constitutive expression of genes encoding all three tgf- isoforms and their receptors . in the time course of the experiment , differential tgf-1 expression after exposure of caco-2 to e. coli lps was observed . lps - stimulated transcription of this gene was remarkably down - regulated by ip6 at that time ( p = 0.01 ) ( figures 1(a ) and 1(b ) ) . endotoxin of s. typhimurium had no influence on tgf-1 mrna level after 3 h treatment ( p = 0.487 ) but longer exposure of cells to it ( 12 h ) caused significant decrease in transcription of the gene ( p < 0.001 ) ( figure 1(a ) ) . treated with both lps and ip6 revealed no statistically significant differences after 3 and 12 h ( p > 0.05 ) ( figure 1(b ) ) . incubation of caco-2 with il-1 for both 3 and 12 h up - regulated tgf-1 gene as compared with untreated cells ( p < 0.05 ) ( figure 1(a ) ) . furthermore , significant decrease in the expression of this gene was revealed in cells exposed to il-1 and ip6 for 12 h ( p = 0.047 ) in comparison to the cultures treated with il-1 only ( figure 1(b ) ) . lps of e. coli gradually down - regulated tgf-2 expression within 312 h ( p < 0.05 ) ( figure 2(a ) ) and ip6 was able to enhance it markedly at both time points in comparison to lps effects only ( p < 0.05 ) ( figure 2(b ) ) . in response to lps of salmonella caco-2 exhibited significantly higher transcription of this gene than control after 3 h ( p = 0.001 ) . ip6 enhanced lps - stimulated transcription of this gene after 3 h ( p < 0.001 ) . subsequently ( 12 h ) , the combination of ip6 and lps gave rise to 2-fold increase in tgf-2 mrna level ( p = 0.002 ) compared to lps - treated cells ( figure 2(b ) ) . the tgf-2 transcript was over 2-fold higher by the treatment with il-1 for 3 h ( p = 0.007 ) and 12 h ( p = 0.001 ) as compared to control ( figure 2(a ) ) . when ip6 was added to il-1-prestimulated cultures , the level of tgf-2 mrna markedly raised at 3 h in comparison to those treated with il-1 alone ( p < 0.0001 ) ( figure 2(b ) ) . e. coli lps diminished transcriptional activity of tgf-3 gene in caco-2 cells in a time - dependent manner . the decrease of the tgf-3 mrna level was statistically significant compared to control at 12 h ( p < 0.0001 ) ( figure 3(a ) ) . ip6 enhanced the expression of this gene after 3 h ( p < 0.0001 ) and 12 h ( p < 0.0001 ) with reference to lps - stimulated cells ( figure 3(b ) ) . cell cultures treated with lps of s. typhi manifested above 2-fold increase in tgf-3 mrna expression compared to control culture at 3 h ( p = 0.016 ) and ip6 markedly enhanced lps - induced expression of this isoform ( p = 0.008 ) . in cells exposed to lps and ip6 statistically significant increase in mrna for tgf-3 in relation to cells treated with s. typhi lps only ( p = 0.005 ) il-1 induced transcriptional activity of this gene by 2-fold after 3 h ( p = 0.009 ) and about 4-fold after 12 h ( p < ip6 modified il-1 effects by significant increasing of tgf-3 mrna expression at 3 h ( p < 0.001 ) ( figure 3(b ) ) . lipopolysaccharide of e. coli downregulated transcriptional activity of the gene encoding type i tgf- receptor in caco-2 at 3 h ( p < 0.001 ) ( figure 4(a ) ) while ip6 strongly induced it ( p < 0.0001 ) ( figure 4(b ) ) . at 12 h , the expression of the gene in control and lps - stimulated cultures was comparable ( p = 0.075 ) , and there were no changes in tri mrna amount in the cells treated with lps and ip6 ( p = 0.479 ) ( figures 4(a ) and 4(b ) ) . caco-2 exposed to lps of salmonella typhi for 3 h produced significantly higher quantity of tri transcript than control ( p < 0.001 ) . the extent of stimulation by this cytokine was 3.9- and 2.6-fold after 3 h and 12 h , respectively , compared to the control ( p < 0.05 ) ( figure 4(a ) ) . over the period of the experiment , a statistically significant decrease in the expression of trii in cultures treated with lps of e. coli in relation to control ( p < 0.05 ) was detected . by comparison , ip6 stimulated lps - decreased transcription of trii for both 3 h ( p = 0.017 ) and 12 h ( p = 0.003 ) ( figures 5(a ) and 5(b ) ) . the transcription of trii did not differ in the control cells and the cells stimulated with lps of salmonella for 3 h ( p = 0.129 ) . exposure of caco-2 to il-1 for both 3 h and 12 h resulted in above 3-fold up - regulation of trii gene as compared with untreated cells ( p < 0.05 ) . in 3 h and 12 h lasting cultures , the expression of triii was significantly lowered by lps of e. coli compared to control cells ( p < 0.05 ) ( figure 6(a ) ) . the considerable , that is , 3.5-fold and 2.6-fold enhancement of triii mrna expression was observed after 3 h and 12 h , respectively , ( p < 0.05 ) ( figures 6(a ) and 6(b ) ) . cells exposed to lps of s. typhi for 3 h produced significantly higher quantity of triii transcript ( p < 0.001 ) which has not been changed by ip6 ( p > 0.05 ) . furthermore , after 12 h , endotoxin of s. typhi reduced transcription of the gene encoding type iii receptor ( p = 0.043 ) , which was markedly up - regulated by ip6 ( p = 0.041 ) . treatment of cells with il-1 for both 3 h and 12 h showed a significant ( p < 0.0001 ) increase in the expression of triii in comparison with control . after 12 h , a marked decrease in il-1-enhanced triii transcript level was observed in response to ip6 ( p < 0.001 ) ( figures 6(a ) and 6(b ) ) . studies conducted in vitro and in vivo have demonstrated that phytochemicals , such as curcumin , resveratrol , and genistein , exert chemopreventive effect by targeting the constituents of inflammatory signal pathways [ 32 , 33 ] . moreover , it appeared to influence the expression of tnf- , proinflammatory cytokine , and its receptors tnfri and tnfrii in colon cancer cells . in the present study , we evaluated the influence of ip6 on the expression of tgf-1 , -2 , and -3 and their receptors tri , trii , and triii in human intestinal cells under inflammatory conditions . the cells of intestinal epithelium are capable of producing and releasing il-1 , il-6 , tnf- , and tgf- , either spontaneously or during the course of intestinal mucosa inflammation [ 35 , 3840 ] . therefore , we used lps derived from e. coli and s. typhi , as well as il-1 as a relevant in vitro model , to study the regulation of tgf- isoforms and their receptors expression in colon epithelial cells . the cell line caco-2 ( enterocyte - like ) utilized in the present experiment is a well - established and widely used model of human intestinal barrier . over the last years , the effects of enteropathogenic and enteroinvasive bacteria and members of the normal intestinal microflora on the expression of tgf-s were examined . showed that the presence and composition of enteric bacteria affects the production of iec - derived tgf- and that the modulatory effect of this cytokine is highly dependent on the bacterial stimulus . studies demonstrated that intestinal epithelial dld1 cells increased tgf-1 and lovo cells increased tgf-2 secretion , at 12 h in response to e. coli lps . considerable upexpression of mrnas for tgf-1 , tgf-2 , and tgf-3 was detected in human intestinal line ht-29 after 3 h coculture with enterotoxigenic e. coli . also , a significant increase in tgf-2 and decrease in tgf-3 mrnas in these cells co - cultured with commensal e. coli were determined . in our study , lps derived from e. coli down - regulated mrna levels of tgf-2 and tgf-3 and all types of receptors in caco-2 cells over the course of the experiment . in the case of tgf-1 , its reduced transcriptional activity was seen after 3 h of incubation . ip6 elicited the opposed to e. coli lps effect by increasing downregulated transcription of the examined genes and by suppressing the expression of tgf-1 at 12 h. then , endotoxin of s. typhi promoted differential expression profile of tgf-s and their receptors in a time - dependent manner . lps to the cell cultures was manifested by higher transcriptional activity of tgf-2 , -3 , tri , and trii at 3 h , but longer stimulation of caco-2 resulted in up - regulation of tgf-1 and all receptors . however , it enhanced lps - increased mrna expression of tgf-2 and -3 . likewise , this agent was capable of activating lps - downregulated transcription of tri , ii and iii after 12 h. according to the published data , the proinflammatory cytokines like il-1 , tnf- , and ifn- increased the production of tgf- isoforms . the main source of il-1 in ibd patients is the monocyte / macrophage system and active il-1 is released into the colonic mucosa . as shown in this study , il-1 stimulation of the epithelial cells up - regulated mrna levels of all tgf- isoforms and their receptors at both 3 h and 12 h. ip6 counteracted the stimulatory effect of il-1 on tgf-1 , trii , and triii genes expression in caco-2 cells by decreasing their mrna levels in 12 h lasting cultures . furthermore , ip6 acted synergistically with il-1 by enhancing the transcription of tgf-2 and -3 isoforms at 3 h. tgb-s exert their effects via activation of heteromeric receptor complexes of tri and trii this receptor acts as an enhancer of tgf-s activities by promoting their access to the signaling receptors , especially that of tgf-2 isoform which has a low affinity for the type ii receptor . the differential impact of ip6 on the expression of mrna tgf-s and their receptors in colon epithelium under inflammatory conditions may be related to the role which these isoforms play . the tgf-2 and -3 isoforms , tgf-2 suppresses ifn- and il-1 at the transcriptional level and plays a critical role in the development of tolerance and the prevention of autoimmunity and anti - inflammatory responses . also , the increased expression of tgf-1 mrna in caco-2 cells treated with ip6 for 3 h following e. coli endotoxin pretreatment may indicate its anti - inflammatory activity in relation to this lps . inositol hexaphosphate exerts influence on cells via phosphatidylinositol-3 kinase ( pi3k ) , mapk , pkc , ap-1 , and nf-b [ 28 , 29 ] . our previously published data demonstrated that ip6 modulated the expression of p65 subunit of nuclear factor b and its ib inhibitor in the intestinal epithelial cells . tgf- , in particular the tgf-1 isoform , is a potent profibrogenic agent inducing collagen synthesis and regulating the balance between matrix - degrading metalloproteinases ( mmps ) and their inhibitors ( timps ) . they investigated the ability of resveratrol , a naturally occurring phytochemical , to decrease inflammation and fibrosis in an animal model of cd and showed the reduction of inflammatory cytokines as a promising trend in decreasing tissue fibrosis . the results of the present study demonstrate that ip6 is able to significantly downregulate tgf-1 , trii , and triii activities in colon epithelial cells stimulated with proinflammatory agents il-1 and e. coli lps . moreover , in the recently published studies , we reported that ip6 influenced constitutive expression of both mmp and timp genes and downregulated il-1-stimulated transcription of some of these genes in the intestinal epithelial cells . , who concluded that ip6 reduced pulmonary inflammation and fibrosis in the respiratory bronchioles of rats . in summary , the present findings suggest that ip6 can suppress the inflammation and exert chemopreventive activity through the modulation of expression of genes encoding tgf-s and their receptors . the current data confirm our previous conclusions that ip6 present in the intestinal milieu may exert immunoregulatory effects on colonic epithelium under inflammatory conditions or during microbe - induced infection / inflammation in order to maintain the colonic mucosa in a noninflammatory state or to counteract infection [ 35 , 37 ] .

enzymatic proteins can exhibit abnormal activity in a wide variety of diseases such as cancer and autoimmune disorders and , therefore , can be exploited for therapeutic and diagnostic purposes . this anomalous activity can help to increase the specificity and selectivity of drugs to diseased sites to reduce the harmful consequences of impacting healthy tissues and cells . the ability to detect precisely these activities could also prove beneficial for early stage diagnosis and enable a more accurate evaluation of disease progression . moreover , imaging of these biomolecules can provide real time information in an noninvasive manner , thus allowing the selection of the most appropriate medical treatments . enzymes play crucial roles in the progression and spread of cancer , being involved in the processes of cancer cell growth , angiogenesis , and metastasis among others . there are many unique aspects of tumor physiology and pathology that can be utilized for targeting and treatment . for example , many tumor types contain leaky , irregularly shaped blood vessels that allow therapeutics and imaging probes to enter the tumor easily ; poor lymphatic drainage then leads to greater retention . for greater selectivity , however , an active targeting strategy is preferred , one that provides a distinct means of distinguishing cancerous tissues from healthy . in this case , targeting of abnormally expressed enzymes could be advantageous , though there are other tumor microenvironment factors that can also be targeted for improved selectivity , including ph , cell - surface receptors , redox potential , hypoxia , and more . enzymes are relevant and effective targets for selective cancer drug and imaging probe delivery due to their substrate specificity and ability to perform biological catalysis . a wide variety of enzyme classes are overexpressed in tumor microenvironments , such as proteases , lipases , oxidoreductases , and phosphatases , and serve as potential targets ; however , our focus here will center on cancer - associated proteases . the proteases that can be used for cancer therapy and imaging are cathepsins , matrix metalloproteinases , caspases , and urokinases . with a specific protease in mind , responsive drugs and imaging probes can be designed for that target to increase selectivity and efficacy . nanomaterials have been used for various medicinal applications and have made a significant impact on the field of drug delivery and diagnosis by improving efficacy and reducing systemic toxicity . a wide variety of platform nanostructures and materials have been developed , including liposomes , dendrimers , inorganic nanoparticles , hydrogels , protein conjugates , and polymeric nanoparticles . several key design features impact their pharmacokinetic profiles and biodistribution , such as shape , size , and surface chemistry , and must be considered when developing new nanomedicines . specifically , in cancer imaging and treatment , nanomaterial - based systems offer many advantages over small molecule drugs and imaging probes . nanomaterials have greater solubility and stability in vivo with longer circulation times and slower clearance rates that allow for sustained delivery . with their ability to be engineered for particular stimuli - responsiveness , enhanced accumulation in tumors , and high loading capacity from high surface - area - to - volume ratios , nanomaterials serve as ideal candidates for combating cancer . certain nanomaterials can also be formed from self - assembling monomers , offering further advantages by simplifying formulation and development through the reduction of unnecessary components . nanomaterials can be designed to interact with enzymes to induce self - assembly or to trigger drug release within the confines of the tumor(s ) , which can improve targeting efficacy and reduce unwanted signals or side - effects in healthy tissues . some nanomaterials have found utility in medicine as imaging probes activatable by proteases , whereby enzymatic cleavage turns the probes on to yield a detectable signal . protease - activated materials have been applied to a wide variety of imaging techniques for disease detection , such as optical / fluorescence imaging , magnetic resonance imaging ( mri ) , nuclear imaging ( pet , spect , and ct ) , and more . recent efforts have pushed for the development of imaging probes with multiple modalities to benefit from the advantages each component offers for more accurate signaling . using protease - sensitive nanomaterials for molecular imaging can improve overall accuracy by enhancing target - site accumulation , increasing detectable signals via enzymatic cleavage , improving resistance to nonspecific degradation , and accelerating clearance from the body to reduce background noise . for cancer , these nanomaterial - based probes can present precise information for early detection , staging , diagnosis , and response monitoring to improve patient care . tissues containing healthy ( pink ) and tumor ( gray ) cells can be treated with various nanomaterials , such as ( from left to right ) liposomes , protein - conjugates , polymeric nanoparticles , hydrogels , dendrimers , and inorganic metal nanoparticles , to deliver imaging agents or anticancer drugs with improved selectivity to tumor cells by incorporation of protease - responsiveness into the design of nanomaterials . the benefits of using protease - responsive nanomaterials for imaging directly translate to efficacious drug delivery , which is also illustrated in figure 1 . in recent years , trends in nanomedicine have been for the development of theranostic agents that are capable of simultaneous or tandem diagnosis and therapy . the combination of imaging and therapeutic capabilities in a nanomaterial - based delivery system offers advantages over imaging and therapeutics alone , with minor trade - offs that can be mitigated by incorporating protease - sensitivity into its design . theranostic probes can reveal when and how drugs are delivered and allow for monitoring of a patient s response to therapy . from the information obtained by directly visualizing the pharmacokinetics of these agents , it can assist healthcare providers in decision - making by revealing optimal therapeutic strategies for that specific patient , paving the way toward personalized medicine in the future . using nanomaterial - based systems as theranostic agents can improve therapeutic efficacy , mitigate off - target toxicity , and ultimately lead to better patient outcomes . integrating protease - sensitivity improves selective accumulation and activation at diseased sites and can help overcome the trade - offs between the different time scales needed for imaging and therapeutics . molecules with intrinsic duality are ideal candidates , as they are simpler to synthesize and do not have to compromise on the extent of loading between the imaging agent and the drug . in this review , we will discuss recent advances in imaging and drug delivery with protease - responsive nanomaterials for cancer with a focus on theranostic systems , paying particular attention to their molecular design . since there has been a conscious effort in nanomedicine to design systems sensitive to multiple environmental stimuli for improved selectivity and signal ratios , our discussion will also include systems that incorporate responsiveness to other tumor microenvironment factors in conjunction with protease sensitivity . nanomaterials have been widely employed for the improvement of already commercially available anticancer therapeutics , where their ability to improve drug solubility and retention in tumors has helped increase efficacy and safety . incorporation of protease sensitivity can further improve selectivity to tumor tissue and mitigate harmful side effects to healthy tissues . of particular interest are the following proteases whose abnormal activity is associated with cancer : cathepsins , matrix metalloproteinases , and urokinase - type plasminogen activators . cathepsins are lysosomal cysteine proteases that play a role in regulating angiogenesis during cancer progression and in initiating and promoting tumor formation , growth , invasion , and metastasis . matrix metalloproteinases ( mmps ) are extracellular zinc - containing extracellular matrix ( ecm ) endopeptidases that play a role in tumor growth , invasion , and metastasis . the urokinase - type plasminogen activator ( upa ) is a serine protease and a member of the upa system on the cell surface ; upa degrades the ecm and activates its substrate plasmin , which is more destructive . its activity plays a role in enhancing cell migration , invasion , and metastasis . although proteases are most often utilized to release drugs from nanomaterial carriers , recent studies have investigated using proteases for inducing formation of nanostructures that may be cytotoxic to cancer cells themselves or to change the shape or size of nanocarriers to impact drug release profiles . in this section , we will discuss recent studies on the design of protease - responsive nanomaterials that release drug cargo or aggregate in tumor microenvironments to yield therapeutic effects , including some examples of systems responsive to other additional microenvironment factors . in one example , bossmann and co - workers worked to develop a protease - sensitive liposome that would address common problems faced by liposome carriers , such as decreasing the amount of leaking from the vesicles , increasing their release kinetics , and being able to target cancer cells more specifically . the liposomes were designed with a cholesterol - anchored , graft copolymer containing a upa - cleavable peptide sequence ( sgrsa ) and poly(acrylic acid ) , and the liposomes used have high osmolarities to make them swell more easily . the liposomes are cross - linked with diamine and ethylenediamine , which causes the liposomes to exhibit significantly increased resistance to osmotic swelling and thus prevents premature leaking of their contents . in the presence of upa , these liposomes are able to deliver their entire payload , indicating their heightened sensitivity to the protease . the focus of this study was on creating and optimizing the design of these liposomes , so no in vitro or in vivo studies were conducted . an optimal design was created by looking at the impact of cross - linking level and degree of polymer - incorporation on release against osmotic pressure , showcasing the numerous factors and importance of nanomaterial design to be efficacious for drug delivery . in another example , he and co - workers developed a mesoporous silica nanoparticle ( msn ) to improve the targeting of the anticancer drug doxorubicin ( dox ) to cancer cells while reducing adverse side effects to healthy cells . msns are suitable drug carriers because they have a large loading capacity , are easily functionalized , have low toxicity , and are chemically inert . this design involves a classic rotaxane structure formed between an alkoxysilane tether and alpha - cyclodextrin ( -cd ) used to anchor onto orifices of msns and act as gatekeeper for doxorubicin release . these are modified with a multifunctional peptide ( azido - gflgr7rgds ) that contains v3 integrin ( overexpressed on the surfaces of different cancer lines ) targeting sequence rgds , cell penetrating peptide sequence r7 , and cathepsin b - cleavable peptide sequence gflg , which function to penetrate tumor cells selectively and release doxorubicin . drug release behavior studies with nanoparticles in pbs showed that the nanoparticles had the greatest release of contents in the presence of cathepsin b at lysosomal ph , indicating the requirement of cathepsin b cleavage for sufficient drug delivery . in vitro studies were conducted with v3-positive hela cancer cells that overexpress cathepsin b and v3-negative cos7 cells that express cathepsin b at relatively low levels . these studies showed higher uptake in hela cells but lower cytotoxicity than free doxorubicin , as shown in figure 2 , by flow cytometry and mtt assay . the difference in cytotoxicity between the msns and free dox is likely due to the slower rate of endocytosis of the msns as opposed to concentration gradient diffusion by free dox ; however , the msns have an increased selectivity from v3-targeting and cathepsin b - triggered drug release that makes them safer alternatives to the free drug . the difference in cell viability between hela cells , v3 receptor - blocked hela cells , and cos7 cells highlights the selectivity of the v3- and cathepsin b - targeting motifs of the msns . although no in vivo study has been conducted with these msns , this study highlights the advantages of targeting other tumor microenvironment factors ( specifically receptor overexpression ) in conjunction with protease overexpression for improving the selectivity of drug delivery and release to cancer cells over healthy cells . ( a f ) functionalization procedure and mechanism of action of the doxorubicin - loaded mesoporous silica nanoparticles ( msns ) , where panel a shows capping of and subsequent release from the msns of doxorubicin , panel b shows the drug - loaded msns as they appear in physiological ph , panel c illustrates targeting to v3 integrins overexpressed on cancer cells by the rgds peptide sequence , panel d portrays endocytosis of msns into a specific tumor cells , panel e shows triggered drug release by cathepsin b activity , and panel f represents the tumor cell undergoing apoptosis . ( g ) cell viability data of cos7 and hela cells incubated in vitro with doxorubicin - loaded msn - gflgr7rgds/-cd in the absence or presence of free rgds peptide ( 2 m ) and ( h ) free doxorubicin , highlighting the comparable cytotoxicity as the free drug but with more specificity to tumor cells . adapted with permission from ref ( 72 ) . co - workers developed a nanoparticle system responsive to both extracellular ph and mmp-2 activity for gene delivery . their design involves dendrigraft poly lysine ( dgl ) that complexes and condenses dna to form a nonviral vector nanoparticle via electrostatic interactions . the nanoparticles are modified with a dual - triggered activatable cell - penetrating peptide ( dtacpp ) , composed of a ph - sensitive masking peptide ( e4k4 , d - amino acids , pi = 6.4 ) , an mmp-2 substrate ( plglag ) , and a polycationic cell - penetrating peptide ( nonarginine ) . the dtacpps are conjugated to the surface of a dgl via -malemidyl--n - hydroxysuccinimidyl polyethylene glycol ( mal - peg - nhs ) to create the gene nanocarrier , dtacpp - peg - dgl ( dtacppd ) . the internalization of the nanoparticles via the cell - penetrating peptide is inhibited until the nanoparticles enter environments with ph values typical of tumors ; here the now positively or neutrally charged ph - sensitive masking peptide can be cleaved by overexpressed mmp-2 to then enhance cellular uptake of the genes to cancer cells . cytotoxicity of the dtacppds was tested using flow cytometry and fluorescent microscopy with bel-7402 hepatocellular carcinoma cells cultured at either ph 6.0 or 7.4 and pretreated with or without mmp-2 , showing uptake as high as 90.6% in the presence of mmp-2 at a ph of 6.0 ( mimicking slightly acidic tumor microenvironment ) . in vivo testing with mice showed progressive accumulation of dtacppds in tumors over time with the smallest accumulation in liver and kidneys in comparison to different control groups . this study further showcases the benefits of multiresponsive targeting to cancer cells and how its incorporation into nanocarrier design can improve overall therapeutic efficacy . in a study by mallik and co - workers , they designed nanovesicles responsive to overexpression of glutathione ( gsh ) and mmp-9 in the tumor microenvironment to deliver efficiently and selectively the anticancer drug gemcitabine ( gem ) . an mmp-9-cleavable , collagen mimetic lipopeptide forms nanovesicles with 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ( popc ) , cholesteryl - hemisuccinate , and the reduction - sensitive , peglyated 1-palmitoyl-2-oleoyl - sn - glycero-2-phospethanolamine lipid ( pope - ss - peg5000 ) . the peglyation helps instill long circulating characteristics to the nanovesicles while also reducing unintended interactions with circulating proteins . once at cancer sites , the peg chains are shed via reduction by gsh , which then exposes the vesicle to mmp-9 degradation , allowing for release of the contents inside . was investigated with panc-1 and miapaca-2 pancreatic cancer cells of gem - loaded nanovesicles , showing lower cell viability with the panc-1 line ( 3035% ) , which has a higher expression of mmp-9 than miapaca-2 ( viability 4550% ) , where dose - dependent cytotoxicity evidence is shown in figure 3 . in a spheroid culture , the cell viability was similar between free and encapsulated gem in panc-1 cells , showing encapsulation does not compromise cytotoxicity while simultaneously improving selectivity . an in vivo xenograft mice model with panc-1 cancer cells showed a more significant reduction in tumor growth for the gem - encapsulated , mmp-9-responsive nanovesicles in comparison to vesicles without an mmp-9 substrate in their design , which can be seen in figure 3 . the difference in tumor growth and the fact that animals remained healthy after treatment illustrates better control of gem release with mmp-9 selectivity , highlighting another case where multiresponsiveness significantly improves selectivity to cancer cells . ( a ) illustration of nanovesicles and their targeting mechanism that are responsive to the elevated levels of extracellular gsh and mmp-9 by incorporating mmp-9 substrate lipopeptides and reduction - sensitive pope - ss - peg on their surface . ( b ) in vitro cell viability data showcasing the concentration dependent decrease in miapaca-2 cell viability when treated with free gemcitabine ( violet ) or gemcitabine - loaded nanovesicles ( orange ) for 72 h. ( c ) tumor volume percentage increase from xenograft mice model , where blue represents test group ( n = 3 ) treated with mmp-9 substrate - incorporated nanovesicles , red represents group ( n = 3 ) treated with nanovesicles without mmp-9 responsiveness , and black is the control group ( n = 3 ) treated with pbs - loaded nanovesicles ( * p < 0.05 , * * p < 0.05 ) . adapted with permission from ref ( 74 ) . although proteases are often targeting to trigger drug release from various nanomaterials , recent work has been done for the development of nanomaterials systems that use proteases to trigger nanostructure formation to change drug release kinetics or to induce cytotoxicity by their assembly . maruyama and co - workers detail their design of a gelator precursor that undergoes intracellular self - assembly to form nanofibers , leading to hydrogelation and inducing cancer cell death after interacting with mmp-7 . the gelator precursor , n - palmitoyl - ggghgplgark - conh2 ( called er - c16 ) , design incorporated a 16-carbon alkyl chain to provide hydrophobic interactions to enhance self - assembly in aqueous solutions attached to a peptide sequence containing the following : the tetrapeptide sequence gggh to facilitate assembly as a hydrogen - bond acceptor and donor , the mmp-7-cleavable tetrapeptide sequence plgl for triggering gelation , and the cationic peptide sequence rk to prevent er - c16 from forming nanofibers until cleaved off by mmp-7 . from tem imaging , the group found that the gelator precursor forms micelle - like structures until exposed to mmp-7 which initiates self - assembly to nanofibers . the cytotoxicity of er - c16 was investigated in vitro with hela cancer cells and mve normal human microvascular endothelial cells , where their coculture with exposure to er - c16 confirmed its selectivity to cancer cells due to increased uptake in hela cells . the decreased levels of cell viability confirmed the relationship between cell death and high intracellular toxicity and provides a therapeutic strategy that cancer cells are unlikely to acquire drug resistance to , highlighting a different but effective use of nanomaterials to selectively target and treat cancer . xu and co - workers have been exploring the concept of enzyme - instructed self - assembly for therapeutics for the past few years , developing a peptide - based system that responds to alkaline phosphatase for the formation of hydrogels . their earlier design involves the conjugation of the anticancer drug taxol to a succinic acid linker to attach the phosphatase substrate and self - assembly motif ( napffkyp ) to form a hydrogelator precursor . after exposure to enzyme activity , the precursors self - assemble into nanofibers and form a supramolecular hydrogel of the taxol derivative , instilling the dual role of delivery vehicle and therapeutic to this molecule . an in vitro study with hela cancer cells was conducted and showed comparable cytotoxicity between the precursor and free taxol . taxol activity is conserved within the hydrogel and the concentration of the precursor molecule can be used to control the release rate . in a more recent example of their work , xu and co - workers demonstrated the importance of precursor design for enzyme - instructed self - assembly ( eisa ) for selective killing of cancer cells . in this study , they designed and synthesized two different d - tetrapeptides ( ffyy and analogues ) containing one or two phosphotyrosine residues capped with a naphthyl group , where dephosphorylation causes the peptides to self - assemble into nanofibers in water and the use of d - amino acids prevents endogenous protease degradation . the napff and napf are residues with great self - assembly promoting motifs because of aromatic aromatic interactions , and the tyrosine residues provide a site for mono- or diphosphorylation to explore the effect of multiple enzymatic triggers on selectivity . tem images showed that the monophosphorylated precursors form nanofibers better due to less solubility than the diphosphorylated precursors and that the peptide sequence fyfy has a higher tendency to self - assemble . in vitro cytotoxicity studies were conducted with hela cervical cancer cells and saos-2 osteosarcoma cells that showed that precursors inhibited growth of both cell lines by eisa , but response to precursors was dependent on expression levels of alkaline phosphatase and mechanism of cell death was dependent on the cell line . although this lab presents an example of eisa using phosphatases , it highlights the utility of nanomaterials , the importance of their design factors , and the variety of other enzymatic targets that can be used for increasing selectivity to cancer cells . this strategy offers an effective means of overcoming drug resistance and treating multiple cancer lines . in another example , ulijn and co - workers demonstrated the use of proteases to induce a morphological change in nanostructures from micelles to nanofibers to impact drug release rates . in their initial design , the nanostructures were loaded with the anticancer drug doxorubicin ( dox ) , and contained the following units : a self - assembly motif for formation of nanofibers that provides a hydrophobic binding region for drug candidates ( phenylacetyl - ffag ) , an mmp-9-cleavable sequence , and a hydrophilic peptide sequence ( ldd ) that favors formation of micelles . mmp-9 cleaves off the hydrophilic unit of phac - ffagldd and confers the micelles into fibers by changing the balance between hydrophobic and hydrophilic interactions , allowing for localized and sustained delivery of dox to cancer cells . they also developed a peptide - based design ( gfflgldd ) that has the same properties as their first design for the reconfiguration of dox - loaded micelles to nanofibers , and both designs are detailed in figure 4 . the reconfiguration of micelles to fibers via mmp-9 hydrolysis was confirmed with afm and tem for both precursor designs , and aggregation was not impacted by the presence of dox . an in vitro study was conducted with mda - mb-231-luc - d3h2ln breast cancer cells that showed significantly reduced cell viability following treatment with both precursors but slightly lower with the peptide - based design ( 35% vs 37.5% viability , data shown in figure 4 ) , and confocal microscopy confirmed uptake of drug by presence of aggregates in cytoplasm and nucleus of cells with larger aggregates outside cells , indicating local and sustained delivery were possible and effective with these precursor molecules . the same cancer cells were used for a xenograft mice model for in vivo efficacy studies , which further validated the selectivity and efficacy of the system for localized and sustained delivery of dox . this study exemplifies the wide - ranging utility of nanomaterials for cancer therapy and highlights the role proteases can play in formation of nanostructures to control drug release , similar to the other works mentioned in this section . ( a ) schematic depiction of the micelle - to - fiber transition the peptide precursors undergo due to the overexpression of mmp-9 by cancer cells , where the anticancer drug doxorubicin is entrapped in the fibrillar structures , thereby creating less mobile depots of the drug . ( b and c ) impact of peptide design and mmp-9 responsiveness on cancer cell growth of mda - mb-231-luc - d3h2ln cells using peptides 1a and 2a ( as shown in panel a ) with 2.5 mm peptides 200 nm doxorubicin . extensive work has been conducted over the past decade on the development of molecular probes with sensitivity to proteases overexpressed in cancer cells . the following proteases whose abnormal activity is associated with cancer or whose activity is an indicator of cell death are of particular interest : cathepsins , matrix metalloproteinases , urokinase - type plasminogen activators , and caspases . cathepsins , mmps , and upa were discussed in the previous section for their use in therapeutics , but this can directly translate for targeting with imaging agents . caspases are cysteine - aspartic proteases whose activity is involved in apoptosis and inflammation and therefore can be utilized as means of visualizing drug activation and efficacy . a wide variety of imaging modalities can be used in cancer diagnostics , such as fluorescence imaging , mri , and pet , each offering their own advantages . however , different imaging techniques do possess their own limitations , and thus different nanomaterial systems have been designed to incorporate more than one modality , allowing for more holistic and accurate imaging . in this section , we will discuss various nanomaterial systems sensitive to these proteases that use various modalities for the imaging and diagnosis of cancer . matrix metalloproteinases ( mmps ) are popular targets for cancer imaging due to their overexpression in many cancer types and easy accessibility based on their location on and around cell surfaces . various imaging modalities have been incorporated into mmp - detectable systems , and utilization of nanomaterials have improved the efficacy of these systems . for example , nir fret - based probes conjugated to gold nanoparticles as a fluorescence quencher have been shown to be effective in detection of mmps and could be used for early diagnosis of cancer . a novel signal - amplifiable self - assembling f nmr / mri probe was developed by hamachi and co - workers for imaging mmp-2 activity , where there is no observable signal when the probes are aggregated as nanoparticles but enzyme cleavage - induced disassembly turns the signal on . the probe itself is composed of an mmp-2 substrate peptide ( gplgvrg ) , with the f nmr imaging moiety ( 3,5-bis(trifluoromethyl)benzene ) attached to a lysine residue at the c - terminal end and a hydrophobic dodecyl ( c12 ) chain on the n - terminal end . the self - assembly into nanoparticles helps resolve issues concerning low sensitivity and poor delivery , whereas the f mri modality has high nmr sensitivity with no background noise in vivo , making this a seemingly effective design for tumor imaging . the probes showed in vitro efficacy with cancer lines known to secrete mmp-2 , but the f mri modality proved to not be as sensitive as available h mri probes , such as those that are gadolinium - based . another example of a nanoparticle system was developed by liu and co - workers , consisting of a novel activatable photoacoustic nanoprobe for in vivo imaging of cancer - associated mmps . the probe is composed of an nir - absorbing copper sulfide ( cus ) nanoparticle connected to a black hole quencher ( bhq-3 ) via an mmp - cleavable peptide linker ( gplgvrgkgg ) , and showed in vitro reactivity to mmp-13 . the bhq-3 molecule and the cus nanoparticle have different absorbance peaks , and comparing signals at these wavelengths can yield photoacoustic imaging of mmp activity . in a mouse model , the nanoparticles are able to detect scc7 breast cancer cells with in vivo photoacoustic imaging , which in comparison to optical imaging , the mechanism of photoacoustic imaging offers distinctly improved in vivo spatial resolution and exhibits significantly improved tissue penetration . this system offers an interesting and unique design for an alternative imaging modality to optical fluorescence and presents its own advantages for tumor detection and imaging . over the past decade , tsien and co - workers have been working on the development of molecular probes for the detection of mmp activity in tumors . the basis of their design is the incorporation of cell - penetrating peptides ( cpps ) , which are integral for overcoming multidrug resistance in tumor cells . in their early design , their molecular probe comprised of a polyarginine - based cpp ( called activatable cpps ) , blocked by an inhibitory peptide sequence with negative charges , and a linker between these two domains , which when cleaved by mmp-2 or mmp-9 , allows for the cpp and its cargo , a far - red fluorophore , cy5 , to enter cancer cells . this design showed early promise with great in vivo contrast ratios and elevated standard uptake values in tumors relative to normal tissue in ht-1080 cancer in mice . further studies showed that these probes can target many xenograft tumor models from different cancer sites and that background uptake into normal tissue could be decreased by attaching inert macromolecules , sparking an investigation into nanomaterial conjugates . the group developed dendrimeric nanoparticles coated with their activatable cpps labeled with either cy5 for fluorescence imaging , gadolinium for mri , or both . the peptide sequence , pldlag , serves as the mmp - cleavable linker , where cleavage separates the inhibitory domain from the loaded , cpp - conjugated polyamidoamine ( pamam ) dendrimer nanoparticle . the schematic of the nanoparticles , in vivo fluorescence images , and time - dependence and biodistribution data are detailed in figure 5 . the nanoparticles had significantly higher uptake in tumors than the activatable cpps alone , allowed for fluorescence detection of tumors as small as 200 m , and deposited high levels of gd in tumors yielding mri t1 contrast that lasts several days after injection . loading of the dendrimer nanoparticles with mri and fluorescence imaging modalities improves its utility by giving it the advantages of both techniques , finding uses in mri - guided staging and fluorescence - guided resection for many different cancer types in various parts of the body . the group has also applied this nanotechnology to the mri and fluoroscence imaging of atherothrombosis and stroke with different enzyme targets , and incorporated integrin v3-targeting in addition to mmp-2 sensitivity for improved sensitivity and selectivity of their probe , showcasing the ubiquity of protease - responsive nanomaterials in the imaging of many diseases . ( a ) schematic illustration of the activatable cell - penetrating peptide dendrimer ( acppds ) , consisting of a dendrimer ( gray circle ) covalently attached to the polycationic segments ( blue ) of the acpps , an mmp-2,-9 cleavable peptide linker , and polyanionic segments ( red ) that are released to allow entry of acppds into cells . the payloads stored inside the dendrimers ( yellow ovals ) can be either cy5 ( acppd - cy5 ) , gd - dota ( acppd - gd ) , or both ( dual acppd ) . ( b d ) fluorescence images taken 48 h after injection into mice ( with skin removed ) with acppd - cy5 or acpp - cy5 , containing 10 nmol cy5 each , where yellow arrows point to tumors and panel d has been brightened to make signal visible , showing enhanced signal from acppds over dendrimers without mmp - sensitivity and the free probe . ( e ) time course of fluorescence signals in tumors in mice viewed through intact skin , demonstrating enhanced fluorescence signal of mmp - sensitive acppds . ( f ) standardized uptake values in solubilized samples of tumor , liver , kidney and muscle 48 h after acppd - cy5 injection and 6 h after acpp - cy5 injection with pairwise p values shown for each organ type . adapted with permission from ref ( 94 ) . many groups have conducted work over the past decade exploring the development of cathepsin - sensitive probes . many probes have incorporated various imaging modalities , such as optical imaging with fluorescence resonance energy transfer ( fret)-based probes or magnetic resonance imaging , and later have been combined with nanomaterials to improve signal ratios and targeting . molecular probes with cathepsin - sensitivity designed by some groups have showed a lot of potential for clinical applications . very recently , the laboratories of kirsch and brigman have developed a near - infrared fret - based probe with sensitivity to various cathepsins , particularly cathepsin s , that have progressed to ex vivo first - in - human phase 1 clinical trials in patients with soft tissue sarcoma or breast cancer . showing that their probe design is safe for use in humans and yields tumor - specific fluorescent signals , these probes could very well make it to a clinical setting . this design , however , lacks the advantages offered by nanomaterials , and successful completion of clinical trials could suggest nanomaterial systems may be just as effective for cancer imaging , if not more . another recent example of a cathepsin - responsive nanomaterial system was developed by cui and co - workers , comprised of molecular probes that self - assembles into a supramolecular structure they called nanobeacons . their nanobeacons are comprised of hydrophobic and hydrophilic domains , where the amphiphilic nature helps to induce the self - assembly of the probes into core the hydrophobic domain consists of a fluorescent green dye , 5-carboxyfluorescein ( 5-fam ) and a black hole quencher , bhq-1 , whereas the hydrophilic domain consists of an hiv-1 derived cell - penetrating peptide sequence , tat4860 , where the charged residues allow the nanobeacons to be responsive to changes in ph . the fluorophore and quencher are held in close proximity for fret by a cathepsin b ( catb ) cleavable linker ( gflg sequence ) . the imaging modality is contained within the nanobeacon after spontaneous assembly into micelles and thus protected from catb cleavage , until the nanobeacons are converted back to their monomeric form by ph or dilution to below its critical micellization concentration ( cmc ) . the nanobeacons proved effective after incubation with mcf-7 human breast cancer cells in vitro , where confocal imaging and flow cytometry showed localization of the fluorescence signal in lysosomes and an increasing signal over time . the group continued studying their nanobeacons by investigating the role that their shape and surface chemistry play on their uptake and activity in vitro . this design incorporates the same quencher , fluorophore , and catb - degradable linker , but instead uses a gnnqqny central assembly sequence derived from sup35713 that can spontaneously associate into different morphologies based on temperature and incubation time in aqueous solutions . this sequence was modified with either three lysine or three glutamic acid residues for either positive or negative surface charge , and the probes were allowed to form either spherical or filamentous shapes . by observing cellular uptake by pc3-flu metastatic human prostate cancer cells , they were able to show that positively charged nanobeacons had much higher uptake efficiency as opposed its monomeric form , making this combination of shape and surface chemistry most - suited for intracellular sensing . the nanobeacon filaments had significantly lower internalization by cancer cells , making them more suitable for sensing extracellular proteases , like mmps and upa . the molecular design , nanobeacon characteristics , and confocal images and flow cytometry data of cellular uptake can be seen in figure 6 . although in vivo work was not conducted , these studies are revealing of a nanoscale system with potential for significant tumor accumulation and accurate sensing based on the optimal shape and surface chemistry for their target protease . ( a ) molecular design of the two studied molecular beacons capable of self - assembling , sfb - k and sfb - e , which are composed of the following design elements : the black hole quencher-1 ( bhq-1 ) , the fluorophore 5-fam , the degradable peptide linker for cathepsin b specificity gflg , and the central assembly regulating sequence gnnqqny terminated with either three lysine ( k ) or glutamic acid ( e ) residues . ( b ) schematic illustration of molecular beacons assembly into spherical or filamentous supramolecular nanobeacons based on assembly temperature and incubation time . ( c ) cellular uptake efficiency by cancer cells of beacons , varying in surface charge , shape , and assembly state , as characterized by flow cytometry : fluorescence intensity measurements ( left ) , flow cytometry spectra comparing fluorescence intensity of sfb - k ( top right ) and sfb - e ( bottom right ) . ( d ) confocal laser scanning microscopy of pc3-flu cells after 1 h incubation with 5 m nanobeacons with different surface charges and shapes ( a f ) . adapted with permission from ref ( 101 ) . urokinase - type plasminogen activator ( upa ) of the urokinase - type plasminogen activator system is an attractive target for cancer sensitivity due to its overexpression in a handful of cancer lines and its presence on the exterior of the cell membrane . over the past few years , molecular probes and nanomaterial systems responsive to upa activity , particularly for breast cancer imaging , have been developed . for example , law and co - workers developed a self - assembled nanofiber from a peg2000-peptide conjugate . the upa substrate , sgrsana , served as a linker between the hydrophobic d - amino acid domain and the hydrophilic peg domain with conjugated fluorescein isothiocyanate ( fitc ) for fluorescence imaging . , where in the self - assembled state although only a proof - of - principle study , this group showed the applicability of their technology to in vivo upa activity imaging for cancer detection . continuing with this concept , law and co - workers developed an nir molecular probe with a similar structure as mentioned before , with a peg - based hydrophilic domain conjugated to the near - ir dye , nir664 , linked by a upa - cleavable peptide sequence to a hydrophobic d - amino acid sequence , which allows for self - assembly into nanofibers in aqueous solution . these nanofibers were able to detect upa activity from several human cancer cell lines in vitro with very high fluorescence signals , giving more evidence to the applicability of these nanofibers for the diagnosis and even treatment of cancer based on different expression levels of upa activity . another interesting nanomaterial design for cancer imaging via upa proteolysis was developed by stevens and co - workers . they designed multiplex assay nanoparticles that are able to detect upa activity and human epidermal growth factor receptor 2 ( her2 ) kinase activity , which are both overexpressed in breast cancer . their system consists of two quantum dot ( qd ) populations with different emission wavelengths and orthogonal surface functionalizations for signal independence between the two different enzymes . the qds serve not only as a reporter molecule but also as a scaffold for conjugates attached via enzyme - specific linker peptide sequences . the qd , qd525 , was used for upa - sensing and was attached to the upa - cleavable linker sequence , sgrsan , which is covalently coupled to a gold nanoparticle that quenches the qd signal until cleaved away by upa . a similar design was implemented for her2 kinase detection , where phosphorylation of the her2 kinase - sensitive linker induces fret - based quenching . this system is the first demonstration of a nanoparticle - based activity assay being able to simultaneously sense the activity of two different classes of enzymes , which can provide prognostic information for breast cancer patients . caspases are useful imaging targets due to their role in programmed cell death , like apoptosis , and inflammation . the information obtained by visualizing when cells undergo apoptosis can elucidate information about the pharmacokinetics of a certain therapeutic and allow for long - term monitoring of drug response in a patient . an example of a caspase - sensitive nanomaterial system is described here for visualization of cancer cell response to different chemotherapeutics . a caspase - sensitive nanoaggregation fluorescent probe , called c - snaf , was developed by rao and co - workers that uses a biorthogonal cyclization reaction that triggers self - assembly and yields an aggregation - induced nir fluorescence signal . the c - snaf probe contains d - cysteine and 2-cyano-6-hyrdoxyquinoline ( chq ) moieties that are linked to an amino luciferin scaffold . additionally , it contains a l - devd capping sequence and a disulfide bond that are required for a two - step activation with caspase-3/7-mediated cleavage and an intracellular thiol - mediated reduction , which consequently promotes aggregation and gives nir signal from cy5.5 dye . figure 7 details the molecular design of c - snaf and the mechanism of nanoaggregation in live versus apoptotic tumor cells . as indicated by fluorescence and 3d - sim imaging in doxorubicin - treated mice tumor models , this probe is able to penetrate into tumor tissue after iv injection and successfully report tumor - cell death induced from chemotherapy in vivo . the probes are rigid and hydrophobic after undergoing cyclization that promotes nanoaggregation , where they are retained in apoptotic cells and give high imaging contrast for detection of therapeutic - response . ( a ) molecular design and the proposed conversion of c - snaf into c - snaf - cycl via reduction and caspase-3/7 activation of the biorthogonal intermolecular cyclization reaction , which is then followed by the self - assembly into nanoaggregates : green represents the capping peptide residues ; dark orange , amino group of d - cysteine ; light orange , thiol group of d - cysteine ; yellow , thioethyl masking group , blue , the chq group ; and red , the red nir fluorophore cy5.5 . ( b ) illustration of the mechanism of c - snaf for in vivo imaging of tumor response following chemotherapy in living versus apoptotic cancer cells . ( c ) chemical structures of probes used as controls for this study . adapted with permission from ref ( 106 ) . a gd - based mri probe , called c - snam , was developed , with prolonged accumulation in chemotherapy - induced apoptotic cells and tumors with significantly brighter contrast between treated and nontreated tumors . additionally , a f - based pet probe was developed which also had significantly higher tumor signal and tumor - to - muscle ratios in murine models in response to tumor therapy . the group also applied this technology for apoptosis imaging with caspase - activity in arthritis , highlighting the vast applicability of this probe design to imaging drug efficacy for many diseases . many promising and efficacious systems have been developed for cancer imaging and drug delivery , which has helped sparked interest in studying and designing theranostic systems . these systems allow for simultaneous imaging and treatment and offer a means of visualizing the pharmacokinetics and biodistribution of therapeutics used for a cancer patient . theranostic nanomaterials can benefit from the incorporation of protease - sensitivity for improved selectivity , but multiresponsive systems that include protease - responsiveness have become popular for designing more efficacious drug delivery and imaging systems . the nanomaterial systems can respond to other factors in the tumor microenvironment , such as cell - surface receptors , ph , and other classes of enzymes , thereby increasing sensitivity and selectivity and thus improving the overall efficacy of the cancer treatment . in this section , we will be discussing theranostic nanomaterial systems responsive to protease activity , including some examples that are sensitive to other factors in the tumor microenvironment . an early example of the development of a theranostic probe was by zheng and co - workers that showcased the possibility and advantages of simultaneous imaging and treatment of tumor cells . their probe is capable of photodynamic therapy for cancer , where photodamage is induced via irradiation , and subsequently gives a near - infrared fluorescence signal to indicate successful induction of apoptosis . the molecular design contains the photosensitizing agent , puropheophorbide a ( pyro ) , dual fluorescence and singlet oxygen quencher , bhq-3 , and a caspase-3-responsive peptide linker sequence , gdevdgsgk . when tumor cells are irradiated with light in the presence of these molecular beacons , the photosensitizer converts oxygen into singlet oxygen , which destroys mitochondrial membranes and triggers apoptosis ; therefore , caspase-3 expression will increase and act to cleave the linker sequence and give a detectable signal to indicate cell death . this design showed efficacy in vitro and was later shown to have in vivo efficacy after the incorporation of folate into the molecular design to target overexpressed folate receptors on the surfaces of cancer cells to induce endocytosis and improve selectivity to tumor tissue . further work was done to improve design by instead using an mmp-7-sensitive linker for targeting to tumor cells , which simplified molecule complexity and synthesis requirements while also yielding comparable selectivity and therapeutic efficacy as earlier designs in vitro and in vivo . these beacons showcased mitigated nonspecific accumulation and enhanced tumor cell death in human breast carcinoma cells ( mt-1 line ) that commonly produce spinal metastases , where risks of spinal cord damage are very high , offering a safe approach of selective photodynamic therapy while preserving critical tissues . their beacon design has shown efficacy for other types of cancer metastases without the incorporation of a nanomaterial , which could be useful in improving tumor accumulation , but regardless , the molecular beacons exemplify the utility of theranostics for cancer treatment . although the previously discussed example detailed an effective theranostic probe , using nanomaterials can prolong circulation time , reduce possibility of nonspecific activation , and increase accumulation at tumor sites . the laboratories of kim and ahn designed gold nanorods with mmp - sensitivity for cancer treatment and imaging . when irradiated , the gold nanorods absorb the near - infrared laser light and convert it into heat as a means of hyperthermal therapy for cancer cells , which are more susceptible to treatment due to their lower heat tolerance from poor blood supply . the near - infrared dye cy5.5 was conjugated to the surface of the nanorods via the mmp - sensitive peptide linker sequence , gplgvrgc , which is responsive to a variety of mmps . gold is a popular material for imaging due to its exhibition of surface plasmon resonance , where it can serve as a fluorescence quencher as it does in this design . these nanorods were studied with hela cells in vitro and with scc-7 tumors in mice , where the nanorods showed the ability to kill cancer cells effectively and very rapidly , as temperatures could increase over 45 c in little as 4 min . however , the external skin of the mice was burned at tumor sites and nonspecific damage seems unavoidable with this design . only the imaging function of this design is sensitive to the overexpressed mmp activity in tumors and the therapeutic function can be delivered regardless of mmp presence ; therefore , further modifications could be added for improving selectivity . however , this does represent a unique therapeutic approach , and it showcases the importance of selectivity and the utility that gold possesses for theranostic applications . in another example of utilizing inorganic nanostructures , cheng and xing report the design of a protease - responsive , core shell , dual - imaging magnetic silica - coated nanoparticles . the anticancer - drug , doxorubicin ( dox ) , is conjugated to a cathepsin b - cleavable peptide sequence ( fk ) with a para - aminobenzyloxycarbonyl ( pabc ) linker , and through click chemistry , they are attached to the surface of uniform silica - coated , superparamagnetic iron oxide nanoparticles via an azido - dpeg4 linker . doxorubicin is a red fluorescent drug , which in conjunction with the iron oxide nanoparticles , allows for dual - modality imaging of both mri and optical imaging . with confocal microscopy and mri spectroscopy , they showed highly efficient release of dox upon interaction with cathepsin b in ht-29 cancer cells in vitro . cell viability was comparable between nanoparticle - treated and free drug - treated cells but was much higher for the nanoparticle - treated negative control cells than free drug , indicating that the nanoparticles function to give selective tumor intracellular drug delivery and imaging while keeping healthy tissues safe . this system , while showcasing dual - modality for imaging , also contains an intrinsically theranostic moiety in dox , which greatly improves efficacy as a theranostic for cancer . expanding upon this notion of intrinsically theranostic systems , by using moieties capable of both imaging and therapy in the design of nanomaterial systems for cancer , there is no longer a need to compromise on the extent of loading between imaging and therapeutic moieties nor long lag time periods present between drug activation and corresponding optical signal . cui and co - workers developed , to the best of their knowledge , the first enzyme - specific dox prodrug conjugated with a dark chromophore quencher capable of both diagnostic and therapeutic functions . their fret - based molecular probe contains red - fluorescent anticancer drug , dox , a black hole quencher ( bhq-2 ) conjugated via a cathepsin b - sensitive peptide linker ( gflg ) , and a cell - penetrating peptide sequence ( r8 ) to help circumvent drug resistance in some cancer lines . confocal images taken of nci / adr - res ovarian cancer cells , which are resistant to dox , indicate the efficacy of the drug - beacons to simultaneously image and treat cancer while overcoming drug resistance , as the drug - beacons showed significantly better cytotoxicity in comparison to free dox . although this system does not incorporate any nanomaterials , the beacons could be modified to self - assemble into nanostructures for in vivo efficacy . the intrinsic theranostic ability of this system simplifies molecular design and offers sufficient drug loading with direct visualization of drug activation . in another example , rao and daldrup - link report the design of a novel , multifunctional theranostic nanoparticle with the capability to release drugs via enzymatic cleavage and to give mr and fluorescence imaging of drug delivery in vivo . the design incorporates a magnetic iron oxide nanoparticle , ferumoxytol , conjugated to an mmp-14-responsive peptide sequence that holds the green - fluorescent dye , fluorescein isothiocyanate ( fitc ) , and a vascular disrupting agent , azademethylcolchicine ( ict ) , creating a theranostic probe they call clio - icts . the molecular design and mechanism of action of clio - icts are detailed in figure 8 . upon treatment of mmp-14-positive mmtv - pymt breast cancer cells with clio - icts , significant cell death was observed in vitro , but not for cells treated with ferumoxytol alone nor mmp-14-negative fibroblasts , indicating mmp-14-sensitivity is needed for effective delivery . tumor - bearing mice were given iv injections of the nanoparticles , and subsequent mr imaging showed significant tumor accumulation of clio - ict and was confirmed with histopathology staining , further demonstrating the selectivity of the nanoparticles to tumors and not healthy tissues . the dual - modality imaging offers the advantages of both techniques in a single probe that also treats cancer , the nanostructure design increases tumor retention , and together , improve the overall antitumor efficacy . ( a ) schematic illustration representing the activation of the theranostic nanoparticles , clio - ict , by mmp-14 cleavage . the iron oxide nanoparticle ( ionp ) is shown in orange ; the prodrug azademethylcolchicine ( ict ) is shown in red , and its product after mmp-14 degradation is shown in magenta ; the mmp-14 sensitive peptide linker is shown in blue ; and the green fluorophore , fluorescein isothiocyanate ( fitc ) , is shown in green . ( b ) synthesis pathway for the clio - ict theranostic nanoparticles , highlighting cross - linking and ict prodrug addition . ( c ) axial t2-weighted mr images of mmtv - pymt mammary tumors before and after a single iv injection of either 0.6 m ( fe ) solution of fermyoxytol , 0.4 m solution of clio - ict , 0.29 mm solution of ict , or pbs , where contrast agent accumulation is indicated as a negative ( dark ) signal enhancement of the tumors . ( d ) corresponding mr signal enhancement data of tumors in panel c quantified as r2 = ( r2pre r2post ) . although protease - responsiveness is an effective means of increasing tumor selectivity , incorporating other factors of the tumor microenvironment alongside protease - activation can further improve cancer targeting . the groups of mao and yang report on the design and effectiveness of urokinase plasminogen activator receptor ( upar)-targeted , cathepsin b - sensitive magnetic iron oxide nanoparticles ( ionps ) that carry the chemotherapeutic drug gemcitabine ( gem ) . the iron oxide nanoparticles are conjugated with an amino - terminal fragment peptide ( atf ) of the receptor - binding domain of upa and gem via a cathepsin b - cleavable peptide linker ( gflg ) on their surfaces , with nanoparticles being called atf - ionp - gem . the nanoparticles showed higher drug release in more acidic surroundings , which is representative of the lysosomal and endosomal environments that contain cathepsins . in vitro and in vivo studies of atf - ionp - gem efficacy with the mia paca-2 human pancreatic cancer cell line showed that free gem only inhibited tumor growth by 30% , whereas atf - ionp - gem exhibited approximately 50% tumor growth inhibition in xenograft mice . the delivery of atf - ionp - gem and presence of residual tumors could then be detected noninvasively by mri using both t2-weighted and t1-weighted ultrasound echo time imaging , allowing for monitoring and assessment of treatment efficacy . the significant difference in tumor treatment between nanoparticles with only cathepsin b - sensitivity and those with upar and cathepsin b - targeting emphasizes the increasing selectivity to tumor tissue by incorporating responsiveness to multiple factors in the tumor microenvironment , which is promising for the development of safer and more personalized medicine in the future via theranostics . ( a ) illustration of the conjugation of the amino - terminal fragment peptides of the receptor - binding domain of upa ( atf ) , the cathepsin b - sensitive linker , and anticancer drug gemcitabine ( gflg - gem ) conjugates to the surface of iron oxide nanoparticles ( ionps ) , forming atf - ionp - gem . ( b ) schematic representation of the release of gemcitabine after cathepsin b - cleavage from atf - ionp - gem . ( c ) data from a cell proliferation assay conducted on mia paca-2 cells after 4 h treatment with free gem , ionp - gem , or atf - ionp - gem followed by 72 h incubation , indicating high reduction in cell viability from conjugation of gem to ionps with targeting for upar and cathepsin b. ( d ) further evidence of improved efficacy of atf - ionp - gem from tumor xenograft mice models by comparing mean tumor weights and individual tumor weight distributions of mice in each group represented by colored symbols . adapted with permission from ref ( 125 ) . copyright 2013 american chemical society . a better understanding of the molecular basis of many diseases has been developed over the years , particularly in regards to cancer , leading to the identification of microenvironment factors and cellular features that are representative of diseased tissues . these factors have been significantly beneficial in improving the efficacy and selectivity of imaging agents and therapeutics to tumor tissues . proteases , which are overexpressed in many cancer types at various cellular locations , play important roles in cancer progression , and therefore represent attractive targets for diagnostics and therapeutics . nanotechnology can further improve the efficacy of imaging agents and pharmaceuticals , where design factors can be modified to influence the characteristics of nanomaterials and impact their delivery properties , primarily through improvement of the pharmacokinetic profile and biodistribution of molecular probes and free drugs . the combination of imaging and therapeutics has opened a new field of medicine called theranostics , producing systems that possess the ability to monitor drug delivery , drug release , and drug efficacy using a single entity . incorporating protease - responsiveness into a theranostic platform can assist with early stage cancer diagnosis , give an accurate evaluation of cancer progression , and noninvasively provide real time information to healthcare providers to choose appropriate medical treatments . such multifunctional platforms can improve clinical outcomes and pave the way toward personalized medicine . in this review , we have discussed recent examples of therapeutic , diagnostic , and theranostic nanomaterial systems that have proven useful for cancer treatment . looking forward , the selection of the proper protease target is critical in theranostic system design , as some proteases may prove more beneficial for imaging or drug delivery based on their location in , on , or around cancer cells . signals and delivery are susceptible to protease location , quantity , and activity , which can vary and are not uniform among all cancer types . multiple modalities for imaging can give the benefits of multiple techniques in a single probe , yielding more information , but this can complicate the manufacturing process and reproducibility of signals . although targeting multiple environmental factors increases selectivity , finding the optimal combination to maximize selectivity and preserve healthy tissues is necessary . additionally , the sequence in which the system will respond to these factors must be considered for the highest efficacy . trade - offs occur when combining imaging and therapy , such as compromising on the loading of one agent over the other , accounting for the difference in the necessary concentration for good imaging contrast and therapeutic response , and considering the optimal circulation times between different agents ( imaging agents benefit from faster clearance whereas therapeutics benefit from sustained delivery ) . these issues can be addressed by using molecules that possess both imaging and therapeutic properties and work should be done to develop these types of theranostic probes . addressing these challenges will yield better clinical outcomes for cancer treatment and bring us closer to personalized medicine .

many diseases can be characterized by the abnormal activity exhibited by various biomolecules , the targeting of which can provide therapeutic and diagnostic utility . recent trends in medicine and nanotechnology have prompted the development of protease - sensitive nanomaterials systems for therapeutic , diagnostic , and theranostic applications . these systems can act specifically in response to the target enzyme and its associated disease conditions , thus enabling personalized treatment and improved prognosis . in this review , we discuss recent advancements in the development of protease - responsive materials for imaging and drug delivery and analyze several representative systems to illustrate their key design principles .

Introduction Protease-responsive nanomaterial systems for therapeutics Protease-responsive nanomaterial systems for diagnostics Protease-responsive nanomaterial systems for theranostics Conclusion

enzymatic proteins can exhibit abnormal activity in a wide variety of diseases such as cancer and autoimmune disorders and , therefore , can be exploited for therapeutic and diagnostic purposes . enzymes play crucial roles in the progression and spread of cancer , being involved in the processes of cancer cell growth , angiogenesis , and metastasis among others . in this case , targeting of abnormally expressed enzymes could be advantageous , though there are other tumor microenvironment factors that can also be targeted for improved selectivity , including ph , cell - surface receptors , redox potential , hypoxia , and more . the proteases that can be used for cancer therapy and imaging are cathepsins , matrix metalloproteinases , caspases , and urokinases . several key design features impact their pharmacokinetic profiles and biodistribution , such as shape , size , and surface chemistry , and must be considered when developing new nanomedicines . nanomaterials can be designed to interact with enzymes to induce self - assembly or to trigger drug release within the confines of the tumor(s ) , which can improve targeting efficacy and reduce unwanted signals or side - effects in healthy tissues . protease - activated materials have been applied to a wide variety of imaging techniques for disease detection , such as optical / fluorescence imaging , magnetic resonance imaging ( mri ) , nuclear imaging ( pet , spect , and ct ) , and more . using protease - sensitive nanomaterials for molecular imaging can improve overall accuracy by enhancing target - site accumulation , increasing detectable signals via enzymatic cleavage , improving resistance to nonspecific degradation , and accelerating clearance from the body to reduce background noise . tissues containing healthy ( pink ) and tumor ( gray ) cells can be treated with various nanomaterials , such as ( from left to right ) liposomes , protein - conjugates , polymeric nanoparticles , hydrogels , dendrimers , and inorganic metal nanoparticles , to deliver imaging agents or anticancer drugs with improved selectivity to tumor cells by incorporation of protease - responsiveness into the design of nanomaterials . the benefits of using protease - responsive nanomaterials for imaging directly translate to efficacious drug delivery , which is also illustrated in figure 1 . in recent years , trends in nanomedicine have been for the development of theranostic agents that are capable of simultaneous or tandem diagnosis and therapy . the combination of imaging and therapeutic capabilities in a nanomaterial - based delivery system offers advantages over imaging and therapeutics alone , with minor trade - offs that can be mitigated by incorporating protease - sensitivity into its design . integrating protease - sensitivity improves selective accumulation and activation at diseased sites and can help overcome the trade - offs between the different time scales needed for imaging and therapeutics . in this review , we will discuss recent advances in imaging and drug delivery with protease - responsive nanomaterials for cancer with a focus on theranostic systems , paying particular attention to their molecular design . of particular interest are the following proteases whose abnormal activity is associated with cancer : cathepsins , matrix metalloproteinases , and urokinase - type plasminogen activators . in this section , we will discuss recent studies on the design of protease - responsive nanomaterials that release drug cargo or aggregate in tumor microenvironments to yield therapeutic effects , including some examples of systems responsive to other additional microenvironment factors . in one example , bossmann and co - workers worked to develop a protease - sensitive liposome that would address common problems faced by liposome carriers , such as decreasing the amount of leaking from the vesicles , increasing their release kinetics , and being able to target cancer cells more specifically . in the presence of upa , these liposomes are able to deliver their entire payload , indicating their heightened sensitivity to the protease . the difference in cytotoxicity between the msns and free dox is likely due to the slower rate of endocytosis of the msns as opposed to concentration gradient diffusion by free dox ; however , the msns have an increased selectivity from v3-targeting and cathepsin b - triggered drug release that makes them safer alternatives to the free drug . although no in vivo study has been conducted with these msns , this study highlights the advantages of targeting other tumor microenvironment factors ( specifically receptor overexpression ) in conjunction with protease overexpression for improving the selectivity of drug delivery and release to cancer cells over healthy cells . ( a f ) functionalization procedure and mechanism of action of the doxorubicin - loaded mesoporous silica nanoparticles ( msns ) , where panel a shows capping of and subsequent release from the msns of doxorubicin , panel b shows the drug - loaded msns as they appear in physiological ph , panel c illustrates targeting to v3 integrins overexpressed on cancer cells by the rgds peptide sequence , panel d portrays endocytosis of msns into a specific tumor cells , panel e shows triggered drug release by cathepsin b activity , and panel f represents the tumor cell undergoing apoptosis . the nanoparticles are modified with a dual - triggered activatable cell - penetrating peptide ( dtacpp ) , composed of a ph - sensitive masking peptide ( e4k4 , d - amino acids , pi = 6.4 ) , an mmp-2 substrate ( plglag ) , and a polycationic cell - penetrating peptide ( nonarginine ) . the internalization of the nanoparticles via the cell - penetrating peptide is inhibited until the nanoparticles enter environments with ph values typical of tumors ; here the now positively or neutrally charged ph - sensitive masking peptide can be cleaved by overexpressed mmp-2 to then enhance cellular uptake of the genes to cancer cells . an mmp-9-cleavable , collagen mimetic lipopeptide forms nanovesicles with 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ( popc ) , cholesteryl - hemisuccinate , and the reduction - sensitive , peglyated 1-palmitoyl-2-oleoyl - sn - glycero-2-phospethanolamine lipid ( pope - ss - peg5000 ) . an in vivo xenograft mice model with panc-1 cancer cells showed a more significant reduction in tumor growth for the gem - encapsulated , mmp-9-responsive nanovesicles in comparison to vesicles without an mmp-9 substrate in their design , which can be seen in figure 3 . although proteases are often targeting to trigger drug release from various nanomaterials , recent work has been done for the development of nanomaterials systems that use proteases to trigger nanostructure formation to change drug release kinetics or to induce cytotoxicity by their assembly . the gelator precursor , n - palmitoyl - ggghgplgark - conh2 ( called er - c16 ) , design incorporated a 16-carbon alkyl chain to provide hydrophobic interactions to enhance self - assembly in aqueous solutions attached to a peptide sequence containing the following : the tetrapeptide sequence gggh to facilitate assembly as a hydrogen - bond acceptor and donor , the mmp-7-cleavable tetrapeptide sequence plgl for triggering gelation , and the cationic peptide sequence rk to prevent er - c16 from forming nanofibers until cleaved off by mmp-7 . although this lab presents an example of eisa using phosphatases , it highlights the utility of nanomaterials , the importance of their design factors , and the variety of other enzymatic targets that can be used for increasing selectivity to cancer cells . in their initial design , the nanostructures were loaded with the anticancer drug doxorubicin ( dox ) , and contained the following units : a self - assembly motif for formation of nanofibers that provides a hydrophobic binding region for drug candidates ( phenylacetyl - ffag ) , an mmp-9-cleavable sequence , and a hydrophilic peptide sequence ( ldd ) that favors formation of micelles . the reconfiguration of micelles to fibers via mmp-9 hydrolysis was confirmed with afm and tem for both precursor designs , and aggregation was not impacted by the presence of dox . this study exemplifies the wide - ranging utility of nanomaterials for cancer therapy and highlights the role proteases can play in formation of nanostructures to control drug release , similar to the other works mentioned in this section . ( a ) schematic depiction of the micelle - to - fiber transition the peptide precursors undergo due to the overexpression of mmp-9 by cancer cells , where the anticancer drug doxorubicin is entrapped in the fibrillar structures , thereby creating less mobile depots of the drug . the following proteases whose abnormal activity is associated with cancer or whose activity is an indicator of cell death are of particular interest : cathepsins , matrix metalloproteinases , urokinase - type plasminogen activators , and caspases . cathepsins , mmps , and upa were discussed in the previous section for their use in therapeutics , but this can directly translate for targeting with imaging agents . in this section , we will discuss various nanomaterial systems sensitive to these proteases that use various modalities for the imaging and diagnosis of cancer . the group has also applied this nanotechnology to the mri and fluoroscence imaging of atherothrombosis and stroke with different enzyme targets , and incorporated integrin v3-targeting in addition to mmp-2 sensitivity for improved sensitivity and selectivity of their probe , showcasing the ubiquity of protease - responsive nanomaterials in the imaging of many diseases . ( a ) schematic illustration of the activatable cell - penetrating peptide dendrimer ( acppds ) , consisting of a dendrimer ( gray circle ) covalently attached to the polycationic segments ( blue ) of the acpps , an mmp-2,-9 cleavable peptide linker , and polyanionic segments ( red ) that are released to allow entry of acppds into cells . many groups have conducted work over the past decade exploring the development of cathepsin - sensitive probes . the molecular design , nanobeacon characteristics , and confocal images and flow cytometry data of cellular uptake can be seen in figure 6 . this system is the first demonstration of a nanoparticle - based activity assay being able to simultaneously sense the activity of two different classes of enzymes , which can provide prognostic information for breast cancer patients . an example of a caspase - sensitive nanomaterial system is described here for visualization of cancer cell response to different chemotherapeutics . additionally , a f - based pet probe was developed which also had significantly higher tumor signal and tumor - to - muscle ratios in murine models in response to tumor therapy . many promising and efficacious systems have been developed for cancer imaging and drug delivery , which has helped sparked interest in studying and designing theranostic systems . these systems allow for simultaneous imaging and treatment and offer a means of visualizing the pharmacokinetics and biodistribution of therapeutics used for a cancer patient . theranostic nanomaterials can benefit from the incorporation of protease - sensitivity for improved selectivity , but multiresponsive systems that include protease - responsiveness have become popular for designing more efficacious drug delivery and imaging systems . the nanomaterial systems can respond to other factors in the tumor microenvironment , such as cell - surface receptors , ph , and other classes of enzymes , thereby increasing sensitivity and selectivity and thus improving the overall efficacy of the cancer treatment . in this section , we will be discussing theranostic nanomaterial systems responsive to protease activity , including some examples that are sensitive to other factors in the tumor microenvironment . an early example of the development of a theranostic probe was by zheng and co - workers that showcased the possibility and advantages of simultaneous imaging and treatment of tumor cells . when tumor cells are irradiated with light in the presence of these molecular beacons , the photosensitizer converts oxygen into singlet oxygen , which destroys mitochondrial membranes and triggers apoptosis ; therefore , caspase-3 expression will increase and act to cleave the linker sequence and give a detectable signal to indicate cell death . the near - infrared dye cy5.5 was conjugated to the surface of the nanorods via the mmp - sensitive peptide linker sequence , gplgvrgc , which is responsive to a variety of mmps . gold is a popular material for imaging due to its exhibition of surface plasmon resonance , where it can serve as a fluorescence quencher as it does in this design . only the imaging function of this design is sensitive to the overexpressed mmp activity in tumors and the therapeutic function can be delivered regardless of mmp presence ; therefore , further modifications could be added for improving selectivity . however , this does represent a unique therapeutic approach , and it showcases the importance of selectivity and the utility that gold possesses for theranostic applications . in another example of utilizing inorganic nanostructures , cheng and xing report the design of a protease - responsive , core shell , dual - imaging magnetic silica - coated nanoparticles . the anticancer - drug , doxorubicin ( dox ) , is conjugated to a cathepsin b - cleavable peptide sequence ( fk ) with a para - aminobenzyloxycarbonyl ( pabc ) linker , and through click chemistry , they are attached to the surface of uniform silica - coated , superparamagnetic iron oxide nanoparticles via an azido - dpeg4 linker . cell viability was comparable between nanoparticle - treated and free drug - treated cells but was much higher for the nanoparticle - treated negative control cells than free drug , indicating that the nanoparticles function to give selective tumor intracellular drug delivery and imaging while keeping healthy tissues safe . expanding upon this notion of intrinsically theranostic systems , by using moieties capable of both imaging and therapy in the design of nanomaterial systems for cancer , there is no longer a need to compromise on the extent of loading between imaging and therapeutic moieties nor long lag time periods present between drug activation and corresponding optical signal . cui and co - workers developed , to the best of their knowledge , the first enzyme - specific dox prodrug conjugated with a dark chromophore quencher capable of both diagnostic and therapeutic functions . their fret - based molecular probe contains red - fluorescent anticancer drug , dox , a black hole quencher ( bhq-2 ) conjugated via a cathepsin b - sensitive peptide linker ( gflg ) , and a cell - penetrating peptide sequence ( r8 ) to help circumvent drug resistance in some cancer lines . the dual - modality imaging offers the advantages of both techniques in a single probe that also treats cancer , the nanostructure design increases tumor retention , and together , improve the overall antitumor efficacy . the iron oxide nanoparticle ( ionp ) is shown in orange ; the prodrug azademethylcolchicine ( ict ) is shown in red , and its product after mmp-14 degradation is shown in magenta ; the mmp-14 sensitive peptide linker is shown in blue ; and the green fluorophore , fluorescein isothiocyanate ( fitc ) , is shown in green . the significant difference in tumor treatment between nanoparticles with only cathepsin b - sensitivity and those with upar and cathepsin b - targeting emphasizes the increasing selectivity to tumor tissue by incorporating responsiveness to multiple factors in the tumor microenvironment , which is promising for the development of safer and more personalized medicine in the future via theranostics . ( a ) illustration of the conjugation of the amino - terminal fragment peptides of the receptor - binding domain of upa ( atf ) , the cathepsin b - sensitive linker , and anticancer drug gemcitabine ( gflg - gem ) conjugates to the surface of iron oxide nanoparticles ( ionps ) , forming atf - ionp - gem . the combination of imaging and therapeutics has opened a new field of medicine called theranostics , producing systems that possess the ability to monitor drug delivery , drug release , and drug efficacy using a single entity . incorporating protease - responsiveness into a theranostic platform can assist with early stage cancer diagnosis , give an accurate evaluation of cancer progression , and noninvasively provide real time information to healthcare providers to choose appropriate medical treatments . in this review , we have discussed recent examples of therapeutic , diagnostic , and theranostic nanomaterial systems that have proven useful for cancer treatment . looking forward , the selection of the proper protease target is critical in theranostic system design , as some proteases may prove more beneficial for imaging or drug delivery based on their location in , on , or around cancer cells . trade - offs occur when combining imaging and therapy , such as compromising on the loading of one agent over the other , accounting for the difference in the necessary concentration for good imaging contrast and therapeutic response , and considering the optimal circulation times between different agents ( imaging agents benefit from faster clearance whereas therapeutics benefit from sustained delivery ) .

one of the major problems that humans face as we enter the twenty - first century is how to feed an overpopulated planet . currently , the world population is approximately six billion people , and it is estimated that with the current birth rate it will double by 2050 . the consequence of such growth is that humans will be forced to carry out agriculture using less , and lower quality , farming land . in order for us to be successful under these adverse conditions , a highly sophisticated knowledge of plant biology will be required that will allow the development of agronomically important species suitable for producing more food per unit farming area . the introduction of arabidopsis as a model plant species fifteen years ago has revolutionized plant biology and provides opportunities for achieving this goal . arabidopsis was adopted as a model organism by plant geneticists because of its small diploid genome , low repetitive dna content , and rapid reproductive cycle . arabidopsis appears to contain homologs of most of the genes found in agronomically important crop plants , including rice , maize , soybean , and tomato . the sequencing of the mustard weed genome , together with the genomes of other eukaryotes - yeast , worm , fly and human , reaffirms the validity of this axiom . it has been only 48 years since the structure of the dna was elucidated and almost 30 years since the first dna molecule was cloned and propagated in escherichia coli . subsequently , recombinant dna technology was developed that allowed biologists to clone and study genes ' one by one ' , laying the foundations for the biotechnology industry . after 20 years , however , the ' one by one ' approach was not revealing information fast enough to allow understanding of the complexity of biological systems . it costs $ 10 to $ 20 per base to sequence your favorite gene in a timely manner . more importantly , genomic sequencing of intensely studied eukaryotic organisms with a small fraction of redundant genes , such as saccharomyces cerevisiae , has indicated that no more than 30% of an organism 's genes can be identified by classical genetic analysis . it was the advent ten years ago of genomics , a new scientific discipline established by the emergence of three independent fields - biology , engineering and computer science - that eliminated the shortcomings of the ' one by one ' approach and opened up new and exciting possibilities for advancing biology when established in 1989 , the human genome project included all the best - known model organisms ( e. coli , yeast , worm , fly , human and mouse ) but omitted the mustard weed , for reasons that are poorly understood . the arabidopsis sequencing project was initiated in 1994 by the european community ( now the european union , eu ) under the leadership of michael bevan at the john innes institute in the uk . funds were secured in 1994 that led to the establishment of a consortium of european laboratories ( essa ) , led by bevan , to sequence chromosome 4 . shortly thereafter , satoshi tabata of the kazusa dna research institute in japan was funded for sequencing chromosome 5 . the flame that initiated the american sequencing effort was kindled one humid night - on july 8 1993 - at the cold spring harbor laboratory ( cshl ) , where the instructors of the arabidopsis molecular genetics course ( joe ecker , joanne chory , and myself ) were entertaining elliot meyerowitz after his evening lecture at blackford hall . while we were drinking beer together with rob martienssen and venkatesan sundaresan , two members of the cshl plant biology group , and gary drews ( another instructor ) and a few students from the course , i remember asking elliot when the sequencing of the arabidopsis genome would be initiated . his answer was , " well , we need a few sequencing machines to be given to each of you and the project can start . " that evening 's conversation was somehow transmitted to jim watson by rob and venkatesan , and the rest is history . jim watson 's deep interest in genome sequencing led to a banbury conference in the spring of 1994 at cshl that convinced the national science foundation ( nsf ) and some prominent skeptics of the urgency and importance of sequencing the arabidopsis genome . the most obvious objections at that time , from some of the leaders in the arabidopsis community , were whether funds would be removed from other aspects of plant biological research and whether it was intellectually stimulating to sequence the arabidopsis genome . following the banbury conference , a workshop on sequencing the arabidopsis genome was held at nsf in the summer of 1994 , where the guidelines were established for finding resources for the project . the resources were allocated in 1995 and the sequencing effort was initiated in the fall of 1996 by three us groups ( see figure 1 ) . an historic meeting was held in the summer of 1996 at nsf , where the six sequencing groups from europe , japan and the us , representatives of the arabidopsis community , nsf , the department of energy ( doe ) and the us department of agriculture ( usda ) met to coordinate an international effort to sequence the 125 mb arabidopsis genome . that meeting established the arabidopsis genome initiative ( agi ) , an international effort to sequence the first plant genome by 2004 ( figure 1 ) . the agi discussed various strategies for sequencing the arabidopsis genome , including the whole - genome shotgun - sequencing approach , which was proposed by ron davis , one of the principal investigators ( pis ) of the spp consortium ( see figure 1 ) . the approach was rejected at that time as risky , and more secure strategies were adopted , such as bac - end sequencing ( see below ) , and approaches based on physical maps . in retrospect , if the shotgun approach had been adopted in 1996 , the discovery of all the genes in the arabidopsis genome would have preceded the corresponding studies of the worm , fly and human genomes . it was decided that chromosomes 4 and 5 would be sequenced using a map - based strategy , built on existing physical maps made using yeast artificial chromosome ( yac ) , cosmid and plasmid p1 clones . chromosomes 1 , 2 , and 3 would be sequenced using the bac - end strategy ( figure 2a ) . in 1996 two bac libraries became available to agi . in a collaborative effort among three agi groups - genoscope , a member of the eu-2 consortium , the institute for genomic research ( tigr ) and spp ( see figure 1 ) - the end - sequences for almost all the bacs ( approximately 18,300 clones , representing 14-fold coverage ) in the two libraries were determined and made publicly available . the bac - end sequencing strategy ( figure 2a ) is based on extension from a few fully sequenced bac clones ( ' seed ' or nucleation points ) using a minimum set of overlapping bac clones selected from a set of end - sequenced bacs . all sequencing groups used a variation of the same strategy for sequencing individual bacs , p1 or cosmid clones , as shown in figure 2b . shotgun , plasmid , or m13 libraries were constructed and an appropriate number of clones were sequenced to 7 - 10-fold coverage ( that is , each base is sequenced an average of 7 - 10 times ) . two major software programs were used for assembly and editing by most of the agi members ( except tigr , which used the in - house ' tigr assembler ' ): phred / phrap for sequence assembly and gap4 or consed9 for viewing and editing . the agi members used almost all the same annotation programs for gene prediction and annotation of the genome : programs such as genefinder , grail , genscan , xpound , trnascan - se , blastn , blastx , gene mark hmm , glimmer a , netgene 2 , splice predictor , pedant and repeat masker ( see ) . the entire genome was also reannotated upon completion , by two agi members , tigr and mips ( a member of the eu consortia ) , to ensure uniformity of the final product . the adopted strategies allowed different groups around the globe to sequence different regions of the various chromosomes at the same time . existing incomplete physical and genetic maps of each of the five chromosomes were used in selecting the seed bacs necessary to initiate the sequencing process . the incomplete physical chromosome maps were supplemented by fingerprint and hybridization data using 24,000 bacs , to yield a complete map of the genome with 99% coverage and resulting in an acceleration of the sequencing process . a few additional crucial decisions were also made during the 1996 nsf meeting , regarding data release policy , acceptable error in the final sequencing product , and acceptable degree of completion . it was agreed that the sequence produced by the agi should be immediately deposited in genbank even before it was finished and annotated . accordingly , phase i genomic sequence - comprising raw sequence containing gaps and of unknown orientation - was available to the plant biology community at the htgs section of genbank a few days after the shotgun sequencing was completed . this immediate - release policy allowed plant molecular geneticists to clone their favorite gene by ' walking ' much faster than before . there are numerous examples for which the availability of unfinished genome sequence allowed the cloning of genes , such as axr3 and shy2 ( two examples that i know about because of my own research interests ) . it was also agreed that the acceptable sequencing error rate would be no more than 1 error per 10 kb , and that the finished sequence should be at least 97% double - strand sequenced , with the remaining 3% to be pseudodouble - stranded ( defined as the sequence of a single clone obtained with two different chemistries or the sequence of two clones with the same chemistry ) . regarding the extent of completion , the agi agreed that the genome sequence would be considered complete when each chromosome was represented by only two contigs - chromosomal arms - separated by the centromeric region . the rdna clusters of chromosomes 2 and 4 were also excluded from the finished product . chromosomes 2 and 4 were published in december 1999 and the remaining chromosomes 1 , 3 and 5 , along with a uniform annotation and analysis of the entire genome , were published in december 2000 . the accelerated pace was primarily due to the acceleration of funding by the nsf two years into the project , thanks to the vision of the nsf administrators , mary clutter , machi dilworth and the late delill nasser . the rapid progress of the project during the first two years , and the excess of sequencing capacity of the participating groups , warranted such an action . the ten chromosomal arms are without gaps , except for three at the bottom arm of chromosome 1 where there are highly repetitive dna sequences . the combined length of the ten arms , which extend from either the telomeres or the ribosomal dna repeats to the 180 base - pair centromeric repeat is approximately 115 mb ( table 1 ) . the unsequenced centromeric and rdna repeat regions are estimated to represent approximately 10 mb , resulting in a genome size of about 125 mb . the overall gc content is low ( 35% ) and uniform across the five chromosomes , which made the dna relatively easy to sequence using the available chemistry . the sequencing error rate is no more than 1 error per 20 kb , better than that agreed upon by the agi in 1996 . the collinearity of the assembled chromosomes was verified using blast alignment analysis of the bac - end sequences against the assembled chromosomal contigs . in addition , the location of a large number of sequenced chromosomal markers on the assembled chromosomes is collinear with their location on the recombinant inbred linkage map . the relationship between physical and genetic distance is around 200 - 250 kb per cm and is uniform across the five chromosomes . per base ( $ 70 million for 115 mb ) , corresponding to approximately 10 ? per citizen of europe , japan and the usa ( a combined population of about 600 million people ) . gene prediction programs and database searches the genome contains 25,500 putative genes at a density of 1 gene per 4 kb ( table 1 ) . arabidopsis contains as many genes as humans and twice as many as the fruitfly ( table 2 ) . thus , almost 50% of the chromosomal dna is covered with genes ( table 1a ) . approximately 20% of the genes are intronless , and some of them have been annotated as hypothetical . for example , chromosome 1 contains a gene ( f5f8.4 ) with 68 exons that encodes a 500 kda polypeptide that is a ubiquitous putative membrane protein found in yeast , worm , fly and human . the same chromosome contains a very small gene that encodes the l41 protein of the 60s ribosomal complex , with only 25 amino acids . at least 15,300 genes ( 60% ) are expressed , as indicated by the presence of corresponding cdnas or expressed sequence tags ( ests ) in various databases ( table 1 ) . genes that are most highly represented by ests include proteins of the photosynthetic and protein synthesis machineries and few metabolic enzymes , such as catalase . the distribution of ests is more or less the same across the length of the chromosomes but decreases dramatically in the regions flanking the centromeres . the chromosomes encode approximately 600 trnas , with chromosome 1 bearing the majority of them ( 236 trnas ) . the trna gene content of chromosome 1 is very similar to that of the entire fly genome . the trna genes are evenly distributed along the chromosomes , except for two regions in chromosome 1 where trna gene clustering is observed ; the two clusters are located at 9.989 mb and 19.282 mb , respectively . the first contains 26 genes encoding trna and the other 27 tandem repeats of the tri - repeat trna- trna- trna . the trna genes of eukaryotes in general occur as multigene families with a diverse arrangement . in some cases they are spread throughout the genome , whereas others are clustered at single chromosomal sites . it has been suggested that trna gene clustering may reflect their tissue - specific co - regulation . the number of homologous genes in the duplicated regions varies considerably , ranging from 20 to 50% . in addition to the detection of the large - scale intra- and inter - chromosomal duplications , analysis reveals a plethora of diverse repetitive elements comprising 10% of the sequence . retroelements include members of the line - like ta11 family of elements , and long - terminal repeat ( ltr ) elements of both the ty3-gypsy and the ty1-copia families . representative members of various transposable element families are scattered throughout the chromosome ; for example , the ac / ds-(hat / mariner ) , en - spm - mutator and tc1 type elements all occur . in addition , a number of simple and low - complexity repeats are found throughout the chromosomes . there is an inverse relationship between gene and retroelement density in the borders of the centromeric region , a hallmark of such chromosomal regions . computational analysis of the chromosomal sequences reveals that they encode 25,500 putative proteins ( table 1b ) . approximately one third ( 28% ) of the proteins are ' hypothetical ' , meaning that they are predicted by various gene - prediction programs but do not have corresponding ests or other evidence of expression . a quarter ( 23% ) have unknown function , but they are known to be transcription - ally active because an est corresponds to each of them . the same analysis also reveals that 70% of the annotated proteins have some similarity to other hypothetical , unknown or putative - function proteins from plants and other eukaryotes , such as yeast , worm , fly and human , or include protein - family signature or motif sequences . table 1b shows a functional classification of the proteins based on the amino acid motifs . the analysis was generated by pedant and shows that almost 30% of this class of proteins participate in cellular metabolism and another 50% are involved in transcription , plant defense , signaling , and growth and development ( table 1b ) . comparison of the predicted proteins of the five chromosomes with other proteins from available complete genome sequences reveals that the absolute number of arabidopsis gene families and singletons ( proteins with no paralogs ) is in the same range as in worm and fly , indicating that a proteome of 11,000 types is sufficient for multicellular life . this analysis also reveals that proteins involved in rna splicing , trna biosynthesis , translation and metabolism are highly conserved throughout the eukaryotic kingdom . although numerous families of proteins are common among all eukaryotes , arabidopsis has 150 unique protein families encoding transcription factors , structural proteins , enzymes and proteins of unknown function . the number of adjacent members per family varies from 2 to 23 ( figure 3 ) . thus , 17% of all arabidopsis genes are arranged in tandem arrays , and these are distributed throughout the chromosomes . for example chromosome 1 contains 300 gene families with tandem gene arrangement of their members ; 175 families are unique , meaning that their members encode a set of defined protein isoforms . the remaining 123 are superfamilies consisting of more than one multigene family , varying in number of members from 2 to 5 . the completion of the first plant genome sequence is a milestone for biology , in general , and for plant sciences in particular . although the sequence provides a wealth of information , considerably more experimental work is required in order for it to contribute to the advancement of plant science . oligonucleotide and cdna chip technology will allow mapping of the transcriptional units , leading to the isolation of full - length cdna clones for all the proteins encoded by the arabidopsis chromosomes - a set that has been dubbed the ' orfeome ' . construction of the orfeome will eliminate the need for cdna library construction , and each transcribed gene will be represented at equimolar concentration in the orfeome . concomitantly , the dna sequence and chip technology will eliminate the need for southern and northern hybridizations in arabidopsis . furthermore , the isolation of t - dna insertional mutants for all the genes in the genome will offer additional resources for elucidating the function of the genes by reverse genetics . while the value of the arabidopsis genome sequence will be greatly enhanced by the resources described above , its full potential will be realized only when the technique of gene transplacement by homologous recombination is developed in plants . all the resources generated in the post - sequencing era will allow the elucidation of the biological and biochemical function of the arabidopsis proteome . more importantly , we will be able to do more productive and meaningful experimentation leading to a deep and genuine understanding of how plant cells function . a crucial task for the future will be the trying to understand the biological significance of the numerous multigene families . this fundamental question applies for gene families with tandem gene arrangement as well as for families with dispersed gene arrangements , such as the acs genes encoding acc synthases ( 1-aminocyclopropane-1-carboxylate synthases ) . this family has two members ( acs2 and acs10 ) on chromosome 1 and eight other members on the other four chromosomes . it has been postulated that multiple acs isoforms reflect tissue - specific expression of each , to satisfy the biochemical properties of the cells / tissues in which each is expressed . for example , if a group of cells or tissues has low concentrations of s - adenosyl methionine ( ado - met ) , then these cells would express a high affinity ( low km ) acs isoform . accordingly , the distinct biological function of each isoform is defined by its biochemical properties , which in turn determine its tissue - specific expression pattern . the most frequent explanation for the presence of large numbers of multigene families in arabidopsis is that it reflects functional redundancy : if something goes wrong with one gene product there is another to take over the lost function . but no two isoforms have completely overlapping functions , and most evolutionary biologists doubt the existence of functional redundancy . john maynard smith has used theoretical considerations to deduce highly contrived situations in which it could occur , but many prominent geneticists consider his arguments strong evidence that it does not occur in nature . and there is experimental evidence to support such a conclusion : individual knockout of any one of the seven different oxysterol - binding protein genes in yeast yields a different expression profile , even though all seven genes have to be knocked out in order to reveal a lethal phenotype . new technological breakthroughs in genomics will be required for elucidating the complex and repetitive structure of the centromeres . furthermore , the tertiary structure of intact chromosomes has to be elucidated in order to understand how the ' packaging ' of chromosomal dna occurs within the nucleus . eventually , we will be able to chemically synthesize new chromosomes consisting of desirable sets of genes , package them in vitro and construct new plant species with superior agronomical properties . numerous genomes will be sequenced as sequencing technologies improve and the cost per base - pair decreases . we sequenced many individual genes during the ' one by one ' era and many genomes will be sequenced in the era of genomics . only sequencing will reveal how plants evolved and will validate the various phylogenetic trees constructed using limited molecular information . i believe that knowing the evolution of plants is just as important to science as knowing the evolutionary history of the human species . the agi proved to be a successful venture and was an appropriate one to achieve such a landmark . thousands of young , bright , individuals have dedicated their intellectual and technical energies over the last five years to complete this project , using state - of - the - art molecular , engineering and computational technologies to achieve their goal . the agi effort led to the development of high - throughput robotic instruments that were used for sequencing chromosome 1 . , such as an m13 template preparation robot , the mantis plaque and colony picker , the revprep 96-well plasmid - preparation robot and the polyplex 96-well oligonucleotide synthesizer , were developed and tested for the arabidopsis sequencing project by the stanford genome and technology center , a member of the spp consortium . the entire effort was a communal undertaking , making a refreshing change from the competitive nature of modern science . the successful outcome of the agi fulfills the expectation of francis crick " you do not win battles by debating exactly what is meant by the word battle . you need to have good troops , good weapons , a good strategy , and then hit the enemy hard . i am proud to have been a part of this battle whose victory holds such potential rewards for future generations . the arabidopsis genome initiative ( agi ) , an international project for sequencing the genome of the flowering plant arabidopsis thaliana . for each chromosome , black represents centromeres ; grey telomeres ; green rdna repeats ; and red contiguous dna sequence . sequencing groups are : tigr , the institute for genomic research ( pis : craig venter and claire fraser ) ; kazusa dna research institute ( pi : satoshi tabata ) ; sppc , the spp consortium , stanford , university of pennsylvania , pgec - usda and university of california berkeley ( pis : ronald davis , joseph ecker , and athanasios theologis ) ; euc-1 and euc-2 , european union consortia for chromosomes 3 , 4 and 5 ( pis : michael bevan and francis quetier ) ; and the csh / wu / abi consortium of cold spring harbor laboratory , washington university and applied biosystems , inc .

the completion of the arabidopsis thaliana ( mustard weed ) genome sequence constitutes a major breakthrough in plant biology . it will revolutionize how we answer questions about the biology and evolution of plants as well as how we confront and resolve world - wide agricultural problems .

Historical perspective The The sequencing strategy The DNA molecules The proteome The future Figures and Tables

one of the major problems that humans face as we enter the twenty - first century is how to feed an overpopulated planet . currently , the world population is approximately six billion people , and it is estimated that with the current birth rate it will double by 2050 . the consequence of such growth is that humans will be forced to carry out agriculture using less , and lower quality , farming land . in order for us to be successful under these adverse conditions , a highly sophisticated knowledge of plant biology will be required that will allow the development of agronomically important species suitable for producing more food per unit farming area . the introduction of arabidopsis as a model plant species fifteen years ago has revolutionized plant biology and provides opportunities for achieving this goal . arabidopsis appears to contain homologs of most of the genes found in agronomically important crop plants , including rice , maize , soybean , and tomato . the sequencing of the mustard weed genome , together with the genomes of other eukaryotes - yeast , worm , fly and human , reaffirms the validity of this axiom . it has been only 48 years since the structure of the dna was elucidated and almost 30 years since the first dna molecule was cloned and propagated in escherichia coli . after 20 years , however , the ' one by one ' approach was not revealing information fast enough to allow understanding of the complexity of biological systems . more importantly , genomic sequencing of intensely studied eukaryotic organisms with a small fraction of redundant genes , such as saccharomyces cerevisiae , has indicated that no more than 30% of an organism 's genes can be identified by classical genetic analysis . it was the advent ten years ago of genomics , a new scientific discipline established by the emergence of three independent fields - biology , engineering and computer science - that eliminated the shortcomings of the ' one by one ' approach and opened up new and exciting possibilities for advancing biology when established in 1989 , the human genome project included all the best - known model organisms ( e. coli , yeast , worm , fly , human and mouse ) but omitted the mustard weed , for reasons that are poorly understood . the arabidopsis sequencing project was initiated in 1994 by the european community ( now the european union , eu ) under the leadership of michael bevan at the john innes institute in the uk . funds were secured in 1994 that led to the establishment of a consortium of european laboratories ( essa ) , led by bevan , to sequence chromosome 4 . shortly thereafter , satoshi tabata of the kazusa dna research institute in japan was funded for sequencing chromosome 5 . the flame that initiated the american sequencing effort was kindled one humid night - on july 8 1993 - at the cold spring harbor laboratory ( cshl ) , where the instructors of the arabidopsis molecular genetics course ( joe ecker , joanne chory , and myself ) were entertaining elliot meyerowitz after his evening lecture at blackford hall . while we were drinking beer together with rob martienssen and venkatesan sundaresan , two members of the cshl plant biology group , and gary drews ( another instructor ) and a few students from the course , i remember asking elliot when the sequencing of the arabidopsis genome would be initiated . that evening 's conversation was somehow transmitted to jim watson by rob and venkatesan , and the rest is history . jim watson 's deep interest in genome sequencing led to a banbury conference in the spring of 1994 at cshl that convinced the national science foundation ( nsf ) and some prominent skeptics of the urgency and importance of sequencing the arabidopsis genome . the most obvious objections at that time , from some of the leaders in the arabidopsis community , were whether funds would be removed from other aspects of plant biological research and whether it was intellectually stimulating to sequence the arabidopsis genome . following the banbury conference , a workshop on sequencing the arabidopsis genome was held at nsf in the summer of 1994 , where the guidelines were established for finding resources for the project . an historic meeting was held in the summer of 1996 at nsf , where the six sequencing groups from europe , japan and the us , representatives of the arabidopsis community , nsf , the department of energy ( doe ) and the us department of agriculture ( usda ) met to coordinate an international effort to sequence the 125 mb arabidopsis genome . that meeting established the arabidopsis genome initiative ( agi ) , an international effort to sequence the first plant genome by 2004 ( figure 1 ) . the agi discussed various strategies for sequencing the arabidopsis genome , including the whole - genome shotgun - sequencing approach , which was proposed by ron davis , one of the principal investigators ( pis ) of the spp consortium ( see figure 1 ) . the approach was rejected at that time as risky , and more secure strategies were adopted , such as bac - end sequencing ( see below ) , and approaches based on physical maps . in retrospect , if the shotgun approach had been adopted in 1996 , the discovery of all the genes in the arabidopsis genome would have preceded the corresponding studies of the worm , fly and human genomes . chromosomes 1 , 2 , and 3 would be sequenced using the bac - end strategy ( figure 2a ) . in a collaborative effort among three agi groups - genoscope , a member of the eu-2 consortium , the institute for genomic research ( tigr ) and spp ( see figure 1 ) - the end - sequences for almost all the bacs ( approximately 18,300 clones , representing 14-fold coverage ) in the two libraries were determined and made publicly available . all sequencing groups used a variation of the same strategy for sequencing individual bacs , p1 or cosmid clones , as shown in figure 2b . two major software programs were used for assembly and editing by most of the agi members ( except tigr , which used the in - house ' tigr assembler ' ): phred / phrap for sequence assembly and gap4 or consed9 for viewing and editing . the agi members used almost all the same annotation programs for gene prediction and annotation of the genome : programs such as genefinder , grail , genscan , xpound , trnascan - se , blastn , blastx , gene mark hmm , glimmer a , netgene 2 , splice predictor , pedant and repeat masker ( see ) . the entire genome was also reannotated upon completion , by two agi members , tigr and mips ( a member of the eu consortia ) , to ensure uniformity of the final product . the adopted strategies allowed different groups around the globe to sequence different regions of the various chromosomes at the same time . existing incomplete physical and genetic maps of each of the five chromosomes were used in selecting the seed bacs necessary to initiate the sequencing process . the incomplete physical chromosome maps were supplemented by fingerprint and hybridization data using 24,000 bacs , to yield a complete map of the genome with 99% coverage and resulting in an acceleration of the sequencing process . a few additional crucial decisions were also made during the 1996 nsf meeting , regarding data release policy , acceptable error in the final sequencing product , and acceptable degree of completion . accordingly , phase i genomic sequence - comprising raw sequence containing gaps and of unknown orientation - was available to the plant biology community at the htgs section of genbank a few days after the shotgun sequencing was completed . this immediate - release policy allowed plant molecular geneticists to clone their favorite gene by ' walking ' much faster than before . there are numerous examples for which the availability of unfinished genome sequence allowed the cloning of genes , such as axr3 and shy2 ( two examples that i know about because of my own research interests ) . regarding the extent of completion , the agi agreed that the genome sequence would be considered complete when each chromosome was represented by only two contigs - chromosomal arms - separated by the centromeric region . the rdna clusters of chromosomes 2 and 4 were also excluded from the finished product . chromosomes 2 and 4 were published in december 1999 and the remaining chromosomes 1 , 3 and 5 , along with a uniform annotation and analysis of the entire genome , were published in december 2000 . the accelerated pace was primarily due to the acceleration of funding by the nsf two years into the project , thanks to the vision of the nsf administrators , mary clutter , machi dilworth and the late delill nasser . the rapid progress of the project during the first two years , and the excess of sequencing capacity of the participating groups , warranted such an action . the ten chromosomal arms are without gaps , except for three at the bottom arm of chromosome 1 where there are highly repetitive dna sequences . the combined length of the ten arms , which extend from either the telomeres or the ribosomal dna repeats to the 180 base - pair centromeric repeat is approximately 115 mb ( table 1 ) . the unsequenced centromeric and rdna repeat regions are estimated to represent approximately 10 mb , resulting in a genome size of about 125 mb . the overall gc content is low ( 35% ) and uniform across the five chromosomes , which made the dna relatively easy to sequence using the available chemistry . the sequencing error rate is no more than 1 error per 20 kb , better than that agreed upon by the agi in 1996 . the collinearity of the assembled chromosomes was verified using blast alignment analysis of the bac - end sequences against the assembled chromosomal contigs . per base ( $ 70 million for 115 mb ) , corresponding to approximately 10 ? gene prediction programs and database searches the genome contains 25,500 putative genes at a density of 1 gene per 4 kb ( table 1 ) . arabidopsis contains as many genes as humans and twice as many as the fruitfly ( table 2 ) . thus , almost 50% of the chromosomal dna is covered with genes ( table 1a ) . approximately 20% of the genes are intronless , and some of them have been annotated as hypothetical . for example , chromosome 1 contains a gene ( f5f8.4 ) with 68 exons that encodes a 500 kda polypeptide that is a ubiquitous putative membrane protein found in yeast , worm , fly and human . the same chromosome contains a very small gene that encodes the l41 protein of the 60s ribosomal complex , with only 25 amino acids . at least 15,300 genes ( 60% ) are expressed , as indicated by the presence of corresponding cdnas or expressed sequence tags ( ests ) in various databases ( table 1 ) . genes that are most highly represented by ests include proteins of the photosynthetic and protein synthesis machineries and few metabolic enzymes , such as catalase . the distribution of ests is more or less the same across the length of the chromosomes but decreases dramatically in the regions flanking the centromeres . the chromosomes encode approximately 600 trnas , with chromosome 1 bearing the majority of them ( 236 trnas ) . the trna gene content of chromosome 1 is very similar to that of the entire fly genome . the trna genes are evenly distributed along the chromosomes , except for two regions in chromosome 1 where trna gene clustering is observed ; the two clusters are located at 9.989 mb and 19.282 mb , respectively . the first contains 26 genes encoding trna and the other 27 tandem repeats of the tri - repeat trna- trna- trna . the trna genes of eukaryotes in general occur as multigene families with a diverse arrangement . in some cases they are spread throughout the genome , whereas others are clustered at single chromosomal sites . in addition to the detection of the large - scale intra- and inter - chromosomal duplications , analysis reveals a plethora of diverse repetitive elements comprising 10% of the sequence . retroelements include members of the line - like ta11 family of elements , and long - terminal repeat ( ltr ) elements of both the ty3-gypsy and the ty1-copia families . representative members of various transposable element families are scattered throughout the chromosome ; for example , the ac / ds-(hat / mariner ) , en - spm - mutator and tc1 type elements all occur . there is an inverse relationship between gene and retroelement density in the borders of the centromeric region , a hallmark of such chromosomal regions . computational analysis of the chromosomal sequences reveals that they encode 25,500 putative proteins ( table 1b ) . approximately one third ( 28% ) of the proteins are ' hypothetical ' , meaning that they are predicted by various gene - prediction programs but do not have corresponding ests or other evidence of expression . a quarter ( 23% ) have unknown function , but they are known to be transcription - ally active because an est corresponds to each of them . the same analysis also reveals that 70% of the annotated proteins have some similarity to other hypothetical , unknown or putative - function proteins from plants and other eukaryotes , such as yeast , worm , fly and human , or include protein - family signature or motif sequences . table 1b shows a functional classification of the proteins based on the amino acid motifs . the analysis was generated by pedant and shows that almost 30% of this class of proteins participate in cellular metabolism and another 50% are involved in transcription , plant defense , signaling , and growth and development ( table 1b ) . comparison of the predicted proteins of the five chromosomes with other proteins from available complete genome sequences reveals that the absolute number of arabidopsis gene families and singletons ( proteins with no paralogs ) is in the same range as in worm and fly , indicating that a proteome of 11,000 types is sufficient for multicellular life . although numerous families of proteins are common among all eukaryotes , arabidopsis has 150 unique protein families encoding transcription factors , structural proteins , enzymes and proteins of unknown function . the number of adjacent members per family varies from 2 to 23 ( figure 3 ) . thus , 17% of all arabidopsis genes are arranged in tandem arrays , and these are distributed throughout the chromosomes . for example chromosome 1 contains 300 gene families with tandem gene arrangement of their members ; 175 families are unique , meaning that their members encode a set of defined protein isoforms . the completion of the first plant genome sequence is a milestone for biology , in general , and for plant sciences in particular . although the sequence provides a wealth of information , considerably more experimental work is required in order for it to contribute to the advancement of plant science . oligonucleotide and cdna chip technology will allow mapping of the transcriptional units , leading to the isolation of full - length cdna clones for all the proteins encoded by the arabidopsis chromosomes - a set that has been dubbed the ' orfeome ' . construction of the orfeome will eliminate the need for cdna library construction , and each transcribed gene will be represented at equimolar concentration in the orfeome . furthermore , the isolation of t - dna insertional mutants for all the genes in the genome will offer additional resources for elucidating the function of the genes by reverse genetics . while the value of the arabidopsis genome sequence will be greatly enhanced by the resources described above , its full potential will be realized only when the technique of gene transplacement by homologous recombination is developed in plants . all the resources generated in the post - sequencing era will allow the elucidation of the biological and biochemical function of the arabidopsis proteome . a crucial task for the future will be the trying to understand the biological significance of the numerous multigene families . this fundamental question applies for gene families with tandem gene arrangement as well as for families with dispersed gene arrangements , such as the acs genes encoding acc synthases ( 1-aminocyclopropane-1-carboxylate synthases ) . it has been postulated that multiple acs isoforms reflect tissue - specific expression of each , to satisfy the biochemical properties of the cells / tissues in which each is expressed . for example , if a group of cells or tissues has low concentrations of s - adenosyl methionine ( ado - met ) , then these cells would express a high affinity ( low km ) acs isoform . but no two isoforms have completely overlapping functions , and most evolutionary biologists doubt the existence of functional redundancy . and there is experimental evidence to support such a conclusion : individual knockout of any one of the seven different oxysterol - binding protein genes in yeast yields a different expression profile , even though all seven genes have to be knocked out in order to reveal a lethal phenotype . new technological breakthroughs in genomics will be required for elucidating the complex and repetitive structure of the centromeres . furthermore , the tertiary structure of intact chromosomes has to be elucidated in order to understand how the ' packaging ' of chromosomal dna occurs within the nucleus . eventually , we will be able to chemically synthesize new chromosomes consisting of desirable sets of genes , package them in vitro and construct new plant species with superior agronomical properties . numerous genomes will be sequenced as sequencing technologies improve and the cost per base - pair decreases . we sequenced many individual genes during the ' one by one ' era and many genomes will be sequenced in the era of genomics . i believe that knowing the evolution of plants is just as important to science as knowing the evolutionary history of the human species . the agi proved to be a successful venture and was an appropriate one to achieve such a landmark . the agi effort led to the development of high - throughput robotic instruments that were used for sequencing chromosome 1 . , such as an m13 template preparation robot , the mantis plaque and colony picker , the revprep 96-well plasmid - preparation robot and the polyplex 96-well oligonucleotide synthesizer , were developed and tested for the arabidopsis sequencing project by the stanford genome and technology center , a member of the spp consortium . the entire effort was a communal undertaking , making a refreshing change from the competitive nature of modern science . the successful outcome of the agi fulfills the expectation of francis crick " you do not win battles by debating exactly what is meant by the word battle . you need to have good troops , good weapons , a good strategy , and then hit the enemy hard . i am proud to have been a part of this battle whose victory holds such potential rewards for future generations . the arabidopsis genome initiative ( agi ) , an international project for sequencing the genome of the flowering plant arabidopsis thaliana . sequencing groups are : tigr , the institute for genomic research ( pis : craig venter and claire fraser ) ; kazusa dna research institute ( pi : satoshi tabata ) ; sppc , the spp consortium , stanford , university of pennsylvania , pgec - usda and university of california berkeley ( pis : ronald davis , joseph ecker , and athanasios theologis ) ; euc-1 and euc-2 , european union consortia for chromosomes 3 , 4 and 5 ( pis : michael bevan and francis quetier ) ; and the csh / wu / abi consortium of cold spring harbor laboratory , washington university and applied biosystems , inc .

haruta et al . discovered some 20 years ago that gold known for its inert nature in the bulk state exhibits a high reactivity as catalyst for oxidation reactions even below ambient temperatures if reduced to the size of a few nanometers . since then , research on au cluster catalysis has become a subject of tremendous interest , and many reviews on this topic are available , e.g. , refs ( 28 ) . the catalytic activity of a variety of nanoparticle / support systems , such as gold on different oxides ( e.g. , au / tio2 , au / mgo , au / feo ) and even on inert supports , has been investigated experimentally as well as by theoretical methods ( e.g. , refs ( 11 , 12 , 20 , and 21 ) ) . however , despite 20 years of intensive research , the role of the ( mostly oxidic ) support as opposed to factors that are associated with intrinsic properties of the clusters remains under debate up to now . generally , the following issues are regarded as highly relevant for the catalytic activity and selectivity of au : ( i ) quantum size effects . in the case of gold clusters on tio2 studied by valden et al . the maximum in the turnover frequency was observed for particles with a lateral extension of 33.5 nm and a thickness of 1 nm , coinciding with a metal - to - insulator transition occurring at this particle size . hutchings and co - workers attributed the high catalytic activity to the presence of even smaller particles ( ca . 0.5 nm diameter or 10 atoms ) . the vertical extension ( thickness ) of the particles was supposed to be of crucial importance , as particles of bilayer height were reported to be particularly active . this factor is intimately connected to the size of the particles : generally , the number of under - coordinated au atoms increases with decreasing size , at least if the cluster shape remains the same . this is expected to be highly relevant for the adsorption and dissociation of oxygen . ( iii ) charge of the clusters ( due to interaction with the support ) . however , it is under debate if zerovalent gold or a negative or a positive charge of the particle is required to obtain an active catalyst system . ( iv ) interface effects , i.e. , adsorption and activation / dissociation of oxygen at the interface between cluster and oxide . contrariwise , the studies of chen and goodman on a catalytically active gold bilayer on ( 8 2)-tiox / mo(112 ) suggest that the interface is not directly involved in the reaction . according to their measurements , the deposited au layer wets the substrate and hence prevents reactant molecules from interaction with the oxide support . the support provides specific sites , e.g. defects , which facilitate nucleation , suppress cluster mobility , and prevent coalescence . also , the number of low coordinated atoms is partially controlled by the support via the cluster density and shape . in the present work we study the adsorption of carbon monoxide and oxygen and their potential reaction on gold clusters grown on a completely different class of template in order to obtain new insights into the role of the support material . recently , it has been shown that carburized w(110 ) surfaces serve as rather universal substrates for the growth of different kinds of noble - metal clusters : the low carbon coverage r(15 12)c / w(110 ) phase is an excellent template for generation of regularly arranged monolayer - height clusters of a rather fixed size ( ca . 7 atoms ) ( see figure 3c ) , while on the high carbon coverage r(15 3)c / w(110 ) template bilayer particles with lateral extensions up to a few nanometers ( depending on au coverage ) are generated ( see figure 3a ) . the proposed cluster / support system seems to be a promising candidate for investigation of the catalytic properties toward co oxidation for several reasons : the substrate a surface carbide is metallic and hence differs from conventionally used insulating / semiconducting oxidic supports . therefore , exploration of the adsorption behavior of oxygen and carbon monoxide as well as the reactivity toward oxidation is expected to yield interesting results concerning the role of the support material . whereas arguments based on the abundant existence of undercoordinated gold sites or on quantum - size effects due to lateral electron confinement are valid on both insulating and metallic substrates , from other arguments such as charge transfer or reaction at the gold / support interface a different behavior furthermore , use of carburized w(110 ) as template allows growth of well - defined clusters of both monolayer height ( au / r(15 12 ) ) and bilayer height ( au / r(15 3 ) ) on rather similar substrates . as already mentioned above , a thickness of two monolayers is regarded essential for the activity of au nanocatalysts by some authors . in addition as gold clusters are reported to catalyze not only oxidation of carbon monoxide but also other oxidation reactions as well the question will be addressed if the gold clusters are also able to catalyze oxidation of the carburized template itself . in order to test the stability of the cluster systems in a gas atmosphere temperature - programmed desorption ( tpd ) experiments after low - temperature ( 90100 k ) adsorption of oxygen or carbon monoxide were carried out on the templates as well as on the cluster - decorated samples . finally , coadsorption / reaction studies for investigation of the catalytic activity were performed . all experiments were carried out in an uhv chamber with a base pressure of 2 10 mbar , equipped with a room - temperature scanning tunneling microscope ( danish micro engineering ) and a quadrupole mass filter ( inficon ) for tpd measurements . high temperatures ( 10002600 k ) required for sample cleaning and preparation of the carbon superstructures were achieved by electron bombardment ; for tpd experiments the crystal was heated ohmically . up to 1200 k ( i.e. , in tpd measurements ) sample temperatures were measured with a type c ( w-5% re / w-26% re ) thermocouple spot - welded to a thin tantalum foil which was clamped to the tungsten sample . because of temperature gradients across the ta foil , at high temperatures the temperature readout from the thermocouple resulted in too low values ( by approximately 100150 at 1200 k ) . hence , beyond 1200 k ( i.e. , during sample preparation ) a two - color pyrometer was used . the surface was cleaned from carbon impurities by heating in oxygen as described in refs ( 30 and 3234 ) . tungsten oxides formed in course of the procedure were removed by flashing the sample to 23002600 k. the carbon superstructures were generated following the preparation routines as described in refs ( 29 , 30 , 35 , and 36 ) . carbon was provided by thermolysis of ethene ( c2h4 ) ( heating for 10 min at 1250 k in 5 10 mbar ) . subsequent annealing at 1250 k in vacuum led to broader terraces . flashing to 1900 k produced sharp r(15 3 ) leed spots , whereas either flashing to 2300 k or annealing for 2 min at 2000 k , both followed by rapid cooling , yielded the typical r(15 12 ) pattern . accordingly , deposition rates in different experiments varied between 0.10 and 0.20 monolayers ( ml ) per minute , as estimated from stm images of extended gold islands generated on w(110 ) . for the production of well - ordered regular arrays of gold monolayer clusters on r(15 12 ) the sample had to be kept at 600700 k. in contrast , to achieve kinetically controlled growth of nanosized bilayer clusters ( rather than growth of larger ribbon - like multilayer structures ) , gold deposition on r(15 3 ) had to be carried out at room temperature . in - situ stm measurements were performed during gas exposure at room temperature , using gas pressures ( either co or o2 ) of 5 10 mbar . unless otherwise noted , for tpd experiments , the sample was kept at 90100 k during adsorption and subsequently heated with rates of 58 k / s . figure 1 shows tpd spectra obtained after saturation co exposure to clean w(110 ) as well as to the two carburized surface phases . the evolution of the various peaks as a function of co exposure is displayed in the supporting information , figures s1s3 . the spectra taken on clean w(110 ) are in agreement with literature data , showing the low - temperature virgin peak slightly above 400 k and the high - temperature double - peak feature ( ) at 10001100 k. whereas the virgin peak stems from molecularly adsorbed carbon monoxide , the high - temperature features are commonly attributed to co molecules adsorbed dissociatively on w(110 ) , although lee and co - workers recently doubted if co molecules are completely dissociated or only strongly tilted . however , our tpd data after oxygen adsorption on the carburized templates strongly support the dissociation model ( see section 5 ) . tpd spectra of co on w(110 ) , r(15 3)c / w(110 ) , and r(15 12)c / w(110 ) . the tpd data for co adsorbed on the r(15 3 ) carbon superstructure differ from that of the clean surface in several respects : ( i ) the low - temperature feature is split into two components , suggesting a coverage - dependent adsorption energy and/or the existence of ( at least ) two different adsorption sites . the latter interpretation seems plausible due to the heterogeneous character of the r(15 3 ) surface ( consisting of c and w atoms ) and the fairly large size of the unit cell ( having a size equivalent to 15 tungsten surface atoms ) . ( ii ) the ( split ) low - temperature feature is shifted to lower desorption temperatures , indicating a weakened adsorption of molecular co on carbon - modified tungsten . if the features arise from dissociated co , an obvious explanation for their suppression is the blocking of adsorption sites for atomic carbon , since the r(15 3 ) surface is already presaturated with carbon . an alternative explanation would be a substantial c - induced increase of the activation barrier for co dissociation . the tpd spectra from the low carbon - coverage r(15 12 ) surface exhibit a more complicated pattern ( see figure 1 ) with ( at least ) six components : four at low temperatures ( 280420 k ) and two at high temperatures ( 10501150 k ) . we interpret the existence of the pronounced 420 k peak as well as the high - temperature double feature ( which all exhibit peak temperatures similar to clean tungsten ) as an indication of co adsorption on sites that are only weakly perturbed by the presence of carbon . as we have shown recently , the w(110 ) surface is not uniformly covered with r(15 12 ) : wide terraces , exhibiting the r(15 12 ) structure , coexist with smaller terraces being essentially free of carbon . furthermore , stm images of the large r(15 12 ) unit cell ( having a size equivalent to 60 tungsten surface atoms ) exhibit bright regions , which were interpreted as being largely free of carbon atoms . the three low - temperature features ( 370 k and below ) are attributed to co molecules adsorbed in parts of the r(15 12 ) unit cell , where co is more strongly influenced by carbon atoms from the template , resulting in desorption temperatures rather similar to that from the high carbon coverage r(15 3 ) phase . the uptake curves ( at 90 k ) for co on the three templates as derived from the integrated tds intensities are summarized in figure 2 . all curves are quite similar , showing a precursor - mediated adsorption behavior with a linear coverage increase for exposures up to 3.4 langmuirs and an almost constant coverage for larger exposures . the achievable saturation coverages on both carburized surfaces are roughly equal to that on clean w(110 ) , for which saturation values between 0.7 and 1.1 ml were derived in the literature . in good agreement with these values , we calculate a saturation coverage of 0.9 ml from the required saturation exposure of 3.4 langmuirs under the assumption of a constant unity sticking coefficient . co uptake curves ( at 90 k ) on w(110 ) , r(15 12)c / w(110 ) , and r(15 3)/c / w(110 ) as obtained from the integrated tpd curves . the present tpd measurements of co adsorption on r(15 3 ) are similar to tpd data of co adsorption on c / w(111 ) and c / w(110 ) by hwu et al . , both showing a double - peak desorption structure around 300 k and a strong reduction of the high - temperature desorption feature stemming from dissociated co. in hwu s work on c / w(110 ) an ordered c - induced leed pattern was not observed , probably due to the relatively low flash preparation temperature of 1200 k. however , from the preparation procedure used and the amount of carbon detected by aes , we conclude that the surface should be carbon - rich and hence similar to our ordered r(15 3 ) phase . analogous to the present findings , measurements on c / w(100 ) revealed that with increasing carbon content of the surface the ability to dissociate co is reduced , while the overall amount of adsorbed co is always roughly the same . it is also quite instructive to compare the adsorption properties of the carburized tungsten surfaces with those of tungsten carbide single crystals . wc(0001 ) surface and found that co adsorbs molecularly below room temperature . around 250 k co desorbs partly , while the remaining co dissociates . this behavior is qualitatively similar to that of clean w(110 ) or r(15 12)c / w(110 ) but clearly differs from that observed for the high - carbon coverage r(15 3 ) phase , where dissociation of co is effectively suppressed . according to the structural studies of the same group , tungsten carbide ( which is a layered hexagonal crystal ) terminates with a tungsten surface layer , on which about 30% of carbon atoms are statistically distributed . this would explain the close similarity to the clean w(110 ) and the low - carbon - coverage r(15 12 ) surface , while the carbon - saturated r(15 3 ) surface behaves differently . in figure 3 , the results of an in - situ stm study of the influence of co on au clusters on r(15 3)c / w(110 ) and r(15 12)c / w(110 ) , respectively , are displayed . as clearly seen from the images , both types of nanoparticles are stable under co exposure at room temperature , irrespective of the type of c / w(110 ) substrate . furthermore , due to the low au coverage on r(15 3 ) , the stm images also demonstrate that the substrate itself visible as faint , protruding lines with an intrinsic height of 0.8 nm is unaffected by co at room temperature . this might have been expected , since co already desorbs around room temperature from the r(15 3 ) surface ( compare figure 1 ) . deposition of au on r(15 3 ) essentially quenches the low - temperature molecular co desorption features located at t 400 k ( see figure 4 ) . this can be understood as a site - blocking effect . it is tempting to attribute this observation to co molecules adsorbed at the periphery or on top of the deposited gold particles . gold interaction , such a high desorption temperature seems to be impossible for molecularly adsorbed co. it appears to be more reasonable to assign this desorption feature to recombinatively desorbing co. this implies the presence of atomic oxygen which can only arise from co dissociation . the microscopic origin for this surprising au - induced co dissociation is unclear at present . stm images of au clusters on r(15 3)c / w(110 ) ( top ) as well as r(15 12)c / w(110 ) ( bottom ) before ( left ) and after ( right ) exposure to co ( b : 75 langmuirs , d : 181 langmuirs ) . au coverages : ( a , b ) 0.5 min ; ( c , d ) 1.5 min . image sizes : ( a , b ) 40 nm 40 nm ; ( c , d ) 80 nm 80 nm . note the growth of extended gold islands on the narrow carbon - poor tungsten terraces , visible on the right - hand side of images c and d. the better resolved inset in ( c ) shows that the line - like structures visible in ( c ) and ( d ) actually consist of individual small gold clusters . finally , we turn to the low - temperature behavior , which is of crucial importance for a potential co oxidation reaction . various other groups who carried out adsorption studies on either au surfaces or au nanoparticles mostly found two co desorption peaks at temperatures around 120 and 190 k , respectively . these peaks were frequently assigned to differently coordinated co molecules ( depending on author either as surface vs step or as step vs kink adsorption ) . clearly , except for a marginal intensity increase at 120 k , au - induced low - temperature features can not be detected in this temperature regime . in the work of freund and co - workers , co desorption temperatures up to 300 k were reported for as - deposited , unannealed small au particles and attributed to co molecules attached to highly uncoordinated au atoms . it might well be that due to the small size of the au particles in the present study a similar desorption feature is hidden underneath the template - induced double - peak desorption feature at temperatures around 250300 k. however , according to freund et al . peak should shift to lower temperatures with increasing particle size ( i.e. , coverage ) and hence become visible as an extra feature in the tds spectra . as this behavior is not observed , we conclude that the au clusters on r(15 3)c / w(110 ) are not or only marginally able to adsorb weakly bound co molecules . thus , our data parallel the findings of outka and madix that co does not adsorb on au(110 ) down to temperatures of 125 k. as pointed out by these authors , this does not necessarily imply that co does not stick on the surface . the residence time of co on gold could just be too low to allow buildup of sufficient amounts of co that could be detected by tds . furthermore , in the present case co attaching to the gold clusters could also spill over to the substrate or the gold / substrate interface , where it is more strongly bound . tpd spectra of au / r(15 3)c / w(110 ) after exposure to 10 langmuirs of co. a deposition time of 1 min corresponds to a coverage of 0.10.2 ml . co tpd spectra with different amounts of au on r(15 12)c / w(110 ) are displayed in figure 5 . deposition of gold leads to an intensity decrease of the desorption features in the temperature region from 300 to 420 k due to blocking of co adsorption sites . the 420 k peak , resulting from co molecules only weakly affected by the presence of carbon , is reduced most strongly , indicating preferential au nucleation in these surface regions . our stm findings concerning the growth behavior of au corroborate this assumption : preferentially , au nucleates on carbon - poor regions of the unit cell ; at elevated deposition temperatures ( i.e. , 700 k ) au atoms in excess of the optimum coverage ( 0.12 ml ) diffuse to w(110 ) terraces always coexisting with the broader carbon - modified ones . hence , tungsten - rich areas on the surface ( clean terraces as well as carbon - poor regions within the unit cell ) are preferentially covered with au atoms leading to a selective decrease of the signal attributed to co desorption from these areas . au / r(15 12)c / w(110 ) after exposure to 10 langmuirs of co. gold deposition rate was 0.10.2 ml / min . the high - temperature features do not follow this behavior but ( for deposition times up to 5 min ) remain rather unaffected in position and peak intensity . however , one should mention that at such high temperatures ( around 1000 k ) the gold clusters do no longer retain their room temperature size and shape . stm investigations performed after flashing the small clusters of figure 3c to 1000 k showed rectangular - shaped clusters with heights beyond one monolayer and lateral extensions up to several nanometers . interestingly , for gold deposition times exceeding 5 min the high - temperature desorption feature changes its appearance drastically : the peak at 1100 k is quenched almost completely ; instead , a strong new peak appears around 900 k. we presume that this change is again related with a structural change . a possible scenario would be that under the presence of sufficient amounts of gold the underlying r(15 12 ) template is altered . a different explanation not necessarily in contradiction to the former one is related to a change in growth behavior at higher gold coverages . as is known from our previous studies of the analogous ag / r(15 12 ) system , atoms deposited in excess of the ideal coverage of the cluster structure ( 0.12 ml ) mostly diffuse to nearby clean tungsten terraces , where they form extended islands . however , at some stage the ag atoms start to fill the space between the clusters and to overgrow them as larger bilayer islands . accordingly , we suppose that the evolution of the strong 900 k peak is associated with the formation of double - layer high gold islands . this explanation is consistent with the tpd experiments on the r(15 3 ) substrate ( figure 4 ) : on this surface bilayer islands are formed already at low au coverages . accordingly , the 900 k peak evolves ( without delay ) already at small gold coverages . stm investigations of co - covered annealed surfaces with various au precoverages are necessary to clarify this issue . as already observed for the r(15 3 ) template in the low - temperature region below 300 k pronounced au - induced features do not appear except for a weak desorption signal at 120 k. however , since in the series of experiments the desorption intensity at 120 k ( i.e. , close to the adsorption temperature ) could not be reproduced perfectly , the assignment of the intensity increase as a au - induced effect is not unambiguous . in any case this indicates that weakly adsorbed co is only present in small amounts if at all on the au clusters on the r(15 12 ) template , which parallels the findings for r(15 3 ) . from these results we can already guess that low - temperature co oxidation on both types of au clusters is unlikely with the present systems . the stability of both carburized templates with and without au clusters against exposure to oxygen atmosphere was investigated by stm movies at room temperature as well as by temperature - programmed desorption experiments after adsorption at 90100 k. in - situ stm study of the influence of oxygen ( p = 5 10 mbar ) on the r(15 3 ) and r(15 12 ) templates covered with au clusters : ( a ) r(15 3 ) , before gas exposure , ( b ) r(15 3 ) after 55 langmuirs of o2 , ( c ) r(15 12 ) , before gas exposure , ( d ) r(15 12 ) after 86 langmuirs of o2 . image sizes : 40 nm 40 nm ( a , b ) and 80 80 nm ( c , d ) . stm experiments on the bare templates showed a rather good stability for both r(15 3 ) and r(15 12 ) . although the images were somewhat deteriorated ( possibly due to tip instabilities in the presence of oxygen ) and although in particular on the r(15 3 ) surface occasional impurities and hole - like defects appeared with prolonged gas exposure and scanning time , the overall periodicities seemed to be conserved . the evolution of the au - covered r(15 3)c / w(110 ) surface upon o2 exposure is shown in figure 6a , b . ( more images are shown in the supporting information , figures s4 and s5 . ) apart from very occasional modifications ( such as the disappearing cluster in the center of the white marker ) the bilayer gold particles obviously are quite stable to o2 . by contrast , pronounced oxygen - induced changes occur for the au clusters on the r(15 12 ) template ( see figure 6c , d ) . with increasing oxygen exposure the originally well - aligned nanodots show strong modifications : increasing oxygen exposure leads to less perfect ordering , the size distribution is broadened significantly , and some bilayer clusters are formed . after an exposure of 90 langmuirs a quite stable state is reached ; the clusters are hardly altered anymore . agglomeration to larger islands , supposedly exhibiting a negative effect on the reactivity of au clusters , is not observed . note that the rather strong oxygen - induced alterations of the gold nanoparticles indicate dissociation of the oxygen molecules on or at least in close vicinity of the au clusters on the r(15 12 ) substrate . tpd experiments performed after oxygen exposure of both the bare and the au - covered carburized templates never revealed an o2 desorption signal . instead , only desorption of co was observed as shown in figures 7 and 8 . the co desorption spectrum from the bare r(15 12 ) surface ( figure 7 ) bears a close resemblance to the high - temperature features observed for co desorption ( after co exposure ) from clean w(110 ) as shown in figure 1 . obviously , oxygen molecules dissociate on the surface and react with carbon atoms from the templates : co is formed and desorbs at high temperatures ( 9001100 k ) . these temperatures are rather similar to that of dissociated -co on clean w(110 ) , thus lending strong support to the interpretation of the desorption peaks as stemming from dissociated co. this observation is in accordance with the findings of viswanath and schmidt that the desorption properties for oxygen and carbon on w(100 ) are essentially identical to those of adsorbed carbon monoxide . addition of gold decreases the intensity of the 2-like peak at 1100 k but increases the intensity of the 1-like feature around 950 k. hence , the presence of au shifts the maximum in the desorption rate by approximately 150 k toward lower temperatures , implying that gold obviously catalyzes the associative reaction of surface carbon with adsorbed oxygen to gaseous carbon monoxide . according to the principle of detailed balance , then the reverse process of dissociative co adsorption should also be catalyzed in agreement with our findings concerning co adsorption in the presence of gold clusters . co desorption spectra of bare and au - covered r(15 12 ) after exposure to 10 langmuirs of oxygen . a similar effect is seen on the r(15 3 ) template ( figure 8) . here , the desorption rate at 900 k increases markedly when gold is present on the surface , while the higher - temperature peaks are reduced in intensity . surprisingly , on both bare and au - covered r(15 3 ) some co formation and desorption occur even around room temperature . tentatively , we assign this feature to carbon atoms in excess of the amount required to form an ideal r(15 3 ) structure , which can be more easily oxidized . the increased desorption signal at even lower temperatures as ( reproducibly ) observed with larger amounts of gold ( 5 min ) also remains without definite explanation . in contrast , the finite desorption signal at and even above 1200 k can be traced back to the onset of segregation and subsequent oxidation of carbon atoms residing in subsurface regions of the r(15 3 ) phase . in summary , the tpd investigations show that oxygen molecules dissociate on both carburized tungsten surfaces . co is formed , and this reaction is promoted by the presence of gold . the stm investigation on the r(15 12 ) surface even prove that oxygen atoms reside at or in close vicinity to the au clusters . however , weakly bound oxygen atoms which recombine and desorb at intermediate temperatures ( around 500 k)as found e.g. on au(110 ) , see ref ( 61)were not observed . we conclude that either oxygen dissociates only at the template itself or that even if oxygen dissociates on or in close vicinity to the au nanoparticles it subsequently spills over to the c / w(110 ) substrate , where it reacts with the surface carbon and desorbs as co. the modifications of the au clusters induced by exposure to o2 as seen by stm could then be explained by a displacement of au atoms due to the competitive adsorption of dissociated oxygen for ( the same ) favorable adsorption sites . because of the large binding energy of oxygen to tungsten ( adsorption energy 4.20 ev / atom ) , we expect that the carbon - poor regions where the au clusters nucleate are also the most favorable areas for oxygen adsorption , thus providing a strong driving force for an oxygen - induced rearrangement of the noble - metal clusters . however , as the oxygen atoms on w(110 ) are bound rather strongly to the surface , we expect that they are not available for the low - temperature formation of co2 , leading to the conclusion that also for this reason the system despite its ability to dissociate molecular oxygen will not be able to perform co combustion . co desorption spectra of bare and gold - covered r(15 3 ) after exposure to 10 langmuirs of co. gold deposition rate was 0.10.2 ml / min . co oxidation studies were performed , looking for co2 formation in temperature - programmed reaction experiments under various reaction conditions : ( i ) alternating chronological order of exposures , ( ii ) low ( 5 10 mbar ) and high ( 1 10 mbar ) total steady - state background pressures , ( iii ) different o2/co ratios ( 1:1 or oxygen surplus in order to avoid blocking of reactive sites by co ) , and ( iv ) utilizing linear as well as stepwise temperature profiles ( both starting at 90 k ) . furthermore , preadsorption of oxygen at 300 k , followed by heating in co atmosphere ( starting at 90 k ) , was also tested , as stm on the r(15 12 ) revealed that at 300 k oxygen adsorbs dissociatively on or close to the au clusters . however , in none of these experiments could the evolution of co2 due to au - catalyzed co oxidation be verified , neither for bilayer clusters on the r(15 3 ) nor for the small monolayer clusters on the r(15 12 ) template , although the gain of the qms was increased up to 100-fold compared to the co and o2 tpd experiments . hence , we conclude that in the systems investigated in the present work co oxidation is not possible , at least not under the present experimental low - pressure conditions . the tpd experiments presented in sections 4 and 5 provide a reasonable explanation for this observation : neither weakly bound carbon monoxide molecules nor oxygen atoms attached to gold clusters , with desorption temperatures below 200 k ( co ) or around 500 k ( oxygen ) , respectively , could be observed . although co oxidation turned out to be unsuccessful , several conclusions on the catalytic activity of au nanoparticles can be drawn from our adsorption / desorption experiments : 1 . the clusters used in the present study were of similar size ( ranging from 7 atoms to diameters of a few nanometers ) as in related studies , where co combustion could be successfully performed ( see for example refs ( 46 ) and references therein ) . furthermore , clusters of monolayer as well as bilayer height were used , with the latter thickness reported to be particularly active for co oxidation . despite these structural similarities , hence , the present study shows that tailoring intrinsic cluster properties , such as small size , high number of undercoordinated au atoms , or suitable thickness ( 2 ml ) , does not suffice to obtain the adsorption behavior required for co oxidation . obviously , the function of the support is not only to provide suitable nucleation sites for generation of well - shaped and anchored au nanoparticles , but in contrast the support has to actively influence the adsorption properties and in turn the catalytic activity . commonly , dissociation of oxygen is considered as the main obstacle in co oxidation . however , with the present material combinations definite evidence for weakly adsorbed co on the gold clusters could not be found . obviously , for the present systems also the co adsorption / desorption behavior might limit the oxidation reaction . as in the present system , co obviously desorbs already below 100 k from the au clusters ; this would require much higher co pressures for successful co2 formation than in other more usual systems . 3 . in all investigated systems dissociation of oxygen occurred but not necessarily always at the au clusters . however , at least for monolayer au clusters on r(15 12)c / w(110 ) the stm images provide strong evidence for oxygen dissociation on or in the immediate vicinity of the au clusters . as an o2 desorption feature characteristic for dissociated oxygen on gold is not observed , we conclude that even if o2 is dissociated at au clusters , the dissociated oxygen atoms spill over to the template , where they are bound strongly and thus are not available for low - temperature reactions . hence , even if adsorption of co on the au clusters was possible , the strong affinity between oxygen atoms and the support would probably lead to a suppression of the catalytic activity . put into more general terms : reductive materials as the presently used c / w templates are not suitable as supports for au - catalyzed co oxidation . hence , we suppose that in order to achieve co oxidation with the present supports , they first have to be heavily oxidized before the final reaction can take place . the in - situ stm investigations revealed considerable differences between mono- and bilayer clusters on c / w(110 ) , indicating oxygen - induced corrosion of the small monolayer - height clusters but not of the larger bilayer particles . nevertheless , tds and tpr studies did not feature strong distinctions between both cluster types . hence , it might also be possible that in both cases oxygen molecules dissociate at the gold clusters , from where the oxygen atoms migrate to the support . we attribute the different stability of the au clusters on the r(15 12 ) and the r(15 3 ) phases to the presence of carbon - poor , clean - tungsten - like adsorption sites underneath the au clusters on the r(15 12 ) surface . as mentioned above , these clean - tungsten - like areas are also favorable adsorption sites for oxygen , leading to a partial displacement of the gold particles on r(15 12 ) . we note that similar oxygen - induced displacement reactions were also observed for silver islands on clean w(110 ) . on the carbon - saturated r(15 accordingly , the driving force for restructuring the gold particles is missing , and the gold clusters remain unaltered . to some extent this parallels electrochemical measurements on wc and w2c films , which show that the carbon - poor w2c is readily oxidized to tungsten oxide , whereas the carbon - richer wc film is stable to higher anode potentials . in contrast to low - temperature co oxidation , which is not possible under our experimental conditions , our study reveals a promoting effect of the deposited gold particles for the high - temperature oxidation of the carburized surfaces toward co. on both templates associative co formation at around 900 k is enhanced by gold , whereas the co desorption features around 1100 k are reduced in intensity . in agreement with that , dissociative co adsorption as the reverse process is also catalyzed by the presence of gold . while on r(15 3 ) this effect can already be observed for small submonolayer gold coverages , on r(15 12 ) larger coverages are required . a tentative explanation for this delayed onset is related to the formation of bilayer au clusters which form already at smallest gold coverages on r(15 3 ) but only at elevated coverages ( roughly around 0.51 ml ) on r(15 12 ) .

adsorption and coadsorption of carbon monoxide and oxygen on different types of au clusters on r(15 3)c / w(110 ) and r(15 12)c / w(110 ) , respectively , are studied with respect to the catalytic behavior for oxidation of co as well as of surface carbon . carburization of the w(110 ) surface results in a weakening of the adsorption bond for molecularly adsorbed co. dissociation of carbon monoxide , which occurs on w(110 ) , is reduced on the low - carbon coverage r(15 12 ) surface and completely suppressed on the carbon - saturated r(15 3 ) phase . deposition of gold results in a blocking of adsorption sites for molecularly adsorbed co and reopening of the dissociation channel . probably the latter is associated with the existence of double - layer gold clusters and islands . at room temperature the gold clusters on both carburized templates are stable in co atmosphere as shown by in - situ stm measurements . in contrast , exposure to oxygen alters the clusters on the r(15 12 ) surface , implying dissociation of oxygen not only on the substrate but also on or in immediate vicinity of the gold clusters . on the au - free carburized templates oxygen adsorbs dissociatively and is released as co at temperatures beyond 800 k due to reaction with carbon atoms from the templates . deposition of gold enhances the desorption rate of the formed co at the low - temperature end of the recombinative co desorption range , indicating a promoting effect of gold for oxidation of surface carbon . in contrast , low - temperature co oxidation catalyzed by the deposited au clusters is not observed . two reasons could be identified : ( 1 ) weakly bound co with desorption temperatures between 100 and 200 k ( as reported for other related systems ) is not observed , and ( 2 ) oxygen atoms are bonded too strongly to the templates .

Introduction Experiment Adsorption of CO on Clean and Carburized W(110) Adsorption of CO on Au-Decorated Carburized W(110) Adsorption of Oxygen Reaction Studies and Conclusions

in the present work we study the adsorption of carbon monoxide and oxygen and their potential reaction on gold clusters grown on a completely different class of template in order to obtain new insights into the role of the support material . recently , it has been shown that carburized w(110 ) surfaces serve as rather universal substrates for the growth of different kinds of noble - metal clusters : the low carbon coverage r(15 12)c / w(110 ) phase is an excellent template for generation of regularly arranged monolayer - height clusters of a rather fixed size ( ca . 7 atoms ) ( see figure 3c ) , while on the high carbon coverage r(15 3)c / w(110 ) template bilayer particles with lateral extensions up to a few nanometers ( depending on au coverage ) are generated ( see figure 3a ) . whereas arguments based on the abundant existence of undercoordinated gold sites or on quantum - size effects due to lateral electron confinement are valid on both insulating and metallic substrates , from other arguments such as charge transfer or reaction at the gold / support interface a different behavior furthermore , use of carburized w(110 ) as template allows growth of well - defined clusters of both monolayer height ( au / r(15 12 ) ) and bilayer height ( au / r(15 3 ) ) on rather similar substrates . in addition as gold clusters are reported to catalyze not only oxidation of carbon monoxide but also other oxidation reactions as well the question will be addressed if the gold clusters are also able to catalyze oxidation of the carburized template itself . in order to test the stability of the cluster systems in a gas atmosphere temperature - programmed desorption ( tpd ) experiments after low - temperature ( 90100 k ) adsorption of oxygen or carbon monoxide were carried out on the templates as well as on the cluster - decorated samples . for the production of well - ordered regular arrays of gold monolayer clusters on r(15 12 ) the sample had to be kept at 600700 k. in contrast , to achieve kinetically controlled growth of nanosized bilayer clusters ( rather than growth of larger ribbon - like multilayer structures ) , gold deposition on r(15 3 ) had to be carried out at room temperature . the spectra taken on clean w(110 ) are in agreement with literature data , showing the low - temperature virgin peak slightly above 400 k and the high - temperature double - peak feature ( ) at 10001100 k. whereas the virgin peak stems from molecularly adsorbed carbon monoxide , the high - temperature features are commonly attributed to co molecules adsorbed dissociatively on w(110 ) , although lee and co - workers recently doubted if co molecules are completely dissociated or only strongly tilted . tpd spectra of co on w(110 ) , r(15 3)c / w(110 ) , and r(15 12)c / w(110 ) . the tpd data for co adsorbed on the r(15 3 ) carbon superstructure differ from that of the clean surface in several respects : ( i ) the low - temperature feature is split into two components , suggesting a coverage - dependent adsorption energy and/or the existence of ( at least ) two different adsorption sites . the latter interpretation seems plausible due to the heterogeneous character of the r(15 3 ) surface ( consisting of c and w atoms ) and the fairly large size of the unit cell ( having a size equivalent to 15 tungsten surface atoms ) . if the features arise from dissociated co , an obvious explanation for their suppression is the blocking of adsorption sites for atomic carbon , since the r(15 3 ) surface is already presaturated with carbon . the tpd spectra from the low carbon - coverage r(15 12 ) surface exhibit a more complicated pattern ( see figure 1 ) with ( at least ) six components : four at low temperatures ( 280420 k ) and two at high temperatures ( 10501150 k ) . we interpret the existence of the pronounced 420 k peak as well as the high - temperature double feature ( which all exhibit peak temperatures similar to clean tungsten ) as an indication of co adsorption on sites that are only weakly perturbed by the presence of carbon . as we have shown recently , the w(110 ) surface is not uniformly covered with r(15 12 ) : wide terraces , exhibiting the r(15 12 ) structure , coexist with smaller terraces being essentially free of carbon . the three low - temperature features ( 370 k and below ) are attributed to co molecules adsorbed in parts of the r(15 12 ) unit cell , where co is more strongly influenced by carbon atoms from the template , resulting in desorption temperatures rather similar to that from the high carbon coverage r(15 3 ) phase . co uptake curves ( at 90 k ) on w(110 ) , r(15 12)c / w(110 ) , and r(15 3)/c / w(110 ) as obtained from the integrated tpd curves . the present tpd measurements of co adsorption on r(15 3 ) are similar to tpd data of co adsorption on c / w(111 ) and c / w(110 ) by hwu et al . , both showing a double - peak desorption structure around 300 k and a strong reduction of the high - temperature desorption feature stemming from dissociated co. in hwu s work on c / w(110 ) an ordered c - induced leed pattern was not observed , probably due to the relatively low flash preparation temperature of 1200 k. however , from the preparation procedure used and the amount of carbon detected by aes , we conclude that the surface should be carbon - rich and hence similar to our ordered r(15 3 ) phase . this behavior is qualitatively similar to that of clean w(110 ) or r(15 12)c / w(110 ) but clearly differs from that observed for the high - carbon coverage r(15 3 ) phase , where dissociation of co is effectively suppressed . this would explain the close similarity to the clean w(110 ) and the low - carbon - coverage r(15 12 ) surface , while the carbon - saturated r(15 3 ) surface behaves differently . in figure 3 , the results of an in - situ stm study of the influence of co on au clusters on r(15 3)c / w(110 ) and r(15 12)c / w(110 ) , respectively , are displayed . as clearly seen from the images , both types of nanoparticles are stable under co exposure at room temperature , irrespective of the type of c / w(110 ) substrate . furthermore , due to the low au coverage on r(15 3 ) , the stm images also demonstrate that the substrate itself visible as faint , protruding lines with an intrinsic height of 0.8 nm is unaffected by co at room temperature . this might have been expected , since co already desorbs around room temperature from the r(15 3 ) surface ( compare figure 1 ) . deposition of au on r(15 3 ) essentially quenches the low - temperature molecular co desorption features located at t 400 k ( see figure 4 ) . stm images of au clusters on r(15 3)c / w(110 ) ( top ) as well as r(15 12)c / w(110 ) ( bottom ) before ( left ) and after ( right ) exposure to co ( b : 75 langmuirs , d : 181 langmuirs ) . as this behavior is not observed , we conclude that the au clusters on r(15 3)c / w(110 ) are not or only marginally able to adsorb weakly bound co molecules . tpd spectra of au / r(15 3)c / w(110 ) after exposure to 10 langmuirs of co. a deposition time of 1 min corresponds to a coverage of 0.10.2 ml . deposition of gold leads to an intensity decrease of the desorption features in the temperature region from 300 to 420 k due to blocking of co adsorption sites . accordingly , we suppose that the evolution of the strong 900 k peak is associated with the formation of double - layer high gold islands . as already observed for the r(15 3 ) template in the low - temperature region below 300 k pronounced au - induced features do not appear except for a weak desorption signal at 120 k. however , since in the series of experiments the desorption intensity at 120 k ( i.e. in any case this indicates that weakly adsorbed co is only present in small amounts if at all on the au clusters on the r(15 12 ) template , which parallels the findings for r(15 3 ) . from these results we can already guess that low - temperature co oxidation on both types of au clusters is unlikely with the present systems . the stability of both carburized templates with and without au clusters against exposure to oxygen atmosphere was investigated by stm movies at room temperature as well as by temperature - programmed desorption experiments after adsorption at 90100 k. in - situ stm study of the influence of oxygen ( p = 5 10 mbar ) on the r(15 3 ) and r(15 12 ) templates covered with au clusters : ( a ) r(15 3 ) , before gas exposure , ( b ) r(15 3 ) after 55 langmuirs of o2 , ( c ) r(15 12 ) , before gas exposure , ( d ) r(15 12 ) after 86 langmuirs of o2 . stm experiments on the bare templates showed a rather good stability for both r(15 3 ) and r(15 12 ) . although the images were somewhat deteriorated ( possibly due to tip instabilities in the presence of oxygen ) and although in particular on the r(15 3 ) surface occasional impurities and hole - like defects appeared with prolonged gas exposure and scanning time , the overall periodicities seemed to be conserved . the evolution of the au - covered r(15 3)c / w(110 ) surface upon o2 exposure is shown in figure 6a , b . by contrast , pronounced oxygen - induced changes occur for the au clusters on the r(15 12 ) template ( see figure 6c , d ) . note that the rather strong oxygen - induced alterations of the gold nanoparticles indicate dissociation of the oxygen molecules on or at least in close vicinity of the au clusters on the r(15 12 ) substrate . the co desorption spectrum from the bare r(15 12 ) surface ( figure 7 ) bears a close resemblance to the high - temperature features observed for co desorption ( after co exposure ) from clean w(110 ) as shown in figure 1 . obviously , oxygen molecules dissociate on the surface and react with carbon atoms from the templates : co is formed and desorbs at high temperatures ( 9001100 k ) . these temperatures are rather similar to that of dissociated -co on clean w(110 ) , thus lending strong support to the interpretation of the desorption peaks as stemming from dissociated co. this observation is in accordance with the findings of viswanath and schmidt that the desorption properties for oxygen and carbon on w(100 ) are essentially identical to those of adsorbed carbon monoxide . addition of gold decreases the intensity of the 2-like peak at 1100 k but increases the intensity of the 1-like feature around 950 k. hence , the presence of au shifts the maximum in the desorption rate by approximately 150 k toward lower temperatures , implying that gold obviously catalyzes the associative reaction of surface carbon with adsorbed oxygen to gaseous carbon monoxide . in contrast , the finite desorption signal at and even above 1200 k can be traced back to the onset of segregation and subsequent oxidation of carbon atoms residing in subsurface regions of the r(15 3 ) phase . the stm investigation on the r(15 12 ) surface even prove that oxygen atoms reside at or in close vicinity to the au clusters . we conclude that either oxygen dissociates only at the template itself or that even if oxygen dissociates on or in close vicinity to the au nanoparticles it subsequently spills over to the c / w(110 ) substrate , where it reacts with the surface carbon and desorbs as co. the modifications of the au clusters induced by exposure to o2 as seen by stm could then be explained by a displacement of au atoms due to the competitive adsorption of dissociated oxygen for ( the same ) favorable adsorption sites . because of the large binding energy of oxygen to tungsten ( adsorption energy 4.20 ev / atom ) , we expect that the carbon - poor regions where the au clusters nucleate are also the most favorable areas for oxygen adsorption , thus providing a strong driving force for an oxygen - induced rearrangement of the noble - metal clusters . however , as the oxygen atoms on w(110 ) are bound rather strongly to the surface , we expect that they are not available for the low - temperature formation of co2 , leading to the conclusion that also for this reason the system despite its ability to dissociate molecular oxygen will not be able to perform co combustion . co oxidation studies were performed , looking for co2 formation in temperature - programmed reaction experiments under various reaction conditions : ( i ) alternating chronological order of exposures , ( ii ) low ( 5 10 mbar ) and high ( 1 10 mbar ) total steady - state background pressures , ( iii ) different o2/co ratios ( 1:1 or oxygen surplus in order to avoid blocking of reactive sites by co ) , and ( iv ) utilizing linear as well as stepwise temperature profiles ( both starting at 90 k ) . furthermore , preadsorption of oxygen at 300 k , followed by heating in co atmosphere ( starting at 90 k ) , was also tested , as stm on the r(15 12 ) revealed that at 300 k oxygen adsorbs dissociatively on or close to the au clusters . however , in none of these experiments could the evolution of co2 due to au - catalyzed co oxidation be verified , neither for bilayer clusters on the r(15 3 ) nor for the small monolayer clusters on the r(15 12 ) template , although the gain of the qms was increased up to 100-fold compared to the co and o2 tpd experiments . the tpd experiments presented in sections 4 and 5 provide a reasonable explanation for this observation : neither weakly bound carbon monoxide molecules nor oxygen atoms attached to gold clusters , with desorption temperatures below 200 k ( co ) or around 500 k ( oxygen ) , respectively , could be observed . obviously , the function of the support is not only to provide suitable nucleation sites for generation of well - shaped and anchored au nanoparticles , but in contrast the support has to actively influence the adsorption properties and in turn the catalytic activity . however , at least for monolayer au clusters on r(15 12)c / w(110 ) the stm images provide strong evidence for oxygen dissociation on or in the immediate vicinity of the au clusters . as an o2 desorption feature characteristic for dissociated oxygen on gold is not observed , we conclude that even if o2 is dissociated at au clusters , the dissociated oxygen atoms spill over to the template , where they are bound strongly and thus are not available for low - temperature reactions . hence , even if adsorption of co on the au clusters was possible , the strong affinity between oxygen atoms and the support would probably lead to a suppression of the catalytic activity . the in - situ stm investigations revealed considerable differences between mono- and bilayer clusters on c / w(110 ) , indicating oxygen - induced corrosion of the small monolayer - height clusters but not of the larger bilayer particles . we attribute the different stability of the au clusters on the r(15 12 ) and the r(15 3 ) phases to the presence of carbon - poor , clean - tungsten - like adsorption sites underneath the au clusters on the r(15 12 ) surface . as mentioned above , these clean - tungsten - like areas are also favorable adsorption sites for oxygen , leading to a partial displacement of the gold particles on r(15 12 ) . on the carbon - saturated r(15 accordingly , the driving force for restructuring the gold particles is missing , and the gold clusters remain unaltered . in contrast to low - temperature co oxidation , which is not possible under our experimental conditions , our study reveals a promoting effect of the deposited gold particles for the high - temperature oxidation of the carburized surfaces toward co. on both templates associative co formation at around 900 k is enhanced by gold , whereas the co desorption features around 1100 k are reduced in intensity . a tentative explanation for this delayed onset is related to the formation of bilayer au clusters which form already at smallest gold coverages on r(15 3 ) but only at elevated coverages ( roughly around 0.51 ml ) on r(15 12 ) .

alzheimer s disease ( ad ) is the most common type of dementia , but a cure for ad has yet to be discovered . however , it may be possible to prevent or delay the onset of ad by reducing modifiable risks . education has been proposed as a protective factor that can reduce the risk of developing dementia . the protective effect of education has been supported by numerous epidemiological , imaging , pathological , and neuropsychological studies [ 13 ] . however , results are not consistent and some conflicting studies have shown no protective effect of education against dementia [ 4 , 5 ] . imaging studies have shown that education has a morphological influence on the brain in both non - demented and ad subjects [ 69 ] . whole brain and ventricular volumes have mainly been used as measures for evaluating the morphological effects of education on brain [ 6 , 7 , 10 ] . recently studies have shown that cortical thickness can give complementary information for evaluating brain morphology and is a promising parameter for early diagnosis of ad [ 11 , 12 ] . with the development of magnetic resonance ( mr ) post - processing technique , it is now possible to quantitatively measure regional cortical thicknesses and volumes in an automated way which minimizes rater - dependent bias . the relationship between education and regional cortical thicknesses and regional volumes of brain structures has not been studied previously in the same population however . the effect of education on brain structures might help our understanding of the mechanism of how education modulates the risk of ad and is of relevance for policy decisions , especially in low and middle - income countries where education provision is limited by restricted resources . the purpose of this study was to explore if regional cortical thickness and volume measures can provide evidence to support the protective effect of education against ad in a large sample from the addneuromed study [ 13 , 14 ] . a total of 121 ad patients ( 50% female ; 74 6 years ) , 121 mild cognitive impairment ( mci ) subjects ( 65% female ; 75 6 years ) , and 113 age - matched healthy controls ( 55% female ; 73 6 years ) were selected from the addneuromed study [ 13 , 14 ] ; a prospective , longitudinal multicenter study to discover biomarkers for ad . data were collected from six medical centers across europe : university of kuopio , finland ; university of perugia , italy ; aristotle university of thessaloniki , greece ; king s college london , united kingdom ; medical university of lodz , poland ; and university of toulouse , france . the length of formal education was 8 4 , 9 4 , and 11 5 years for the ad patients , mci subjects , and controls , respectively . clinical dementia rating scale ( cdr ) and mini - mental state examination ( mmse ) were assessed for each subject . the apoe genotype was determined in 114 ad , 110 mci , and 108 controls . informed consent was obtained for all subjects and protocols and procedures were approved by the relevant institutional review board at each data acquisition site and the data coordination site . none of the mci and ad subjects had other neurological or psychiatric disease , significant unstable systemic illness or organ failure , and alcohol or substance misuse . the diagnosis of dementia was based on the criteria of the diagnostic and statistical manual of mental disorders , 4th edition ( dsm - iv ) and the diagnosis of ad on the national institute of neurologic and communicative disorders and stroke and alzheimer s disease and related disorders association ( nincds - adrda ) criteria . at baseline all mci subjects fulfilled the following criteria , in line with consensus criteria for amnestic mci [ 19 , 20 ] : i.e. , ( 1 ) memory complaint by patient , family , or physician ; ( 2 ) normal activities of daily living ; ( 3 ) normal global cognitive function as measured by the mmse ( score range between 2430 ) ; ( 4 ) geriatric depression scale score less than or equal to 5 ; ( 5 ) subject aged 65 years or above ; ( 6 ) cdr memory score of 0.5 or 1 ; and ( 7 ) absence of dementia according to the dsm - iv and nincds - adrda criteria for ad . the controls did not have any neurological or psychiatric disorders and were not taking any psychoactive medication . classification of the controls and mci subjects was based on cdr score and clinician s judgment , rather than on cognitive tests . data acquisition took place using six different 1.5-t mr systems ( four general electric , one siemens , and one picker ) . at each site a quadrature birdcage coil was used for rf transmission and reception . a high - resolution sagittal 3d t1-weighted mprage volume was acquired using a custom pulse sequence specifically designed for the alzheimer s disease neuroimaging initiative ( adni ) study to ensure compatibility across scanners . the parameters were : tr = 913 ms , te = 3.04.1 ms , ti = 1,000 ms , flip angle = 8 , voxel size detailed quality control was carried out on all mr images using previously published criteria [ 21 , 22 ] . first , mri scans were regularly acquired at each of the sites using a urethane test phantom developed by the us - based adni consortium . this allowed precise quantification of the geometric accuracy of a scan , together with other quality control measures , including signal - to - noise ratio and image uniformity using the imageowl web - based automated quality control system ( http://www.imageowl.com ) to ensure good performance of each of the scanners during the duration of the study . second , two volunteers were scanned at each site , and their images were processed using the same analysis tools as the cohort images to ensure compatibility of data . finally , the mri sites and data coordination center worked closely together with continuous feedback as data were acquired . all mri centers were trained to perform quality control at their site at the time the images were acquired with particular emphasis on full brain coverage , image wrap around , subject motion artifacts , contrast between gray and white matter , and image non uniformities . all scanners remained within specification for the adni phantom throughout the course of the study . the coefficient of variation for whole brain measures for the two volunteers scanned at each of the sites was 1.7% and the mean ( sd ) of the coefficients of variation for the smaller anatomical regions reported in this paper was 3.4 ( 1.9)% . all mr images received a clinical read by an on - site radiologist in order to exclude any subjects with non - ad - related pathologies . the freesurfer structural mri image processing pipeline ( version 4.5.0 ) which was developed by fischl and his colleagues was utilized for data analysis [ 23 , 24 ] . cortical reconstruction and volumetric segmentation included removal of non - brain tissue using a hybrid watershed / surface deformation procedure , automated talairach transformation , segmentation of the subcortical white matter and deep gray matter volumetric structures ( including the hippocampus , amygdala , caudate , putamen , and ventricles ) [ 24 , 26 ] , intensity normalization , tessellation of the gray matter white matter boundary , automated topology correction , and surface deformation following intensity gradients to optimally place the gray / white and gray / cerebrospinal fluid borders at the location where the greatest shift in intensity defines the transition to the other tissue class . surface inflation was followed by registration to a spherical atlas which utilized individual cortical folding patterns to match cortical geometry across subjects and parcellation of the cerebral cortex into units based on gyral and sulcal structure . this method uses both intensity and continuity information from the entire three - dimensional mr volume in segmentation and deformation procedures to produce representations of cortical thickness , calculated as the closest distance from the gray / white boundary to the gray / csf boundary at each vertex on the tessellated surface . the maps are created using spatial - intensity gradients across tissue classes and are therefore not simply reliant on absolute signal intensity . the maps produced are not restricted to the voxel resolution of the original data and are thus capable of detecting submillimeter differences between groups . procedures for the measurement of cortical thickness have been validated against histological analysis and manual measurements [ 33 , 34 ] . these procedures have been demonstrated to show good test retest reliability across scanner manufacturers and across field strengths . a sample of scatter plot from our data shows good comparable across the different centers ( fig . 1 ) . 1years of education was inversely correlated with regional cortical thickness of the inferior parietal gyrus . the data from different countries and scanners were comparable years of education was inversely correlated with regional cortical thickness of the inferior parietal gyrus . the data from different countries and scanners were comparable the cortical thicknesses and regional cortical / subcortical volumes from the automatic processing output were used in the present study , including 33 regional cortical thicknesses and 15 regional volumes . the averaged values of left and right hemispheres were used in the analysis , unless otherwise noted . the correlation between years of schooling and regional cortical thicknesses / volumes was tested with the partial correlation coefficient for each diagnostic group . to allow for the possibility that education may not correlate linearly with regional thickness / volume , we further dichotomized the subjects into more and less educated groups . since the years of compulsory education in the countries from which the subjects was recruited varied from 911 years , and the median of years of schooling in our study population was 9 years , we chose this as a threshold to separate subjects with more and less education . the difference in presence of apoe4 between subjects with more and less education was tested with the chi square test . the analysis of covariance ( ancova ) test was used to compare mri measures between subjects with more and less education . the intracranial volume ( for volume measures , but not for cortical thickness measures ) , country of origin , age , and gender were used as covariates in the partial correlation analysis and ancova . previous studies have shown that higher education is related with better cognitive performance , and that cognitive performance is sensitive to brain atrophy . to control for the possible influence of cognitive performance on regional cortical thickness / volume , mmse was also used as a covariate in the analysis . as the cortical thickness and volumes were measured from a large number of locations , it was necessary to correct for multiple testing . false discovery rate ( fdr ) < 0.05 [ 38 , 39 ] was used to control for type 1 error in the correlation analysis and ancova with the qvalue software package ( http://genomics.princeton.edu/storeylab/qvalue ) . a q value for fdr analysis less than 0.05 was considered as statistically significant . a total of 121 ad patients ( 50% female ; 74 6 years ) , 121 mild cognitive impairment ( mci ) subjects ( 65% female ; 75 6 years ) , and 113 age - matched healthy controls ( 55% female ; 73 6 years ) were selected from the addneuromed study [ 13 , 14 ] ; a prospective , longitudinal multicenter study to discover biomarkers for ad . data were collected from six medical centers across europe : university of kuopio , finland ; university of perugia , italy ; aristotle university of thessaloniki , greece ; king s college london , united kingdom ; medical university of lodz , poland ; and university of toulouse , france . the length of formal education was 8 4 , 9 4 , and 11 5 years for the ad patients , mci subjects , and controls , respectively . clinical dementia rating scale ( cdr ) and mini - mental state examination ( mmse ) were assessed for each subject . the apoe genotype was determined in 114 ad , 110 mci , and 108 controls . informed consent was obtained for all subjects and protocols and procedures were approved by the relevant institutional review board at each data acquisition site and the data coordination site . none of the mci and ad subjects had other neurological or psychiatric disease , significant unstable systemic illness or organ failure , and alcohol or substance misuse . the diagnosis of dementia was based on the criteria of the diagnostic and statistical manual of mental disorders , 4th edition ( dsm - iv ) and the diagnosis of ad on the national institute of neurologic and communicative disorders and stroke and alzheimer s disease and related disorders association ( nincds - adrda ) criteria . at baseline all mci subjects fulfilled the following criteria , in line with consensus criteria for amnestic mci [ 19 , 20 ] : i.e. , ( 1 ) memory complaint by patient , family , or physician ; ( 2 ) normal activities of daily living ; ( 3 ) normal global cognitive function as measured by the mmse ( score range between 2430 ) ; ( 4 ) geriatric depression scale score less than or equal to 5 ; ( 5 ) subject aged 65 years or above ; ( 6 ) cdr memory score of 0.5 or 1 ; and ( 7 ) absence of dementia according to the dsm - iv and nincds - adrda criteria for ad . the controls did not have any neurological or psychiatric disorders and were not taking any psychoactive medication . classification of the controls and mci subjects was based on cdr score and clinician s judgment , rather than on cognitive tests . data acquisition took place using six different 1.5-t mr systems ( four general electric , one siemens , and one picker ) . at each site a quadrature birdcage coil was used for rf transmission and reception . a high - resolution sagittal 3d t1-weighted mprage volume was acquired using a custom pulse sequence specifically designed for the alzheimer s disease neuroimaging initiative ( adni ) study to ensure compatibility across scanners . the parameters were : tr = 913 ms , te = 3.04.1 ms , ti = 1,000 ms , flip angle = 8 , voxel size detailed quality control was carried out on all mr images using previously published criteria [ 21 , 22 ] . first , mri scans were regularly acquired at each of the sites using a urethane test phantom developed by the us - based adni consortium . this allowed precise quantification of the geometric accuracy of a scan , together with other quality control measures , including signal - to - noise ratio and image uniformity using the imageowl web - based automated quality control system ( http://www.imageowl.com ) to ensure good performance of each of the scanners during the duration of the study . second , two volunteers were scanned at each site , and their images were processed using the same analysis tools as the cohort images to ensure compatibility of data . finally , the mri sites and data coordination center worked closely together with continuous feedback as data were acquired . all mri centers were trained to perform quality control at their site at the time the images were acquired with particular emphasis on full brain coverage , image wrap around , subject motion artifacts , contrast between gray and white matter , and image non uniformities . all scanners remained within specification for the adni phantom throughout the course of the study . the coefficient of variation for whole brain measures for the two volunteers scanned at each of the sites was 1.7% and the mean ( sd ) of the coefficients of variation for the smaller anatomical regions reported in this paper was 3.4 ( 1.9)% . all mr images received a clinical read by an on - site radiologist in order to exclude any subjects with non - ad - related pathologies . the freesurfer structural mri image processing pipeline ( version 4.5.0 ) which was developed by fischl and his colleagues was utilized for data analysis [ 23 , 24 ] . cortical reconstruction and volumetric segmentation included removal of non - brain tissue using a hybrid watershed / surface deformation procedure , automated talairach transformation , segmentation of the subcortical white matter and deep gray matter volumetric structures ( including the hippocampus , amygdala , caudate , putamen , and ventricles ) [ 24 , 26 ] , intensity normalization , tessellation of the gray matter white matter boundary , automated topology correction , and surface deformation following intensity gradients to optimally place the gray / white and gray / cerebrospinal fluid borders at the location where the greatest shift in intensity defines the transition to the other tissue class . surface inflation was followed by registration to a spherical atlas which utilized individual cortical folding patterns to match cortical geometry across subjects and parcellation of the cerebral cortex into units based on gyral and sulcal structure . this method uses both intensity and continuity information from the entire three - dimensional mr volume in segmentation and deformation procedures to produce representations of cortical thickness , calculated as the closest distance from the gray / white boundary to the gray / csf boundary at each vertex on the tessellated surface . the maps are created using spatial - intensity gradients across tissue classes and are therefore not simply reliant on absolute signal intensity . the maps produced are not restricted to the voxel resolution of the original data and are thus capable of detecting submillimeter differences between groups . procedures for the measurement of cortical thickness have been validated against histological analysis and manual measurements [ 33 , 34 ] . these procedures have been demonstrated to show good test retest reliability across scanner manufacturers and across field strengths . a sample of scatter plot from our data shows good comparable across the different centers ( fig . 1 ) . 1years of education was inversely correlated with regional cortical thickness of the inferior parietal gyrus . the data from different countries and scanners were comparable years of education was inversely correlated with regional cortical thickness of the inferior parietal gyrus . the data from different countries and scanners were comparable the cortical thicknesses and regional cortical / subcortical volumes from the automatic processing output were used in the present study , including 33 regional cortical thicknesses and 15 regional volumes . the averaged values of left and right hemispheres were used in the analysis , unless otherwise noted . the correlation between years of schooling and regional cortical thicknesses / volumes was tested with the partial correlation coefficient for each diagnostic group . to allow for the possibility that education may not correlate linearly with regional thickness / volume , we further dichotomized the subjects into more and less educated groups . since the years of compulsory education in the countries from which the subjects was recruited varied from 911 years , and the median of years of schooling in our study population was 9 years , we chose this as a threshold to separate subjects with more and less education . the difference in presence of apoe4 between subjects with more and less education was tested with the chi square test . the analysis of covariance ( ancova ) test was used to compare mri measures between subjects with more and less education . the intracranial volume ( for volume measures , but not for cortical thickness measures ) , country of origin , age , and gender were used as covariates in the partial correlation analysis and ancova . previous studies have shown that higher education is related with better cognitive performance , and that cognitive performance is sensitive to brain atrophy . to control for the possible influence of cognitive performance on regional cortical thickness / volume , mmse was also used as a covariate in the analysis . as the cortical thickness and volumes were measured from a large number of locations , it was necessary to correct for multiple testing . false discovery rate ( fdr ) < 0.05 [ 38 , 39 ] was used to control for type 1 error in the correlation analysis and ancova with the qvalue software package ( http://genomics.princeton.edu/storeylab/qvalue ) . a q value for fdr analysis less than 0.05 was considered as statistically significant . the partial correlation coefficients between years of schooling and regional cortical thicknesses and regional volumes are summarized in table 2 , and the regional cortical thicknesses and regional volumes of subjects with more and less education for each group are summarized in table 3 . there were no significant differences in the presence of apoe4 between subjects with more and less education ( p values 0.57 , 0.72 , and 0.12 in ad , mci , and control groups , respectively).table 1demographics and cognitive test results of the ad , mci , and control subjectscontrol ( n = 113)mci ( n = 121)ad ( n = 121)age73 674 675 6female / male ( % female)62/51 ( 55%)60/61 ( 50%)79/42 ( 65%)education years ; mean sd ( range)11 5 ( 225)9 4 ( 020)8 4 ( 022)apoe4 carrier / non - carrier32/7640/7064/50cdr00.51.2 0.5mmse29 127 221 5table 2correlation between years of schooling and regional cortical thicknesses and volumes after controlling for intracranial volume , age , gender , mmse , and country of origincontrolmciadrpqrpqrpqregional cortical thicknessentorhinal cortex0.1080.2660.4880.0850.3670.7320.1790.0540.089parahippocampal gyrus0.0670.4890.6000.0330.7230.8210.0960.3070.327middle temporal gyrus0.1650.0880.3480.1610.0840.6820.2830.0020.022superior temporal gyrus0.1080.2660.4880.1240.1840.6820.2620.0050.041inferior temporal gyrus0.0670.4890.6000.0780.4020.7320.2370.0100.046temporal pole0.1650.0880.3480.0460.6250.7930.1900.0410.087transverse temporal cortex0.0200.8390.9060.1690.0700.6820.1560.0940.124insula0.0670.4910.6000.0610.5170.7320.1490.1100.140fusiform gyrus0.1450.1330.3660.0001.0001.0000.2270.0140.046medial orbitofrontal cortex0.2210.0210.2060.0550.5610.7410.1650.0770.110lateral orbitofrontal cortex0.2690.0050.1650.0270.7710.8210.1440.1220.146orbital operculum0.2150.0250.2060.1240.1840.6820.1660.0760.110frontal operculum0.1330.1700.3830.1150.2200.7260.1830.0500.087superior frontal gyrus0.0500.6110.7200.0710.4500.7320.2080.0250.069pars triangularis0.1510.1190.3570.0700.4530.7320.1090.2430.267frontal pole0.1620.0950.3480.0290.7530.8210.0430.6440.664rostral middle frontal gyrus0.0070.9390.9520.1460.1170.6820.1970.0340.080caudal middle frontal gyrus0.2310.0160.2060.1000.2860.7260.2340.0120.046precentral gyrus0.0750.4430.6000.1240.1860.6820.1820.0500.087superior parietal gyrus0.1620.0930.3480.0360.7040.8210.2290.0130.046post central gyrus0.1540.1110.3570.1900.0410.6820.1440.1240.146paracentral gyrus0.0410.6710.7640.0300.7500.8210.1880.0440.087inferior parietal cortex0.0740.4440.6000.0750.4230.7320.2940.0010.017supramarginal gyrus0.0750.4380.6000.1010.2800.7260.1850.0470.087pericalcarine cortex0.0060.9520.9520.1780.0560.6820.2310.0130.046precuneus cortex0.1320.1740.3830.0790.3990.7320.2300.0130.046lingual gyrus0.1040.2860.49670.0120.8960.9240.1590.0870.120cuneus cortex0.0180.8510.9060.0810.3860.7320.2140.0210.063lateral occipital cortex0.0980.3130.5160.0630.5010.7320.2970.0010.017isthmus of cingulate cortex0.1370.1570.3830.1060.2580.7260.1730.0640.101caudal anterior cingulate cortex0.1080.2660.4880.0670.4770.7320.0210.8200.820rostral anterior cingulate0.0670.4890.6000.0590.5320.7320.1120.2300.262posterior cingulate cortex0.1650.0880.3480.1410.1300.6820.2030.0290.074regional volumehippocampus0.0820.4000.7500.0810.3870.8930.0950.3120.736amygdala0.0340.7270.8390.0520.5760.8930.0800.3960.736caudate0.1150.2370.7500.0630.5000.8930.0380.6860.736accumbens0.0900.3550.7500.0730.4330.8930.0650.4860.736putamen0.0390.6850.8390.0130.8930.8930.0320.7330.736pallidum0.0370.7050.8390.1430.1240.8930.0880.3500.736brain stem0.0990.3100.7500.1210.1960.8930.0320.7360.736cerebral cortex0.0510.6010.8390.0240.7970.8930.0810.3870.736cerebellum cortex0.0560.5680.8390.0850.3620.8930.1470.1160.736thalamus0.1770.0660.5180.0910.3290.8930.0340.7210.736corpus callosum anterior0.1150.2350.7500.0290.7600.8930.0430.6440.736corpus callosum central0.0160.8660.9080.0320.7330.8930.0410.6650.736corpus callosum mid - anterior0.1760.0690.5180.1380.1400.8930.0470.6180.736corpus callosum mid - posterior0.1010.2980.7500.0160.8660.8930.0520.5810.736corpus callosum posterior0.0110.9080.9080.0430.6430.8930.0620.5060.736indicates significant correlation ( false discovery rate < 0.05)table 3regional cortical thicknesses and absolute regional volumes of more and less educated subjects in ad , mci , and control groupscontrolmciadless educated ( n = 41)more educated ( n = 72)less educated ( n = 69)more educated ( n = 52)less educated ( n = 77)more educated ( n = 44)regional cortical thickness ( mm)entorhinal cortex3.3 0.53.4 0.33.0 0.53.1 0.52.7 0.52.7 0.5parahippocampal gyrus2.5 0.32.6 0.32.4 0.32.4 0.32.2 0.32.2 0.3middle temporal gyrus2.7 0.22.7 0.22.6 0.22.7 0.22.5 0.32.4 0.3superior temporal gyrus2.5 0.22.5 0.12.4 0.22.5 0.22.3 0.22.2 0.2inferior temporal gyrus2.7 0.22.7 0.12.6 0.22.7 0.22.5 0.32.4 0.3temporal pole3.5 0.33.6 0.23.3 0.43.4 0.43.1 0.53.1 0.5transverse temporal cortex2.0 0.22.1 0.21.9 0.22.0 0.31.9 0.31.8 0.3insula2.8 0.22.9 0.22.7 0.22.8 0.22.6 0.32.6 0.2fusiform gyrus2.5 0.22.5 0.22.4 0.22.5 0.22.3 0.32.3 0.2medial orbitofrontal cortex2.3 0.22.3 0.22.3 0.22.3 0.22.1 0.32.1 0.2lateral orbitofrontal cortex2.5 0.12.5 0.12.4 0.22.4 0.22.3 0.22.3 0.2orbital operculum2.5 0.22.6 0.22.5 0.22.5 0.22.4 0.22.4 0.2frontal operculum2.3 0.22.3 0.22.2 0.22.3 0.22.2 0.32.2 0.2superior frontal gyrus2.5 0.22.5 0.22.4 0.22.5 0.22.4 0.22.3 0.2pars triangularis2.2 0.12.2 0.22.2 0.22.2 0.22.1 0.22.1 0.2frontal pole2.6 0.22.7 0.32.6 0.22.6 0.32.5 0.32.5 0.3rostral middle frontal gyrus2.1 0.12.2 0.12.1 0.12.2 0.22.1 0.32.0 0.2caudal middle frontal gyrus2.3 0.22.3 0.22.2 gyrus2.2 0.22.2 0.22.1 0.22.2 0.22.1 0.42.0 0.2superior parietal gyrus2.0 0.12.0 0.21.9 0.22.0 0.21.9 0.41.8 0.2post central gyrus1.8 0.21.8 0.11.7 0.11.8 0.11.7 0.31.7 0.2paracentral gyrus2.1 0.22.1 0.22.0 0.22.0 0.31.9 0.2inferior parietal cortex2.2 0.22.3 0.22.2 0.22.2 0.22.1 0.22.0 0.2supramarginal gyrus2.3 0.22.3 0.22.2 0.22.2 0.22.1 0.32.1 0.2pericalcarine cortex1.4 0.11.4 0.11.4 0.11.4 0.11.4 0.11.3 0.1precuneus cortex2.0 0.22.1 0.22.0 0.12.0 0.21.9 0.31.9 0.2lingual gyrus1.7 0.11.8 0.11.7 0.11.8 0.11.7 0.21.7 0.1cuneus cortex1.6 0.11.7 0.11.6 0.11.7 0.11.7 0.21.6 0.1lateral occipital cortex2.0 0.12.0 0.12.0 0.12.0 0.12.0 0.21.9 0.2isthmus of cingulate cortex2.3 0.22.2 0.32.1 0.3caudal anterior cingulate cortex2.6 0.22.6 0.22.6 0.32.6 0.22.5 0.32.5 0.3rostral anterior cingulate2.8 0.22.9 0.22.8 0.32.8 0.22.7 0.32.7 0.3posterior cingulate cortex2.3 0.22.4 0.22.3 0.12.3 0.22.2 0.22.2 0.2regional volume ( ml)hippocampus3.5 0.53.6 0.43.2 0.63.3 0.52.6 0.52.6 0.4amygdala1.4 0.21.4 0.21.2 0.31.2 0.31.0 0.21.0 0.2caudate3.3 0.63.3 0.53.3 0.53.3 0.63.3 0.73.2 0.7accumbens0.5 0.10.5 0.10.4 0.10.5 0.10.4 0.10.4 0.1putamen4.6 0.54.7 0.54.7 0.64.5 1.14.5 0.64.5 0.9pallidum1.5 0.21.5 0.21.5 0.21.5 0.21.4 0.41.4 0.2brain stem19.4 2.319.8 1.919.4 2.219.6 2.618.9 3.118.8 2.1cerebral cortex198.1 22.4198.9 16.1192.5 18.7196.2 18.3179.5 21.7180.8 22.7cerebellum cortex45.6 5.446.2 4.545.6 5.346.1 5.344.2 6.445.6 5.8thalamus5.9 0.75.9 0.55.8 0.65.9 0.76.0 2.7.5.7 0.7corpus callosum anterior0.8 0.10.8 0.10.7 0.10.8 0.10.8 0.40.7 0.1corpus callosum central0.3 0.10.3 0.10.3 0.10.4 0.10.4 0.70.3 0.1corpus callosum mid - anterior0.3 0.10.4 0.10.3 0.10.4 0.10.5 0.90.3 0.1corpus callosum mid - posterior0.3 0.10.3 0.10.3 0.10.3 0.10.3 0.10.8 0.10.8 0.10.9 0.10.8 0.20.8 0.1indicates significant difference ( fdr < 0.05 ) between more and less educated subjects after adjusting for age , gender , country of origin , and mmse . the intracranial volume was also adjusted for volumes demographics and cognitive test results of the ad , mci , and control subjects correlation between years of schooling and regional cortical thicknesses and volumes after controlling for intracranial volume , age , gender , mmse , and country of origin indicates significant correlation ( false discovery rate < 0.05 ) regional cortical thicknesses and absolute regional volumes of more and less educated subjects in ad , mci , and control groups indicates significant difference ( fdr < 0.05 ) between more and less educated subjects after adjusting for age , gender , country of origin , and mmse . the intracranial volume was also adjusted for volumes after adjusting for confounders and multiple testing , no significant correlation between years of schooling and regional cortical thicknesses / volumes was found . however , when the controls were dichotomized into more and less educated groups ( years of schooling 14 3 vs. 6 2 years ) , the thicknesses of the temporal pole , transverse temporal gyrus , and isthmus of cingulate cortex were significantly larger in the subjects with more education than in those with less education ( fig . 2the healthy controls with more education had greater cortical thickness than those with less education at the transverse temporal cortex , isthmus cingulate , and insula the healthy controls with more education had greater cortical thickness than those with less education at the transverse temporal cortex , isthmus cingulate , and insula after adjusting for confounders and multiple testing , no significant correlation between years of schooling and regional cortical thicknesses / volumes was found . the regional cortical thicknesses / volumes did not differ significantly between the subjects with more education than those with less education ( years of schooling 13 3 vs. 6 2 years ) . after controlling for confounders and multiple testing , in the ad group , the years of schooling were inversely correlated with regional cortical thicknesses of the inferior , middle and superior temporal gyri , fusiform gyrus , caudal middle frontal gyrus , inferior and superior parietal gyri , pericalcarine cortex , precuneus cortex , and lateral occipital cortex . we further separately analyzed the correlation between years of schooling and regional cortical thickness for the left and right hemispheres . the regional cortex was significantly thinner in the inferior , superior , and middle temporal gyri , superior and inferior parietal gyri , and lateral occipital cortex in the patients with more education than those with less education ( years of schooling 12 3 vs. 5 2 years).fig . 3after controlling for confounders and multiple testing , in the ad group , the years of schooling were inversely correlated with regional cortical thicknesses of the bilateral lateral occipital cortex , middle and superior temporal gyri , left fusiform gyrus , and right caudal middle and superior frontal gyri , inferior and superior parietal gyri , inferior temporal gyrus , posterior cingulate cortex , and precuneus cortex after controlling for confounders and multiple testing , in the ad group , the years of schooling were inversely correlated with regional cortical thicknesses of the bilateral lateral occipital cortex , middle and superior temporal gyri , left fusiform gyrus , and right caudal middle and superior frontal gyri , inferior and superior parietal gyri , inferior temporal gyrus , posterior cingulate cortex , and precuneus cortex after adjusting for confounders and multiple testing , no significant correlation between years of schooling and regional cortical thicknesses / volumes was found . however , when the controls were dichotomized into more and less educated groups ( years of schooling 14 3 vs. 6 2 years ) , the thicknesses of the temporal pole , transverse temporal gyrus , and isthmus of cingulate cortex were significantly larger in the subjects with more education than in those with less education ( fig . 2).fig . 2the healthy controls with more education had greater cortical thickness than those with less education at the transverse temporal cortex , isthmus cingulate , and insula the healthy controls with more education had greater cortical thickness than those with less education at the transverse temporal cortex , isthmus cingulate , and insula after adjusting for confounders and multiple testing , no significant correlation between years of schooling and regional cortical thicknesses / volumes was found . the regional cortical thicknesses / volumes did not differ significantly between the subjects with more education than those with less education ( years of schooling 13 3 vs. 6 2 years ) . after controlling for confounders and multiple testing , in the ad group , the years of schooling were inversely correlated with regional cortical thicknesses of the inferior , middle and superior temporal gyri , fusiform gyrus , caudal middle frontal gyrus , inferior and superior parietal gyri , pericalcarine cortex , precuneus cortex , and lateral occipital cortex . we further separately analyzed the correlation between years of schooling and regional cortical thickness for the left and right hemispheres . the regional cortex was significantly thinner in the inferior , superior , and middle temporal gyri , superior and inferior parietal gyri , and lateral occipital cortex in the patients with more education than those with less education ( years of schooling 12 3 vs. 5 2 years).fig . 3after controlling for confounders and multiple testing , in the ad group , the years of schooling were inversely correlated with regional cortical thicknesses of the bilateral lateral occipital cortex , middle and superior temporal gyri , left fusiform gyrus , and right caudal middle and superior frontal gyri , inferior and superior parietal gyri , inferior temporal gyrus , posterior cingulate cortex , and precuneus cortex after controlling for confounders and multiple testing , in the ad group , the years of schooling were inversely correlated with regional cortical thicknesses of the bilateral lateral occipital cortex , middle and superior temporal gyri , left fusiform gyrus , and right caudal middle and superior frontal gyri , inferior and superior parietal gyri , inferior temporal gyrus , posterior cingulate cortex , and precuneus cortex two kinds of reserve against brain damage have been advocated : brain reserve and cognitive reserve . the concept of brain reserve was first introduced by roth , tomlinson , and blessed and was elaborated upon by satz . it refers to individuals with larger brain volumes who can sustain brain damage before reaching a threshold for clinical symptoms . cognitive reserve refers to the ability of the brain to compensate for brain damage by using preexisting cognitive processing approaches or recruiting compensatory approaches . to date the concept of brain reserve has received very limited support ( for review see ) . it is likely that both brain and cognitive reserves are involved in providing reserve against brain damage and cognitive decline . one of the most interesting finding in the present study is that after adjusting for cognitive performance ( mmse ) , i.e. , at similar cognitive performance , some areas of regional cortex were significantly thinner in ad patients with high education than those with less education ( regional cortical thicknesses of middle and superior temporal gyri , inferior parietal cortex , and lateral occipital cortex ) . this implies that the ad patients with more education have better ability than the patients with less education to compensate for the effects of atrophy . this finding strongly supports the concept of an increased cognitive reserve in the subjects with more education . the thinner regional cortex in ad patients with more education might be one reason to explain why deterioration progresses more rapidly in ad patients with more education . this finding is supported by a diffusion tensor imaging study which showed that years of schooling are associated with reduced white matter integrity of media temporal lobe and is in line with the study of querbes et al . who found that people with more education have a thinner cortex than people with less education . the finding that ad patients had smaller regional cortical thicknesses is further supported by a recent study which showed that education is associated with cortical thinning in the frontal , temporal , and parietal association cortexes ; precuneus ; medial , frontal , and basal temporal regions ; orbitofrontal ; and cuneus . however , morphological influence of education on the brain in non - demented people is controversial in the literature . in previous volumetric studies , non - demented people with more education or better socioeconomic status have been shown to have more atrophy [ 6 , 7 ] . piras et al . reported that years of schooling are significantly positively correlated with regional brain volumes and inversely correlated with mean diffusivity of several deep gray matter structures , indicating larger regional volumes and higher level of neural reserve in non - demented people . a recent study showed that people with high early life iq had larger brains than people with low iq in the aged healthy group . in our study , after controlling for confounders and multiple testing , there was no significant correlation between years of schooling and regional cortical thicknesses / volumes in the healthy control group , which is supported by a recent study which showed no effect of education on regional cortical thickness in a linear regression analysis in healthy people . however , when dichotomizing the controls into more and less educated groups with a cutoff of 9 years of schooling , the thicknesses of the temporal pole , transverse temporal gyrus , and isthmus of cingulate cortex were significantly larger in the subjects with more education than those with less education . it is interesting that the healthy controls with more education had larger regional cortical thicknesses than those with less education after controlling for confounding factors , but that they did not show any significant correlation between regional cortical thicknesses and years of schooling . however , this potential nonlinear effect should be replicated in a population with the same education system to be truly robust . in the present study , the controls with more education had thicker regional cortices , but there was no significant difference in the mci subjects . in ad patients , a recent study showed that in non - demented people , higher socioeconomic status is associated with more rapid brain volume loss in those who developed dementia later . this might account for the different finding in our diagnostic groups . it is notable that there were positive findings for regional cortical thickness measures , but a lack of positive findings for regional volume measures . in previous studies , a significant correlation was found between education and whole brain volume or volume of peripheral cerebrospinal fluid [ 6 , 7 ] . however , in the study of coffey et al . , there was no significant correlation between regional volumes and education . in a recent study , seo et al . had similar findings to ours using regional cortical thicknesses as a morphological proxy . it thus seems that regional cortical thickness is a better parameter for studying brain and cognitive reserve than brain volume , especially when results are divided into subregions of the brain . the frequency of presence of apoe4 was different among the three groups and it is important to bear in mind that apoe4 genotype is a significant contribution to cortical thinning in ad and mci . the difference in the frequency of the presence of apoe4 might confound the results of the different groups . we do not have regional cortical thicknesses / volumes at the subject s schooling ages , so it is not possible to exclude the presence of selection bias due to individual difference in innate brain volume . therefore , the finding that healthy aged people with more education have larger regional cortices needs to be confirmed . using 9 years as a cutoff value to dichotomize less or more educated subjects might be one reason for the discrepancy between the results of the correlation analysis and group comparison . we did not control for cardiovascular disease and cardiovascular risk factors ( hypertension and diabetes ) , occupational attainment , and unhealthy life - styles . studies have demonstrated that each of these confounding factors independently contributes to reserve [ 3 , 45 , 47 , 48 ] . the mmse has very low variance and ceiling effects in control subjects might affect the correlation analysis in the control group . education appears to have a differential impact on cortical thickness in healthy controls and ad patients . it may increase regional cortical thickness in healthy controls , leading to increased brain reserve , as well as helping ad patients to cope better with the effects of brain atrophy by increasing cognitive reserve . this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .

introductionthe aim of this study was to determine whether years of schooling influences regional cortical thicknesses and volumes in alzheimer s disease ( ad ) , mild cognitive impairment ( mci ) , and healthy age - matched controls.methodsusing an automated image analysis pipeline , 33 regional cortical thickness and 15 regional volumes measures from mri images were determined in 121 subjects with mci , 121 patients with ad , and 113 controls from addneuromed study . correlations with years of schooling were determined and more highly and less highly educated subjects compared , controlling for intracranial volume , age , gender , country of origin , cognitive status , and multiple testing.resultsafter controlling for confounding factors and multiple testing , in the control group , subjects with more education had larger regional cortical thickness in transverse temporal cortex , insula , and isthmus of cingulate cortex than subjects with less education . however , in the ad group , the subjects with more education had smaller regional cortical thickness in temporal gyrus , inferior and superior parietal gyri , and lateral occipital cortex than the subjects with less education . no significant difference was found in the mci group.conclusioneducation may increase regional cortical thickness in healthy controls , leading to increased brain reserve , as well as helping ad patients to cope better with the effects of brain atrophy by increasing cognitive reserve .

Introduction Methods Subjects Data acquisition Image analysis Statistical analysis Results Control group MCI group AD group Discussion Conclusion Conflict of interest Open Access

there were no significant differences in the presence of apoe4 between subjects with more and less education ( p values 0.57 , 0.72 , and 0.12 in ad , mci , and control groups , respectively).table 1demographics and cognitive test results of the ad , mci , and control subjectscontrol ( n = 113)mci ( n = 121)ad ( n = 121)age73 674 675 6female / male ( % female)62/51 ( 55%)60/61 ( 50%)79/42 ( 65%)education years ; mean sd ( range)11 5 ( 225)9 4 ( 020)8 4 ( 022)apoe4 carrier / non - carrier32/7640/7064/50cdr00.51.2 0.5mmse29 127 221 5table 2correlation between years of schooling and regional cortical thicknesses and volumes after controlling for intracranial volume , age , gender , mmse , and country of origincontrolmciadrpqrpqrpqregional cortical thicknessentorhinal cortex0.1080.2660.4880.0850.3670.7320.1790.0540.089parahippocampal gyrus0.0670.4890.6000.0330.7230.8210.0960.3070.327middle temporal gyrus0.1650.0880.3480.1610.0840.6820.2830.0020.022superior temporal gyrus0.1080.2660.4880.1240.1840.6820.2620.0050.041inferior temporal gyrus0.0670.4890.6000.0780.4020.7320.2370.0100.046temporal pole0.1650.0880.3480.0460.6250.7930.1900.0410.087transverse temporal cortex0.0200.8390.9060.1690.0700.6820.1560.0940.124insula0.0670.4910.6000.0610.5170.7320.1490.1100.140fusiform gyrus0.1450.1330.3660.0001.0001.0000.2270.0140.046medial orbitofrontal cortex0.2210.0210.2060.0550.5610.7410.1650.0770.110lateral orbitofrontal cortex0.2690.0050.1650.0270.7710.8210.1440.1220.146orbital operculum0.2150.0250.2060.1240.1840.6820.1660.0760.110frontal operculum0.1330.1700.3830.1150.2200.7260.1830.0500.087superior frontal gyrus0.0500.6110.7200.0710.4500.7320.2080.0250.069pars triangularis0.1510.1190.3570.0700.4530.7320.1090.2430.267frontal pole0.1620.0950.3480.0290.7530.8210.0430.6440.664rostral middle frontal gyrus0.0070.9390.9520.1460.1170.6820.1970.0340.080caudal middle frontal gyrus0.2310.0160.2060.1000.2860.7260.2340.0120.046precentral gyrus0.0750.4430.6000.1240.1860.6820.1820.0500.087superior parietal gyrus0.1620.0930.3480.0360.7040.8210.2290.0130.046post central gyrus0.1540.1110.3570.1900.0410.6820.1440.1240.146paracentral gyrus0.0410.6710.7640.0300.7500.8210.1880.0440.087inferior parietal cortex0.0740.4440.6000.0750.4230.7320.2940.0010.017supramarginal gyrus0.0750.4380.6000.1010.2800.7260.1850.0470.087pericalcarine cortex0.0060.9520.9520.1780.0560.6820.2310.0130.046precuneus cortex0.1320.1740.3830.0790.3990.7320.2300.0130.046lingual gyrus0.1040.2860.49670.0120.8960.9240.1590.0870.120cuneus cortex0.0180.8510.9060.0810.3860.7320.2140.0210.063lateral occipital cortex0.0980.3130.5160.0630.5010.7320.2970.0010.017isthmus of cingulate cortex0.1370.1570.3830.1060.2580.7260.1730.0640.101caudal anterior cingulate cortex0.1080.2660.4880.0670.4770.7320.0210.8200.820rostral anterior cingulate0.0670.4890.6000.0590.5320.7320.1120.2300.262posterior cingulate cortex0.1650.0880.3480.1410.1300.6820.2030.0290.074regional volumehippocampus0.0820.4000.7500.0810.3870.8930.0950.3120.736amygdala0.0340.7270.8390.0520.5760.8930.0800.3960.736caudate0.1150.2370.7500.0630.5000.8930.0380.6860.736accumbens0.0900.3550.7500.0730.4330.8930.0650.4860.736putamen0.0390.6850.8390.0130.8930.8930.0320.7330.736pallidum0.0370.7050.8390.1430.1240.8930.0880.3500.736brain stem0.0990.3100.7500.1210.1960.8930.0320.7360.736cerebral cortex0.0510.6010.8390.0240.7970.8930.0810.3870.736cerebellum cortex0.0560.5680.8390.0850.3620.8930.1470.1160.736thalamus0.1770.0660.5180.0910.3290.8930.0340.7210.736corpus callosum anterior0.1150.2350.7500.0290.7600.8930.0430.6440.736corpus callosum central0.0160.8660.9080.0320.7330.8930.0410.6650.736corpus callosum mid - anterior0.1760.0690.5180.1380.1400.8930.0470.6180.736corpus callosum mid - posterior0.1010.2980.7500.0160.8660.8930.0520.5810.736corpus callosum posterior0.0110.9080.9080.0430.6430.8930.0620.5060.736indicates significant correlation ( false discovery rate < 0.05)table 3regional cortical thicknesses and absolute regional volumes of more and less educated subjects in ad , mci , and control groupscontrolmciadless educated ( n = 41)more educated ( n = 72)less educated ( n = 69)more educated ( n = 52)less educated ( n = 77)more educated ( n = 44)regional cortical thickness ( mm)entorhinal cortex3.3 0.53.4 0.33.0 0.53.1 0.52.7 0.52.7 0.5parahippocampal gyrus2.5 0.32.6 0.32.4 0.32.4 0.32.2 0.32.2 0.3middle temporal gyrus2.7 0.22.7 0.22.6 0.22.7 0.22.5 0.32.4 0.3superior temporal gyrus2.5 0.22.5 0.12.4 0.22.5 0.22.3 0.22.2 0.2inferior temporal gyrus2.7 0.22.7 0.12.6 0.22.7 0.22.5 0.32.4 0.3temporal pole3.5 0.33.6 0.23.3 0.43.4 0.43.1 0.53.1 0.5transverse temporal cortex2.0 0.22.1 0.21.9 0.22.0 0.31.9 0.31.8 0.3insula2.8 0.22.9 0.22.7 0.22.8 0.22.6 0.32.6 0.2fusiform gyrus2.5 0.22.5 0.22.4 0.22.5 0.22.3 0.32.3 0.2medial orbitofrontal cortex2.3 0.22.3 0.22.3 0.22.3 0.22.1 0.32.1 0.2lateral orbitofrontal cortex2.5 0.12.5 0.12.4 0.22.4 0.22.3 0.22.3 0.2orbital operculum2.5 0.22.6 0.22.5 0.22.5 0.22.4 0.22.4 0.2frontal operculum2.3 0.22.3 0.22.2 0.22.3 0.22.2 0.32.2 0.2superior frontal gyrus2.5 0.22.5 0.22.4 0.22.5 0.22.4 0.22.3 0.2pars triangularis2.2 0.12.2 0.22.2 0.22.2 0.22.1 0.22.1 0.2frontal pole2.6 0.22.7 0.32.6 0.22.6 0.32.5 0.32.5 0.3rostral middle frontal gyrus2.1 0.12.2 0.12.1 0.12.2 0.22.1 0.32.0 0.2caudal middle frontal gyrus2.3 0.22.3 0.22.2 gyrus2.2 0.22.2 0.22.1 0.22.2 0.22.1 0.42.0 0.2superior parietal gyrus2.0 0.12.0 0.21.9 0.22.0 0.21.9 0.41.8 0.2post central gyrus1.8 0.21.8 0.11.7 0.11.8 0.11.7 0.31.7 0.2paracentral gyrus2.1 0.22.1 0.22.0 0.22.0 0.31.9 0.2inferior parietal cortex2.2 0.22.3 0.22.2 0.22.2 0.22.1 0.22.0 0.2supramarginal gyrus2.3 0.22.3 0.22.2 0.22.2 0.22.1 0.32.1 0.2pericalcarine cortex1.4 0.11.4 0.11.4 0.11.4 0.11.4 0.11.3 0.1precuneus cortex2.0 0.22.1 0.22.0 0.12.0 0.21.9 0.31.9 0.2lingual gyrus1.7 0.11.8 0.11.7 0.11.8 0.11.7 0.21.7 0.1cuneus cortex1.6 0.11.7 0.11.6 0.11.7 0.11.7 0.21.6 0.1lateral occipital cortex2.0 0.12.0 0.12.0 0.12.0 0.12.0 0.21.9 0.2isthmus of cingulate cortex2.3 0.22.2 0.32.1 0.3caudal anterior cingulate cortex2.6 0.22.6 0.22.6 0.32.6 0.22.5 0.32.5 0.3rostral anterior cingulate2.8 0.22.9 0.22.8 0.32.8 0.22.7 0.32.7 0.3posterior cingulate cortex2.3 0.22.4 0.22.3 0.12.3 0.22.2 0.22.2 0.2regional volume ( ml)hippocampus3.5 0.53.6 0.43.2 0.63.3 0.52.6 0.52.6 0.4amygdala1.4 0.21.4 0.21.2 0.31.2 0.31.0 0.21.0 0.2caudate3.3 0.63.3 0.53.3 0.53.3 0.63.3 0.73.2 0.7accumbens0.5 0.10.5 0.10.4 0.10.5 0.10.4 0.10.4 0.1putamen4.6 0.54.7 0.54.7 0.64.5 1.14.5 0.64.5 0.9pallidum1.5 0.21.5 0.21.5 0.21.5 0.21.4 0.41.4 0.2brain stem19.4 2.319.8 1.919.4 2.219.6 2.618.9 3.118.8 2.1cerebral cortex198.1 22.4198.9 16.1192.5 18.7196.2 18.3179.5 21.7180.8 22.7cerebellum cortex45.6 5.446.2 4.545.6 5.346.1 5.344.2 6.445.6 5.8thalamus5.9 0.75.9 0.55.8 0.65.9 0.76.0 2.7.5.7 0.7corpus callosum anterior0.8 0.10.8 0.10.7 0.10.8 0.10.8 0.40.7 0.1corpus callosum central0.3 0.10.3 0.10.3 0.10.4 0.10.4 0.70.3 0.1corpus callosum mid - anterior0.3 0.10.4 0.10.3 0.10.4 0.10.5 0.90.3 0.1corpus callosum mid - posterior0.3 0.10.3 0.10.3 0.10.3 0.10.3 0.10.8 0.10.8 0.10.9 0.10.8 0.20.8 0.1indicates significant difference ( fdr < 0.05 ) between more and less educated subjects after adjusting for age , gender , country of origin , and mmse . it may increase regional cortical thickness in healthy controls , leading to increased brain reserve , as well as helping ad patients to cope better with the effects of brain atrophy by increasing cognitive reserve .

the impact of nutrition during development is recognized as an important underlying risk factor for an individual s susceptibility to disease in later life . studies have consistently demonstrated that altering the nutrient supply of pregnant animals induces profound changes in the function of body systems , including raised blood pressure , fewer kidney nephrons , and impaired pancreatic -cell development ( 14 ) . in humans , much of the evidence has focused on the inverse associations observed between birth weight and a range of chronic diseases in later life , including hypertension and diabetes ( 5 , 6 ) . these inverse associations are often interpreted as revealing the importance of nutrient supply to the developing fetus , although many other factors may underlie impaired uterine growth retardation and subsequent disease susceptibility . a range of factors affect the supply of nutrients to the developing fetus , including placental function and maternal nutrient stores as well as the maternal diet . of these , nutritional intake during pregnancy is the most easily manipulated and may thus have the greatest public health importance . direct evidence from humans on the impact of diet during pregnancy on offspring cardiovascular disease ( cvd ) risk is limited and inconclusive ( 7 ) . moreover , the evidence base is dominated by observational studies that are likely to suffer from confounding due to a range of unmeasured characteristics . the follow - up of maternal supplementation trials to investigate the impact of nutrition during pregnancy on offspring cvd risk represents a powerful resource within this research field , taking advantage of the epidemiological strengths of trial design ( 7 ) . it has been suggested that the developmental origins of chronic disease may be particularly relevant to lower - income countries where nutrition and epidemiological transitions are occurring against a background of generational cycles of undernutrition and infant growth retardation ( 8) . it is thus of particular interest to understand the impact of nutritional supplementation in pregnancy in these settings to explore the long - term consequences of these widely promoted interventions . a few studies have now published data on the impact of supplementation trials in resource - poor settings on offspring cvd risk factors . the provision of protein - energy supplements during pregnancy in the gambia was found to be unrelated to any of the cvd risk factors studied , including body composition , blood pressure , and lipid profile in adolescents ( 9 ) . no other studies to our knowledge have looked solely at protein - energy supplements during pregnancy . multiple - micronutrient supplements ( mmss ) provided in pregnancy compared with iron and folate supplements were marginally associated with lower offspring systolic , but not diastolic , blood pressure at 2 y of age in one nepalese study ( 10 ) but were unrelated to offspring blood pressure at 68 y in a separate nepalese trial where mmss were compared with vitamin a supplementation ( 11 ) . in this latter trial , there was also no impact of maternal iron , zinc , and folate supplementation on offspring blood pressure compared with vitamin a supplementation ( 11 ) . here , we present information on blood pressure and kidney function for individuals born as part of the maternal and infant nutrition intervention in the matlab ( minimat ; isrctn16581395 ) trial in rural bangladesh . this trial was a large , combined food and multiple - micronutrient intervention for pregnant women aimed at improving birth weight and neonatal health ( 12 ) . this paper presents follow - up data on blood pressure and kidney function of the children born during the trial when they reached 4.5 y of age . the minimat trial was conducted by the international centre for diarrheal disease research , bangladesh ( icddr , b ) between november 2001 and october 2003 in the rural matlab region of bangladesh , 57 km southeast of the capital dhaka . , women were recruited early in pregnancy through regular surveillance of the demographic area covered by icddr , b . consenting women were randomly assigned to 2 separate nutritional interventions in pregnancy : access to food supplementation and receipt of a micronutrient supplement . for the food intervention , women were randomly assigned to receive encouragement to attend government - sponsored , local community nutrition centers either early in pregnancy ( around 9 wk of gestation ) or at a time of their choosing ( usually around 20 wk of gestation ) . food supplements that provided 608 kcal / d energy and 18 g / d of vegetable protein were available to all attending women . women participating in the minimat trial were also randomly assigned to receive 1 of 3 micronutrient supplements with identical appearance : 30 mg of iron and 400 g of folate ( fe30f)/d , 60 mg of iron and 400 g of folate ( fe60f)/d , or the unimap combination of 15 micronutrients at or above the rda ( 12 ) , which contained 30 mg iron and 400 g folate ( mms)/d . the micronutrient supplements were provided to participants on a monthly basis in special bottles ( edem , aprex ) containing 35 capsules , which was more than sufficient for the daily tablet allowance . compliance was assessed by the reported number of food packages received and the number of micronutrient bottle openings ( recorded by the edem device ) from enrollment to wk 30 gestation . in total , 4436 pregnant women were enrolled in the trial and there were 3560 live singleton births ( fig . the primary outcomes of the original trial were birth anthropometry and neonatal survival ; no effect was observed on birth size , but the combined early invitation to food supplementation and mmss were associated with decreased childhood mortality ( 12 ) . flow diagram of offspring born during the minimat trial and those recruited into the current follow - up study . diagram represents the flow of trial participants from enrollment through birth to the current follow - up at 4.5 y of age . all individuals who were live singleton births during the trial and for whom birth anthropometry was available were eligible for the current follow - up study . details of loss to follow - up between randomization and birth can be found elsewhere ( 12 ) . bp : number of individuals recruited for whom blood pressure was measured , mm hg . kidney volume : number of individuals recruited for kidney size measurements by ultrasound , cm / m . preterm : individuals born before 37 wk of gestation were excluded at the analysis stage . cysc , number of individuals recruited for cystatin c measurements ; fe30f , 30 mg iron and 400 g folate ; fe60f , 60 mg iron and 400 g folate ; mms , multiple micronutrient supplement ; vol , volume . a detailed follow - up of children born during the minimat trial was conducted between may 2007 and february 2009 when they were 4.5 y old . the 3267 children who were live singleton births and had measured birth anthropometry were eligible for the follow - up study and informed consent was obtained by their parents or guardians . scientific approval was granted by the research review committee of icddr , b and ethical approval from the icddr , b ethical review committee as well as the ethical committees of the participating universities ( london school of hygiene and tropical medicine , uppsala university , and the university of tsukuba ) ; separate approval was granted for the follow - up study and the original trial . a range of outcomes was assessed during the follow - up , including blood pressure and kidney function , which are presented here , and child growth and body composition , which are published elsewhere ( 15 ) . blood pressure was assessed on all individuals recruited into the 4.5-y follow - up . for cost and logistic considerations , measurements of kidney function were conducted on a subset of participants ; glomerular filtration rate ( gfr ) assessment was restricted to individuals born during the first year of the minimat trial ( june 2002june 2003 ) and the kidney biometric study was restricted to a different subsample of individuals , born during the second half ( june 2003june 2004 ) of the trial to minimize participant burden . blood pressure was measured on all individuals in triplicate using an automated oscillometric device ( omron 705it , morton medical ) . the first measurement was taken after the participant had been seated at rest for 5 min and there was 1 min between each subsequent measurement . kidney function was assessed by calculation of the gfr estimated from plasma cystatin c ( cysc ) , which was analyzed from stored , frozen samples using the immunoturbidimetric analysis ( 16 ) in uppsala , sweden . gfr was calculated from cysc using a prediction equation generated in swedish patients and applicable for use in children ( 17 ) . kidney volume was assessed by ultrasonography conducted using a 3.75-mhz convex scanner ( toshiba ssa- 510a / p3 , famio-5 , toshiba medical systems ) . intra - observer error ( expressed as the sd of the difference of the first and second measurement ) ranged from 1.86 to 3.36 cm and the inter - observer error ( evaluated by the sd of the difference ) ranged from 1.64 to 3.83 cm ; both measurement errors were within the range reported in the literature for studies conducted by technical staff and medical doctors ( 18 ) . the right and left kidney volumes were calculated by fitting a best - fit ellipsoid shape that was converted into an estimate of volume using internal software . this method was correlated ( r = 0.92 ; p < 0.001 ) with the alternative length , width , depth method of assessing volume . the mean of right and left kidney volume was then adjusted for body surface area calculated from the haycock formula ( 19 ) . in addition to the main outcomes specified above , a variety of anthropometric measurements was conducted during the 4.5-y follow - up , including weight ( digital scale : tanita , chasmors ) and height ( stadiometer : chasmors ) . nutritional status was assessed by comparison with uk reference data ( 20 ) and a cutoff of < 2 z - scores was used to define wasting and stunting based on weight - for - height and height - for - age indices , respectively . total body composition was assessed using a foot - to - foot bioelectrical impedance analyzer ( tanita tbf-300ma , chasmors ) , applying equations previously generated in this population using deuterium dilution as the reference method in children aged 410 y ( 21 ) . parental socioeconomic status was assessed by a continuous wealth index previously generated in this population that included data on land ownership , the construction materials of house walls , ownership of household assets , ownership of sarees or shalwer - kameez for ceremonial use , and pairs of shoes or sandals owned ( 22 ) . all statistical analysis was conducted using stata 11 ( stata corportation ) ; p values < 0.05 were regarded as significant . the effects of the prenatal interventions were assessed by intention - to - treat analysis , which focused on the effect of food and micronutrient supplementation separately before assessing any interaction between the interventions . independent t tests and tests were used to assess any differences in characteristics from the original trial between those recruited and those lost to follow - up . linear regression was used to investigate the effect of the maternal interventions on offspring blood pressure and kidney function . three stages of models were used : adjusted for interventions only ( model 1 ) , adjusted for covariates unrelated to the maternal intervention but related to blood pressure / kidney function ( model 2 ) , and as model 2 but additionally adjusted for covariates that could be associated with the maternal interventions . the analysis of the food supplementation intervention compared the 2 arms : early and usual invitation to access food supplementation . the unbalanced design of the minimat trial allowed for the assessment of 2 important research questions : is there an impact of mms and is there an impact of providing a high iron dose ? the analysis was conducted by creating dummy variables for the mms and fe60f interventions and comparing these with the fe30f group as the reference ( table 1 ) . the linear regression models were fitted with both terms to assess their independent effects , but the coefficients are reported separately in the results . an as - treated analysis was conducted to assess the impact of adherence to the intervention irrespective of randomization . the reported total number of food packets consumed during pregnancy was assessed as the exposure for the food invitation intervention and the pill count obtained from the edem technology provided an estimate of micronutrient tablet consumption . linear regression models were fitted adjusted for covariates relating to blood pressure and kidney function as appropriate . categorizing the micronutrient intervention from the minimat trial for the purposes of analysis original micronutrient arm of the intervention . fe30f , 30 mg iron and 400 g folate ; fe60f , 60 mg iron and 400 folate ; mms , multiple micronutrient supplement . variable is recoded to represent multiple micronutrients or high - iron dose : individuals were coded 0 ( control ) or 1 ( receiving intervention ) and fitted together in regression models of intention - to - treat analysis . all statistical analysis was conducted using stata 11 ( stata corportation ) ; p values < 0.05 were regarded as significant . the effects of the prenatal interventions were assessed by intention - to - treat analysis , which focused on the effect of food and micronutrient supplementation separately before assessing any interaction between the interventions . independent t tests and tests were used to assess any differences in characteristics from the original trial between those recruited and those lost to follow - up . linear regression was used to investigate the effect of the maternal interventions on offspring blood pressure and kidney function . three stages of models were used : adjusted for interventions only ( model 1 ) , adjusted for covariates unrelated to the maternal intervention but related to blood pressure / kidney function ( model 2 ) , and as model 2 but additionally adjusted for covariates that could be associated with the maternal interventions . the analysis of the food supplementation intervention compared the 2 arms : early and usual invitation to access food supplementation . the unbalanced design of the minimat trial allowed for the assessment of 2 important research questions : is there an impact of mms and is there an impact of providing a high iron dose ? the analysis was conducted by creating dummy variables for the mms and fe60f interventions and comparing these with the fe30f group as the reference ( table 1 ) . the linear regression models were fitted with both terms to assess their independent effects , but the coefficients are reported separately in the results . an as - treated analysis was conducted to assess the impact of adherence to the intervention irrespective of randomization . the reported total number of food packets consumed during pregnancy was assessed as the exposure for the food invitation intervention and the pill count obtained from the edem technology provided an estimate of micronutrient tablet consumption . linear regression models were fitted adjusted for covariates relating to blood pressure and kidney function as appropriate . categorizing the micronutrient intervention from the minimat trial for the purposes of analysis original micronutrient arm of the intervention . fe30f , 30 mg iron and 400 g folate ; fe60f , 60 mg iron and 400 folate ; mms , multiple micronutrient supplement . variable is recoded to represent multiple micronutrients or high - iron dose : individuals were coded 0 ( control ) or 1 ( receiving intervention ) and fitted together in regression models of intention - to - treat analysis . the current follow - up study recruited 2526 children who were born during the original trial , representing 77% of the eligible cohort ( fig . recruitment rates were similar across the 6 supplement arms of the trial and the reasons for loss to follow - up were also similar in their distributions . the main cause of loss to follow - up was individuals who could not be located during the follow - up study . among recruited individuals , maternal baseline characteristics there were minor differences in characteristics of mothers whose children were recruited into the current study and those lost to follow - up ( table 2 ) . children lost to follow - up were more likely to have been the first born and their mothers were on average 9 mo younger and had spent 6 mo longer in education . recruited individuals who were born before 37 wk of gestation ( preterm , n = 191 ) were excluded from the analysis , leaving a sample size of 2335 . the mean age at follow - up was 4.6 0.1 y and 50.5% of the recruited individuals were boys . at follow - up , the average bmi - for - age z - score was 1.7 1.0 and the height - for - age z - score was 1.5 1.0 ; 28% of boys and 33% of girls were classified as stunted ( table 3 ) . all 3 blood pressure measurements were correlated ( r > 0.65 ; p = < 0.001 ) and the mean of 3 readings was used as an estimate of average blood pressure . the mean systolic blood pressure was 91.1 7.5 mm hg for girls and 91.4 7.7 mm hg for boys . mean diastolic blood pressure was 55.0 6.4 mm hg for girls and 53.8 6.5 mm hg for boys . mean gfr was 158.2 35.1 ml/(min 1.73 m ) with no difference between boys and girls . neither kidney volume nor gfr was associated with blood pressure ( data not shown ) . difference in maternal and household characteristics between children recruited into the minimat trial follow - up at 4.5 y and those not recruited values are means sds or percentage where indicated . asterisks indicate different from those recruited : * p < 0.05 , * * p < 0.01 . usual food : maternal randomization to access food at the usual time in pregnancy ; fe30f , 30 mg iron and 400 g folate ; fe60f , 60 mg iron and 400 folate ; mms , multiple micronutrient supplement . all individuals recruited into current follow - up study . measured during wk 8 of gestation . the food invitation intervention successfully produced 2 groups of women who received different amounts of food during pregnancy . the median reported food packet consumption in the early invitation group was 103 packets ( iqr : 67 , 128 ) compared with 70 packets ( iqr : 39 , 92 ) in the usual invitation arm . the invitation to early food supplementation was associated with lower offspring diastolic blood pressure by a mean of 0.74 mm hg [ ( 95% ci : 0.18 , 1.30 ) ; p = 0.01 ] in the fully adjusted analysis ( model 3 ) compared with the offspring of women in the usual invitation arm ( table 4 ) . there was no association of this intervention with systolic blood pressure or with either kidney volume or gfr . anthropometry and body composition of offspring born during the minimat trial , bangladesh at 4.5 y of age values are means sds . fat - free mass and fat mass assessed by bioelectrical impedance analysis ( tanita , tbf-300ma ) using population - specific prediction equations ( 21 ) . the mean number of tablets consumed by women in the 3 micronutrient intervention groups was 81 ( fe30f ) , 80 ( fe60f ) , and 76 ( mms ) , respectively . in the unadjusted intention - to - treat analysis , there was no association between the micronutrient intervention and offspring blood pressure or kidney function ( table 5 ) . however , in the adjusted analysis , there was an effect of the micronutrient intervention on diastolic blood pressure , with a mean of 0.65 mm hg [ ( 95% ci : 0.06 , 1.24 ) ; p = 0.03 ] higher diastolic blood pressure for children whose mothers received mmss during pregnancy compared with iron and folate . there was no effect of the high- compared with low - iron intervention on offspring blood pressure ( table 6 ) . there was also no effect of the iron intervention on offspring kidney function in unadjusted analysis , but in the adjusted analysis , individuals whose mothers had received 60 mg of iron during pregnancy had a mean 4.98 ml/(min 1.73 m ) [ ( 95% ci : 0.30 , 9.67 ) ; p = 0.04 ] higher gfr at 4.5 y of age than those whose mothers had received 30 mg of iron during pregnancy . effect of maternal food intervention on offspring blood pressure and kidney function at 4.5 y in bangladesh values are regression coefficients ( ) showing the difference in mean blood pressure ( 95% ci ) for individuals born to women invited to receive food supplements early in pregnancy ( coded 0 ) compared with the usual time ( coded 1 ) , derived from linear regression analysis . model 2 , additionally adjusted for age , sex , wealth index , tertiles of maternal wk 8 of gestation blood pressure ( blood pressure models only ) , and season of birth fitted as fourier terms ( 36 ) . model 3 , as model 2 but additionally adjusted for height , bmi , fat free mass , diarrhea in the past 2 wk , and feeling well on the study day . effect of maternal multiple micronutrient supplementation on offspring blood pressure and kidney function at 4.5 y in bangladesh values are regression coefficients ( ) showing the difference in mean blood pressure ( 95% ci ) for individuals born to women in the mms compared with the fe30f intervention group , derived from linear regression analysis . fe30f , 30 mg iron and 400 g of folate ; gfr , glomerular filtration rate ; mms , multiple micronutrient supplement . model 2 , additionally adjusted for age , sex , wealth index , tertiles of maternal blood pressure ( blood pressure models only ) , and season of birth . model 3 , as model 2 but additionally adjusted for height , bmi , fat - free mass , diarrhea in past 2 wk , and feeling well on the study day . the combined effect of the food and micronutrient interventions was assessed by introducing 2 interactions terms , one between the food and mms intervention and one between the food and fe intervention . there appeared to be no interaction between the interventions in relation to offspring blood pressure or kidney function ( supplemental table 1 ) . there were no interactions between any of the potential effect modifiers tested ( sex , wealth index , or maternal baseline bmi ) and any of the 3 interventions on either child blood pressure or kidney function ( data not shown ) . the analyses of both the food and micronutrient interventions were repeated using an as - treated rather than intention - to - treat design . there was no association between the number of food packets consumed and offspring blood pressure , kidney volume , or gfr ( supplemental table 2 ) . similarly , there was no association between tablet consumption as measured by bottle opening and offspring blood pressure , kidney volume , or gfr ( supplemental table 3 ) . this study is one of the first to report follow - up data on offspring blood pressure and kidney function as a result of a combined pregnancy nutritional intervention . there is evidence that invitation to access food supplements early in pregnancy was associated with a slightly lower offspring diastolic blood pressure at 4.5 y compared with the standard care in later pregnancy . in contrast , mmss were associated with slightly higher diastolic blood pressure compared with iron and folate supplementation alone and a high- compared with low - iron supplement was associated with a higher offspring gfr . no other effects of the food or micronutrient interventions on offspring blood pressure , kidney volume , or gfr were observed and there was no interaction between the interventions on the outcomes studied . previous studies of protein - energy supplementation trials do not provide a direct comparison with the minimat intervention , partly because they lack a temporal variation in food supplementation . the protein - energy supplements provided from 20 wk of gestation to pregnant women in the gambia were not associated with offspring blood pressure at 1017 y of age ( 23 ) , and those provided during pregnancy and early childhood in guatemala were similarly not associated with offspring blood pressure at 2029 y of age ( 24 ) . in india , although a community - based cereal meal intervention was not associated with offspring blood pressure in adolescence , children born in intervention areas had a lower measure of global arterial stiffness ( the augmentation index ) ( 25 ) . in the current study , no association was observed between food packet consumption and offspring diastolic blood pressure irrespective of the treatment arm , which cautions against the overinterpretation of these findings . in addition , the relatively small effect size and the lack of an effect on systolic blood pressure may question the validity of the findings . however , these data do suggest that researchers may wish in future studies to assess the timing as well as the type of pregnancy intervention . multiple - micronutrient supplementation in pregnancy was associated with a marginal increase in offspring diastolic blood pressure , although the association was only strongly apparent in the fully adjusted analysis . two other trials , both from nepal , of multiple - micronutrient supplementation in pregnancy have published data on offspring blood pressure . in contrast to the minimat trial , both the nepalese trials reported an increase in birth weight in the multiple - micronutrient arms of the trial ( 26 , 27 ) . ( 10 ) reported lower systolic blood pressure at 2.5 y of age for children born to women who had received multiple micronutrients in pregnancy . in contrast , stewart et al . ( 11 ) found no effect of mmss on child blood pressure at 68 y , consistent with the results presented here . one interpretation of the first nepalese trial is a detrimental effect of the high - iron dose in the comparison group ( 10 ) . the design of the minimat trial allows for this to be tested and the analysis presented here has shown no effect of high- compared with low - iron dose on offspring blood pressure in this population . it has been suggested that a reduction in nephron number as a result of inadequate fetal nutrition represents a potential mechanism linking low birth weight and hypertension , with supportive evidence from animal models ( 3 , 2830 ) . in humans , low birth weight has been associated with lower kidney volume ( by ultrasound ) ( 31 ) and chronic kidney disease in later life ( 32 ) . few human studies have investigated the association between the maternal diet during pregnancy and offspring kidney function . in the second nepalese trial reported above , the control group ( vitamin a supplements only ) had a higher risk of urinary microalbuminuria ( microalbumin : creatinine ratio 3.4 mg / mmol ) compared with individuals whose mothers received folic acid or folic acid , iron , and zinc in addition to vitamin a ( 11 ) . here , we found weak evidence for an increased gfr in children exposed to high - iron supplementation during in utero development , which is challenging to interpret . some authors have suggested that fewer nephrons will initially be associated with compensatory hyperfiltration ( 33 ) , which could be consistent with a higher gfr . in later life , these same individuals may demonstrate relatively low gfr , which is a standard marker of kidney disease ( 34 ) , but further studies will be required to unravel this association and assess validity . effect of maternal iron supplementation on offspring blood pressure and kidney function at 4.5 y in bangladesh values are regression coefficients ( ) showing the difference in mean blood pressure and kidney function ( 95% ci ) for individuals born to women in the fe60f compared to fe30f intervention group , derived from linear regression analysis . fe60f , 60 mg of iron and 400 g of folate ; gfr , glomerular filtration rate ; mms , multiple micronutrient supplement . model 2 , additionally adjusted for age , sex , wealth index , tertiles of maternal blood pressure ( blood pressure models only ) , and season of birth . model 3 , as model 2 but additionally adjusted for height , bmi , fat - free mass , diarrhea in past 2 wk , and feeling well on the study day . there are a number of important strengths of the current study , including the large sample size and good retention of participants ; 77% of eligible individuals born during the maternal trial were successfully recruited at 4.5 y of age . loss to follow - up is a well - recognized issue for long - term follow - up studies within this research field and these rates of loss are relatively low ( 35 ) . the lack of differential follow - up rates between the intervention groups and the marginal differences between recruited individuals and those lost to follow - up suggests limited selection bias for the current study . a related issue is the reduction in sample size as a consequence of participant attrition , which affects study power . in this study , the precision estimate for the effect of maternal mms supplementation on offspring systolic blood pressure ranged from 0.57 to 1.02 mm hg and effects within this range can not therefore be discounted . however , it is arguable whether effect sizes of this magnitude would be meaningful from a public health standpoint . cost and logistic considerations resulted in the measurements of kidney volume and gfr being conducted on a subset of individuals at the 4.5-y follow - up , but the sample size remained large and the precision of effect estimates high . although it was a limitation that only a small number of individuals had both kidney function measurements , this would not be expected to have influenced the analysis of the intervention effect ; the sample size for both outcomes remained large and there is no reason to think that the intervention would have a different effect in these subgroups . the main limitation with the original study design is that women were only randomly assigned to encouragement to access food provision rather than being provided with the food supplement itself . some women would therefore be unlikely to access the supplement at all , although only 54 individuals reported not having had a single food packet during their pregnancy and the majority ( 76% ) of these were in the usual food invitation arm . the intervention was designed to evaluate the existing government nutritional provision , but as a result lacked a true control arm . however , data on food packet consumption during pregnancy did demonstrate that the intervention successfully created 2 distinct groups ; women in the early invitation arm accessed the service much earlier in pregnancy and consumed on average 30 more packets during pregnancy . the micronutrient intervention benefitted from being a double - blind randomized trial but again lacked a control group that could be provided with a placebo owing to the ethics of withdrawing fefol from this antenatal population , which receives 60 mg of iron and 400 g folate as standard . this intervention is further compromised by its unbalanced design ; there is no multiple micronutrient arm that also contains 60 mg of iron . as a consequence , any effect of the mms arm could be confounded by the iron treatment arm and it is not possible to look at the interaction between these different treatments . however , because only limited intervention effects have been observed , this limitation is of minor concern . a final important limitation relates to the age of study participants who may be too young for differences in cvd risk factors to emerge . it will be important to study this cohort into the future to assess any prolonged effects of the pregnancy intervention , particularly as groups of offspring become exposed to different environments as they age . in conclusion , this is one of the first studies to include a follow - up of a combined food and micronutrient intervention in pregnancy . there is some evidence that access to food supplements early in pregnancy is associated with reduced offspring diastolic blood pressure in childhood , albeit with a relatively small effect size . weaker evidence was also found for an impact of mmss on increased offspring diastolic blood pressure and of high- compared with low - iron supplements on increase offspring gfr . overall , there was limited evidence for long - lasting impacts of pregnancy supplementation on offspring blood pressure or markers of kidney function in this population .

observational evidence suggests nutritional exposures during in utero development may have long - lasting consequences for health ; data from interventions are scarce . here , we present a trial follow - up study to assess the association between prenatal food and micronutrient supplementation and childhood blood pressure and kidney function . during the minimat trial in rural bangladesh , women were randomly assigned early in pregnancy to receive an early or later invitation to attend a food supplementation program and additionally to receive either iron and folate or multiple micronutrient tablets daily . the 3267 singleton birth individuals with measured anthropometry born during the trial were eligible for a follow - up study at 4.5 y old . a total of 77% of eligible individuals were recruited and blood pressure , kidney size by ultrasound , and glomerular filtration rate ( gfr ; calculated from plasma cystatin c ) were assessed . in adjusted analysis , early invitation to food supplementation was associated with a 0.72-mm hg [ ( 95% ci : 0.16 , 1.28 ) ; p = 0.01 ] lower childhood diastolic blood pressure and maternal mms supplementation was associated with a marginally higher [ 0.87 mm hg ( 95% ci : 0.18 , 1.56 ) ; p = 0.01 ] childhood diastolic blood pressure . there was also some evidence that a supplement higher in iron was associated with a higher offspring gfr . no other effects of the food or micronutrient interventions were observed and there was no interaction between the interventions on the outcomes studied . these marginal associations and small effect sizes suggest limited public health importance in early childhood .

Introduction Methods Statistical analysis. Results Discussion Supplementary Material

studies have consistently demonstrated that altering the nutrient supply of pregnant animals induces profound changes in the function of body systems , including raised blood pressure , fewer kidney nephrons , and impaired pancreatic -cell development ( 14 ) . in humans , much of the evidence has focused on the inverse associations observed between birth weight and a range of chronic diseases in later life , including hypertension and diabetes ( 5 , 6 ) . the follow - up of maternal supplementation trials to investigate the impact of nutrition during pregnancy on offspring cvd risk represents a powerful resource within this research field , taking advantage of the epidemiological strengths of trial design ( 7 ) . it is thus of particular interest to understand the impact of nutritional supplementation in pregnancy in these settings to explore the long - term consequences of these widely promoted interventions . the provision of protein - energy supplements during pregnancy in the gambia was found to be unrelated to any of the cvd risk factors studied , including body composition , blood pressure , and lipid profile in adolescents ( 9 ) . multiple - micronutrient supplements ( mmss ) provided in pregnancy compared with iron and folate supplements were marginally associated with lower offspring systolic , but not diastolic , blood pressure at 2 y of age in one nepalese study ( 10 ) but were unrelated to offspring blood pressure at 68 y in a separate nepalese trial where mmss were compared with vitamin a supplementation ( 11 ) . in this latter trial , there was also no impact of maternal iron , zinc , and folate supplementation on offspring blood pressure compared with vitamin a supplementation ( 11 ) . here , we present information on blood pressure and kidney function for individuals born as part of the maternal and infant nutrition intervention in the matlab ( minimat ; isrctn16581395 ) trial in rural bangladesh . this paper presents follow - up data on blood pressure and kidney function of the children born during the trial when they reached 4.5 y of age . the minimat trial was conducted by the international centre for diarrheal disease research , bangladesh ( icddr , b ) between november 2001 and october 2003 in the rural matlab region of bangladesh , 57 km southeast of the capital dhaka . , women were recruited early in pregnancy through regular surveillance of the demographic area covered by icddr , b . consenting women were randomly assigned to 2 separate nutritional interventions in pregnancy : access to food supplementation and receipt of a micronutrient supplement . for the food intervention , women were randomly assigned to receive encouragement to attend government - sponsored , local community nutrition centers either early in pregnancy ( around 9 wk of gestation ) or at a time of their choosing ( usually around 20 wk of gestation ) . women participating in the minimat trial were also randomly assigned to receive 1 of 3 micronutrient supplements with identical appearance : 30 mg of iron and 400 g of folate ( fe30f)/d , 60 mg of iron and 400 g of folate ( fe60f)/d , or the unimap combination of 15 micronutrients at or above the rda ( 12 ) , which contained 30 mg iron and 400 g folate ( mms)/d . in total , 4436 pregnant women were enrolled in the trial and there were 3560 live singleton births ( fig . the primary outcomes of the original trial were birth anthropometry and neonatal survival ; no effect was observed on birth size , but the combined early invitation to food supplementation and mmss were associated with decreased childhood mortality ( 12 ) . flow diagram of offspring born during the minimat trial and those recruited into the current follow - up study . diagram represents the flow of trial participants from enrollment through birth to the current follow - up at 4.5 y of age . all individuals who were live singleton births during the trial and for whom birth anthropometry was available were eligible for the current follow - up study . kidney volume : number of individuals recruited for kidney size measurements by ultrasound , cm / m . cysc , number of individuals recruited for cystatin c measurements ; fe30f , 30 mg iron and 400 g folate ; fe60f , 60 mg iron and 400 g folate ; mms , multiple micronutrient supplement ; vol , volume . a detailed follow - up of children born during the minimat trial was conducted between may 2007 and february 2009 when they were 4.5 y old . the 3267 children who were live singleton births and had measured birth anthropometry were eligible for the follow - up study and informed consent was obtained by their parents or guardians . scientific approval was granted by the research review committee of icddr , b and ethical approval from the icddr , b ethical review committee as well as the ethical committees of the participating universities ( london school of hygiene and tropical medicine , uppsala university , and the university of tsukuba ) ; separate approval was granted for the follow - up study and the original trial . a range of outcomes was assessed during the follow - up , including blood pressure and kidney function , which are presented here , and child growth and body composition , which are published elsewhere ( 15 ) . blood pressure was assessed on all individuals recruited into the 4.5-y follow - up . for cost and logistic considerations , measurements of kidney function were conducted on a subset of participants ; glomerular filtration rate ( gfr ) assessment was restricted to individuals born during the first year of the minimat trial ( june 2002june 2003 ) and the kidney biometric study was restricted to a different subsample of individuals , born during the second half ( june 2003june 2004 ) of the trial to minimize participant burden . kidney function was assessed by calculation of the gfr estimated from plasma cystatin c ( cysc ) , which was analyzed from stored , frozen samples using the immunoturbidimetric analysis ( 16 ) in uppsala , sweden . this method was correlated ( r = 0.92 ; p < 0.001 ) with the alternative length , width , depth method of assessing volume . in addition to the main outcomes specified above , a variety of anthropometric measurements was conducted during the 4.5-y follow - up , including weight ( digital scale : tanita , chasmors ) and height ( stadiometer : chasmors ) . the effects of the prenatal interventions were assessed by intention - to - treat analysis , which focused on the effect of food and micronutrient supplementation separately before assessing any interaction between the interventions . independent t tests and tests were used to assess any differences in characteristics from the original trial between those recruited and those lost to follow - up . linear regression was used to investigate the effect of the maternal interventions on offspring blood pressure and kidney function . three stages of models were used : adjusted for interventions only ( model 1 ) , adjusted for covariates unrelated to the maternal intervention but related to blood pressure / kidney function ( model 2 ) , and as model 2 but additionally adjusted for covariates that could be associated with the maternal interventions . the analysis of the food supplementation intervention compared the 2 arms : early and usual invitation to access food supplementation . the unbalanced design of the minimat trial allowed for the assessment of 2 important research questions : is there an impact of mms and is there an impact of providing a high iron dose ? linear regression models were fitted adjusted for covariates relating to blood pressure and kidney function as appropriate . categorizing the micronutrient intervention from the minimat trial for the purposes of analysis original micronutrient arm of the intervention . the effects of the prenatal interventions were assessed by intention - to - treat analysis , which focused on the effect of food and micronutrient supplementation separately before assessing any interaction between the interventions . independent t tests and tests were used to assess any differences in characteristics from the original trial between those recruited and those lost to follow - up . linear regression was used to investigate the effect of the maternal interventions on offspring blood pressure and kidney function . three stages of models were used : adjusted for interventions only ( model 1 ) , adjusted for covariates unrelated to the maternal intervention but related to blood pressure / kidney function ( model 2 ) , and as model 2 but additionally adjusted for covariates that could be associated with the maternal interventions . the analysis of the food supplementation intervention compared the 2 arms : early and usual invitation to access food supplementation . the unbalanced design of the minimat trial allowed for the assessment of 2 important research questions : is there an impact of mms and is there an impact of providing a high iron dose ? linear regression models were fitted adjusted for covariates relating to blood pressure and kidney function as appropriate . categorizing the micronutrient intervention from the minimat trial for the purposes of analysis original micronutrient arm of the intervention . the current follow - up study recruited 2526 children who were born during the original trial , representing 77% of the eligible cohort ( fig . recruitment rates were similar across the 6 supplement arms of the trial and the reasons for loss to follow - up were also similar in their distributions . the main cause of loss to follow - up was individuals who could not be located during the follow - up study . among recruited individuals , maternal baseline characteristics there were minor differences in characteristics of mothers whose children were recruited into the current study and those lost to follow - up ( table 2 ) . the mean age at follow - up was 4.6 0.1 y and 50.5% of the recruited individuals were boys . all 3 blood pressure measurements were correlated ( r > 0.65 ; p = < 0.001 ) and the mean of 3 readings was used as an estimate of average blood pressure . mean diastolic blood pressure was 55.0 6.4 mm hg for girls and 53.8 6.5 mm hg for boys . neither kidney volume nor gfr was associated with blood pressure ( data not shown ) . difference in maternal and household characteristics between children recruited into the minimat trial follow - up at 4.5 y and those not recruited values are means sds or percentage where indicated . usual food : maternal randomization to access food at the usual time in pregnancy ; fe30f , 30 mg iron and 400 g folate ; fe60f , 60 mg iron and 400 folate ; mms , multiple micronutrient supplement . all individuals recruited into current follow - up study . the invitation to early food supplementation was associated with lower offspring diastolic blood pressure by a mean of 0.74 mm hg [ ( 95% ci : 0.18 , 1.30 ) ; p = 0.01 ] in the fully adjusted analysis ( model 3 ) compared with the offspring of women in the usual invitation arm ( table 4 ) . there was no association of this intervention with systolic blood pressure or with either kidney volume or gfr . anthropometry and body composition of offspring born during the minimat trial , bangladesh at 4.5 y of age values are means sds . in the unadjusted intention - to - treat analysis , there was no association between the micronutrient intervention and offspring blood pressure or kidney function ( table 5 ) . however , in the adjusted analysis , there was an effect of the micronutrient intervention on diastolic blood pressure , with a mean of 0.65 mm hg [ ( 95% ci : 0.06 , 1.24 ) ; p = 0.03 ] higher diastolic blood pressure for children whose mothers received mmss during pregnancy compared with iron and folate . there was no effect of the high- compared with low - iron intervention on offspring blood pressure ( table 6 ) . there was also no effect of the iron intervention on offspring kidney function in unadjusted analysis , but in the adjusted analysis , individuals whose mothers had received 60 mg of iron during pregnancy had a mean 4.98 ml/(min 1.73 m ) [ ( 95% ci : 0.30 , 9.67 ) ; p = 0.04 ] higher gfr at 4.5 y of age than those whose mothers had received 30 mg of iron during pregnancy . effect of maternal food intervention on offspring blood pressure and kidney function at 4.5 y in bangladesh values are regression coefficients ( ) showing the difference in mean blood pressure ( 95% ci ) for individuals born to women invited to receive food supplements early in pregnancy ( coded 0 ) compared with the usual time ( coded 1 ) , derived from linear regression analysis . model 3 , as model 2 but additionally adjusted for height , bmi , fat free mass , diarrhea in the past 2 wk , and feeling well on the study day . effect of maternal multiple micronutrient supplementation on offspring blood pressure and kidney function at 4.5 y in bangladesh values are regression coefficients ( ) showing the difference in mean blood pressure ( 95% ci ) for individuals born to women in the mms compared with the fe30f intervention group , derived from linear regression analysis . fe30f , 30 mg iron and 400 g of folate ; gfr , glomerular filtration rate ; mms , multiple micronutrient supplement . model 3 , as model 2 but additionally adjusted for height , bmi , fat - free mass , diarrhea in past 2 wk , and feeling well on the study day . the combined effect of the food and micronutrient interventions was assessed by introducing 2 interactions terms , one between the food and mms intervention and one between the food and fe intervention . there appeared to be no interaction between the interventions in relation to offspring blood pressure or kidney function ( supplemental table 1 ) . there were no interactions between any of the potential effect modifiers tested ( sex , wealth index , or maternal baseline bmi ) and any of the 3 interventions on either child blood pressure or kidney function ( data not shown ) . the analyses of both the food and micronutrient interventions were repeated using an as - treated rather than intention - to - treat design . there was no association between the number of food packets consumed and offspring blood pressure , kidney volume , or gfr ( supplemental table 2 ) . similarly , there was no association between tablet consumption as measured by bottle opening and offspring blood pressure , kidney volume , or gfr ( supplemental table 3 ) . this study is one of the first to report follow - up data on offspring blood pressure and kidney function as a result of a combined pregnancy nutritional intervention . there is evidence that invitation to access food supplements early in pregnancy was associated with a slightly lower offspring diastolic blood pressure at 4.5 y compared with the standard care in later pregnancy . in contrast , mmss were associated with slightly higher diastolic blood pressure compared with iron and folate supplementation alone and a high- compared with low - iron supplement was associated with a higher offspring gfr . no other effects of the food or micronutrient interventions on offspring blood pressure , kidney volume , or gfr were observed and there was no interaction between the interventions on the outcomes studied . the protein - energy supplements provided from 20 wk of gestation to pregnant women in the gambia were not associated with offspring blood pressure at 1017 y of age ( 23 ) , and those provided during pregnancy and early childhood in guatemala were similarly not associated with offspring blood pressure at 2029 y of age ( 24 ) . in the current study , no association was observed between food packet consumption and offspring diastolic blood pressure irrespective of the treatment arm , which cautions against the overinterpretation of these findings . in addition , the relatively small effect size and the lack of an effect on systolic blood pressure may question the validity of the findings . multiple - micronutrient supplementation in pregnancy was associated with a marginal increase in offspring diastolic blood pressure , although the association was only strongly apparent in the fully adjusted analysis . two other trials , both from nepal , of multiple - micronutrient supplementation in pregnancy have published data on offspring blood pressure . in contrast to the minimat trial , both the nepalese trials reported an increase in birth weight in the multiple - micronutrient arms of the trial ( 26 , 27 ) . ( 10 ) reported lower systolic blood pressure at 2.5 y of age for children born to women who had received multiple micronutrients in pregnancy . the design of the minimat trial allows for this to be tested and the analysis presented here has shown no effect of high- compared with low - iron dose on offspring blood pressure in this population . few human studies have investigated the association between the maternal diet during pregnancy and offspring kidney function . in the second nepalese trial reported above , the control group ( vitamin a supplements only ) had a higher risk of urinary microalbuminuria ( microalbumin : creatinine ratio 3.4 mg / mmol ) compared with individuals whose mothers received folic acid or folic acid , iron , and zinc in addition to vitamin a ( 11 ) . here , we found weak evidence for an increased gfr in children exposed to high - iron supplementation during in utero development , which is challenging to interpret . some authors have suggested that fewer nephrons will initially be associated with compensatory hyperfiltration ( 33 ) , which could be consistent with a higher gfr . effect of maternal iron supplementation on offspring blood pressure and kidney function at 4.5 y in bangladesh values are regression coefficients ( ) showing the difference in mean blood pressure and kidney function ( 95% ci ) for individuals born to women in the fe60f compared to fe30f intervention group , derived from linear regression analysis . fe60f , 60 mg of iron and 400 g of folate ; gfr , glomerular filtration rate ; mms , multiple micronutrient supplement . there are a number of important strengths of the current study , including the large sample size and good retention of participants ; 77% of eligible individuals born during the maternal trial were successfully recruited at 4.5 y of age . loss to follow - up is a well - recognized issue for long - term follow - up studies within this research field and these rates of loss are relatively low ( 35 ) . the lack of differential follow - up rates between the intervention groups and the marginal differences between recruited individuals and those lost to follow - up suggests limited selection bias for the current study . in this study , the precision estimate for the effect of maternal mms supplementation on offspring systolic blood pressure ranged from 0.57 to 1.02 mm hg and effects within this range can not therefore be discounted . cost and logistic considerations resulted in the measurements of kidney volume and gfr being conducted on a subset of individuals at the 4.5-y follow - up , but the sample size remained large and the precision of effect estimates high . although it was a limitation that only a small number of individuals had both kidney function measurements , this would not be expected to have influenced the analysis of the intervention effect ; the sample size for both outcomes remained large and there is no reason to think that the intervention would have a different effect in these subgroups . the main limitation with the original study design is that women were only randomly assigned to encouragement to access food provision rather than being provided with the food supplement itself . however , data on food packet consumption during pregnancy did demonstrate that the intervention successfully created 2 distinct groups ; women in the early invitation arm accessed the service much earlier in pregnancy and consumed on average 30 more packets during pregnancy . it will be important to study this cohort into the future to assess any prolonged effects of the pregnancy intervention , particularly as groups of offspring become exposed to different environments as they age . in conclusion , this is one of the first studies to include a follow - up of a combined food and micronutrient intervention in pregnancy . there is some evidence that access to food supplements early in pregnancy is associated with reduced offspring diastolic blood pressure in childhood , albeit with a relatively small effect size . weaker evidence was also found for an impact of mmss on increased offspring diastolic blood pressure and of high- compared with low - iron supplements on increase offspring gfr . overall , there was limited evidence for long - lasting impacts of pregnancy supplementation on offspring blood pressure or markers of kidney function in this population .

styrene was listed in the twelfth edition of the national toxicology program 's ( ntp 's ) report on carcinogens ( ntp 2011 ) . in its report , ntp ( 2011 ) states : styrene is reasonably anticipated to be a human carcinogen based on limited evidence of carcinogenicity from studies in humans , sufficient evidence of carcinogenicity from studies in experimental animals , and supporting data on mechanisms of carcinogenesis . we submitted comments to ntp regarding its weight - of - evidence analysis assessing whether styrene should be considered a human carcinogen . these included an alternative weight - of - evidence analysis for each cancer type noted by ntp , with a comparison of risk estimates across exposure categories and studies , and a systematic evaluation of consistency and coherence with experimental and mechanistic studies ( goodman 2008 , 2009 ; rhomberg 2008 , 2009 ) . in this commentary , we summarize the findings of our analysis . we found that several requirements for limited or sufficient evidence are not considered by ntp , however , and the mode - of - action data do not support human carcinogenicity . as described in more detail below , the effects of styrene observed in experimental animals do not meet the standard of sufficient evidence in animals and the underlying mode of action is species - specific and not applicable to humans . in addition , the human data do not consistently show increased incidence of or mortality from cancer , and the epidemiology studies collectively do not support the standard of limited evidence . finally , there is no concordance among the human , experimental animal , and mode - of - action data . thus , based on ntp 's stated criteria , the evidence shows that styrene does not meet the standard for reasonably anticipated to be a human carcinogen and ntp ( 2011 ) states the following criteria for limited evidence of carcinogenicity in humans : there is limited evidence of carcinogenicity from studies in humans , which indicates that causal interpretation is credible , but that alternative explanations , such as chance , bias , or confounding factors , could not adequately be excluded . there are no precisely defined standards , however , for when the limited criterion is to be deemed satisfied . it is important to realize that limited evidence still requires a positive finding that a causal explanation is credible ; it is not simply applied when the data are inconsistent or inconclusive , and the mere presence of some positive evidence in some studies is not by itself grounds to conclude that a causal explanation is credible . instead , when results are mixed or inconsistent , an evaluation of all of the data must consider whether it is credible to hold that a truly causal relationship exists ( and the studies failing to show it do so because of chance , low statistical power , or because the true responses are somehow obscured by extraneous factors ) or whether it is more credible that there is no causal effect ( and the studies appearing to show an effect of exposure are in fact only showing chance findings or the effects of biases or confounding factors ) . making such a judgment requires a thorough and systematic evaluation of the evidence and an evaluation of the relative plausibility of the competing explanations actual causality partially obscured by chance or bias on the one hand versus bias and confounding creating the spurious appearance of apparent effects on the other . that is , the limited evidence category does not simply consist of cases for which there are some positive and some negative results ; it is only when a case for a credible ( albeit unproven ) causative effect can be made that the limited evidence characterization should be applied . in our view , when all the human evidence is evaluated , and the low numbers of observed cases and the lack of consistent patterns in cancer outcomes within and across cohorts , combined with concerns about co - exposures and confounding are considered , one comes to the conclusion that a causal relationship between styrene exposure and human cancer is not credible , and the standards of limited evidence are not met . ntp ( 2011 ) also states the following criteria for sufficient evidence of carcinogenicity in experimental animals : there is an increased incidence of malignant and/or a combination of malignant and benign tumors ( 1 ) in multiple species or at multiple tissue sites , ( 2 ) by multiple routes of exposure , or ( 3 ) to an unusual degree with regard to incidence , site , or type of tumor , or age at onset . as described in more detail below , the data show that the ntp criteria for sufficient evidence in animals are not met for styrene . the ntp profile for styrene suggests that epidemiology studies of workers exposed to styrene show increased incidence of or mortality from lymphohematopoietic cancer and that some studies in the reinforced plastics industry provide suggestive evidence for increased incidence of or mortality due to pancreatic and esophageal tumors ( ntp 2011 ) . when considering the epidemiology evidence as a whole , however , there are no consistent associations between styrene exposure and any specific cancer type either within or among studies . according to the background document ( ntp 2008 ) , the major epidemiology studies of styrene focus on 10 cohorts from the reinforced plastics and composites ( rpc ) , styrene - butadiene latex rubber ( sbr ) , and styrene / polystyrene ( ps ) industries , an occupational cohort in finland reporting urinary concentrations of a styrene metabolite ( anttila et al . 1998 ) , and a cohort of students who attended high school adjacent to facilities that produced synthetic styrene - butadiene ( loughlin et al . 1999 ; ntp 2008 ) . the highest exposures occurred in the rpc industry , followed by the sbr industry , and then the ps industry ( ntp 2008 ) . workers in the sbr industry were co - exposed to 1,3-butadiene , an established carcinogen . abbreviations used : ppm = parts per million ; ppm - years = ppm per day x number of years exposed ; twa = time - weighted average ; dmdtc = dimethyldithiocarbamate in certain studies , there were some statistically significant associations noted with some styrene exposure metrics for lymphohematopoietic , esophageal , and pancreatic cancers , but the risk estimates were not markedly large ( i.e. , most were below 2 ) and were far outnumbered by null associations for each cancer type . in addition , most analyses were based on a small number of observed cases , which resulted in unstable estimates , vis - - vis wide confidence intervals that either included or were generally close to 1 . furthermore , there were significant and non - significant negative associations reported for certain cancer types that were often as strong as positive associations reported for others . just as it is unlikely that these negative associations are reflective of a protective mechanism for styrene , the few positive associations are unlikely to reflect a causal association . we have demonstrated this for each cancer type ntp considered in our submitted comments ( goodman 2008 , 2009 ; rhomberg 2008 , 2009 ) . tables 2 and 3 , which summarize the leukemia data , are provided here as an example . the weight of evidence suggests that , if there are any associations between styrene exposure and leukemia , they are not evident in the high exposure industry ( rpc ) . there are no consistent associations seen across studies of the same cohort in the sbr industry , and co - exposure to 1,2-butadiene likely confounded results . if styrene were associated with any cancer type , then one would expect an exposure - response relationship within studies and among industries . studies of rpc workers should carry the greatest weight in an assessment of the epidemiology data because these workers have the highest styrene exposures . studies of sbr workers should carry less weight because the styrene exposures are far lower , and 1,3-butadiene , even when adjusted for , can not be completely ruled out as a con - founder . as noted by boffetta et al . ( 2009 ) : the excess leukemia mortality in the sbr industryis in line with what would be expected from exposure to the established carcinogen , 1,3-butadiene with no evidence for an amplified effect from the co - exposure to styrene . stronger associations between styrene exposure and cancer risk were not observed in rpc workers . in addition , there was no indication of an increased cancer risk with increased styrene exposure within studies . thus , when weighting these studies accordingly , the evidence for a lack of an effect becomes even stronger . although the ntp ( 2011 ) profile for styrene suggests increased risks for lymphohematopoietic cancers , each of these cancer types is unique , with an independent mode of action ( schottenfeld and fraumeni 2006 ) , and there were no consistent associations with any specific lymphohematopoietic cancer either within or across studies . risks of other types of cancer , such as pancreatic and esophageal cancer , were also not consistently observed among studies . taken together , the evidence does not support the profile 's suggestion that styrene exposure is associated with increased incidence of , or mortality from , lymphohematopoietic , pancreatic , or esophageal cancer and does not meet the ntp criteria for limited evidence of carcinogenicity in humans . the ntp profile for styrene states : styrene caused lung tumors in several strains of mice and by two different routes of exposure ( ntp 2011 ) . there is inconsistency in the tumor incidence among different strains of mice , however , and each study that reports lung tumors in mice also suffers from limitations , as described below . risk estimates were reported as one of the following : standardized mortality ratio ( smr ) , standardized incidence ratio ( sir ) , relative risk ( rr ) , or standardized risk ratio ( srr ) . statistically significant findings indicated in bold . if 95% ci was not provided , statistical significance was indicated in study . the total includes the entire study cohort , it is not the sum of all the observed cases by exposure category . compared to the us population . smr not calculated by authors if observed <3 . in the only chronic inhalation study of styrene in mice , with exposures of 0 , 20 , 40 , 80 , or 160 ppm styrene vapor , increased incidence rates of bronchioalveolar adenomas ( benign tumors ) were observed in male and female cd-1 mice , but only at the end of the 24-month study period and with no dose response pattern ( cruzan et al . 2001 ) . females exposed to the highest dose also had an increased incidence ( 14% ) of bronchioalveolar carcinomas ( malignant tumors ) at the end of the study . the historical control incidence rates of lung tumors for female cd-1 mice ranged from 04% for the laboratory , based on five different studies , and from 013.5% for the animal supplier ( charles river ) , based on nine different studies ( cruzan et al . the adenomas and carcinomas did not differ in tumor morphology between control and treated mice , and histopathologic changes were observed in the terminal bronchioles at all exposure concentrations at 12- , 18- , and 24-month time points . these changes included decreased eosinophilic staining of clara cells and epithelial hyperplasia that extended into the alveolar ducts . abbreviations used : obs = observed cases ; sir = standardized incidence ratio ; 95% ci = 95% confidence interval ; smr = standardized mortality ratio ; rr = relative risk ; ppm = parts per million ; ppm - yr = ppm per day x number of years exposed values were divided by 100 for comparison . the 1-year cutoff was used in the 1993 study ; the 2-year cutoff was used in the 1994 study . in a chronic oral gavage study , male b6c3f1 mice were treated with 0 , 150 , or 300 mg / kg - day styrene in corn oil ( nci 1979 ) . males treated with the highest dose of styrene showed an increased incidence ( 18% ) of combined bronchioalveolar adenomas and carcinomas . the tumors were only observed at the end of the 21-month study period , and there was no increase in tumor incidence in females in any dose group . no lung tumors were observed in the 20 vehicle controls , even though the historical control incidence for untreated controls at the laboratory ranged from 020% . nci determined that the historical control data were insufficient for vehicle - treated controls . the background document ( ntp 2008 ) examined vehicle controls from two studies in the same laboratory ( litton bionetics ) , as well as 14 studies from a nearby laboratory ( hazleton laboratories ) , and concluded that the tumor incidence in high dose males in the styrene study could be considered as outside the historical control range . the use of historical controls from a different testing laboratory is not scientifically supported , however . other investigators have recommended that historical control comparisons should only use controls from the same testing laboratory because of the statistically significant inter - laboratory variability that has been observed in control mouse lung tumor incidence ( haseman et al . thus , the new historical control data used by ntp are not valid for assessing the nci study . because of the discrepancy in control incidence and the tumor response at only one site and in one sex , nci ( 1979 ) concluded that under the conditions of this bioassay , no convincing evidence for the carcinogenicity of the compound was obtained in b6c3f1 mice of either sex . in other chronic oral gavage studies , an increased incidence of combined adenomas and carcinomas was observed in the lungs of male and female o20 mice treated with 1,350 mg / kg styrene in olive oil ( ponomarkov and tomatis 1978 ) . treatment with this dose of styrene was associated with the presence of severe lung congestion and early mortality ; the average age of death for treated males and females was 32 and 49 weeks , respectively , compared to 88 and 85 weeks , respectively , for the male and female vehicle controls . lung tumors were observed in 20/23 ( 87% ) of styrene - treated males and 32/32 ( 100% ) of styrene - treated females , compared to 8/19 ( 42% ) of male and 14/21 ( 67% ) of female vehicle controls , after adjusting for early mortality . lung tumors were also observed in 34/53 ( 64% ) of male and 25/47 ( 53% ) female untreated controls , indicating that the o20 mouse strain is particularly sensitive to the induction of lung tumors . the incidence in the styrene - treated males was significantly higher than the vehicle controls only , whereas in styrene - treated females , the incidence was significantly higher than both control groups . the increased incidence and early appearance of lung tumors could possibly indicate a carcinogenic effect for styrene in o20 mice . this experiment , however , has severe limitations , since the dose used was obviously very high , causing severe toxic effects and an early mortality . results from additional studies are needed before a final evaluation of the carcinogenicity of styrene in rodents can be made the same authors , using a similar study design , and a dose of 300 mg / kg styrene reported no increase in the incidence of tumors of any type in styrene - treated male and female c57b1 mice ( ponomarkov and tomatis 1978 ) . in addition to the inhalation and oral gavage studies , styrene carcinogenicity was examined in mice after parenteral exposure . although not a two - year carcinogenicity study , intraperitoneal injection of styrene into a / j mice for seven weeks did not result in an increased incidence of lung or other types of tumors at sacrifice 20 weeks later ( brunnemann et al . there were no increased tumor incidence rates reported in seven chronic rat studies of styrene that used various rat strains and exposure routes , including inhalation exposure in sprague - dawley ( sd ) rats ( cruzan et al . 1998 ; conti et al . 1988 ; jersey et al . 1978 ) , oral gavage in sd , f344 , and bd iv rats ( conti et al . 1988 ; nci 1979 ; ponomarkov and tomatis 1978 ) , and drinking water in sd rats ( beliles et al . 1985 ) . the ntp profile for styrene states : the evidence from studies in rats is insufficient for reaching a conclusion concerning the carcinogenicity of styrene ( ntp 2011 ) . that is , there is a lack of generality of the tumorigenic response among rodent species . it is unclear why ntp did not consider negative rat bioassay results as evidence against styrene 's general carcinogenicity . in summary , there is evidence that styrene causes an increased incidence of lung tumors in mice after inhalation exposure . other studies in mice that used oral gavage as the exposure route were equivocal , and tumor incidence was not increased in one study that used intraperitoneal injection . thus , an increased tumor incidence caused by styrene exposure has only been observed in one experimental animal species and at one tissue site . the tumors observed in mice after inhalation of styrene were mostly benign and occurred at the end of the chronic study period , causing no early mortality . in addition , the tumors were observed in the presence of lung toxicity in mouse strains with a high background incidence of lung tumors ( cruzan et al . , the animal data for styrene do not meet the ntp criteria for sufficient evidence of carcinogenicity in experimental animals . neither lung toxicity nor lung tumors have been observed in humans exposed to styrene and , as described below , the proposed mode of action for styrene - induced lung tumors in mice is not applicable to humans , or even to other rodent species . as noted above , styrene induces lung toxicity in mice , but not in rats or humans . in this section , the mode - of - action data that indicate the species - specific origin of this toxicity and how this toxicity is the likely cause of tumor formation in the mouse lung are described . these data include the results of studies examining differences in styrene metabolism across species and localized metabolism of styrene in the mouse lung , as well as genotoxicity data that strongly suggest a non - genotoxic mode of action for styrene - induced lung tumors in mice . inhalation exposures to styrene induce lung toxicity and subsequent tumors in mice and nasal toxicity without tumor development in mice and rats ( cruzan et al . lung toxicity has been reported as decreased cytoplasmic staining and increased replication of clara cells of the mouse bronchiolar epithelium , cell crowding in the terminal bronchioles , decreased eosinophilia of the mouse bronchiolar epithelium , and hyperplasia of clara cells in the terminal bronchioles ( cruzan et al . 2001 ) observed a progression from decreased eosinophilia to hyperplasia of the terminal bronchiolar epithelium , and finally , to hyperplasia that extended into the alveolar ducts . with increasing duration , the exposure concentration at which effects were seen also decreased , such that mice in each dose group were affected by the end of the study . the differences in styrene - induced toxicity among mice , rats , and humans can be explained by differences in styrene metabolism . styrene metabolism occurs mainly in the liver and the lung , and results primarily in the formation of the weakly genotoxic metabolite , styrene-7,8-oxide ( so ) ( iarc 2002 ) . so can be detoxified by glutathione conjugation or by conversion to styrene glycol by microsomal epoxide hydrolase ( iarc 2002 ) . styrene can also be metabolized by conversion to pheny - lacetic and phenylaceturic acids ( paa pathway ) , or by oxidation of its benzene ring , which can lead to the formation of 4-vinylphenol ( 4-vp ) ( iarc 2002 ) . based on measurements of urinary metabolites , humans metabolize styrene almost exclusively via the so - epoxide hydrolase pathway ( johanson et al . the ring oxidation and paa pathways play very small roles in styrene metabolism in humans and are used much more in the metabolism of styrene in mice than in rats , indicating that there are major species differences in the overall metabolism of styrene ( johanson et al . the formation of so from styrene is catalyzed by cytochrome p450 . while cyp2e1 is the predominant enzyme for styrene metabolism in the liver , metabolism by cyp2f ( 2f1 in humans , 2f2 in mice , and 2f4 in rats ) is necessary for toxicity to occur in the lung ( hynes et al . there are significant species differences with respect to the activities and concentrations of the cyp2f enzymes in the lung . in mice and rats , the cyp2f enzymes readily metabolize styrene , whereas in humans , cyp2f1 does not appear to metabolize styrene at all ( green et al . clara cells are the major cell type in the lung that metabolizes styrene to so following inhalation exposure , and in mice , these cells are numerous and are spread throughout the airways ( green 2000 ) . in rats , they are significantly fewer in number , particularly in the terminal bronchiolar region ( green 2000 ) . in humans , clara cells are rare and can be found in small numbers in the distal bronchioles ( green 2000 ) . ( 1999 ) demonstrated that mouse clara cells produce five times more so than rat clara cells . thus , the cells that metabolize styrene in the lung differ in their number , location , and specificity among mice , rats , and humans . physiologically - based pharmacokinetic ( pbpk ) modeling has shown that the target tissue concentration of so is primarily due to localized tissue metabolism of styrene rather than delivery of so to the lung via the arterial blood ( sarangapani et al . this model also predicted that at a given airborne concentration of styrene , the level of total so in mouse terminal bronchioles is approximately 10 times higher than in rats and 100 times higher than in humans ( sarangapani et al . 2002 ) . evidence that local metabolism of styrene is responsible for the lung toxicity of styrene is given by the fact that circulating levels of so do not correlate with lung tumor incidence . blood levels of so were much higher in rats at non - tumorigenic concentrations than in mice at levels associated with an increased incidence of lung tumors ( cruzan et al . in addition , metabolism and cytotoxicity occur in the mouse lung after oral exposure to styrene ( green et al . 2001b ) , indicating that systemically absorbed concentrations of styrene are preferentially metabolized in the mouse lung . a recent study using cyp2f2 knockout mice demonstrated that both styrene and so toxicity in the mouse lung require cyp2f2 metabolism ( cruzan et al . studies in mice using a cyp2f2 inhibitor have also shown that metabolism of styrene by cyp2f2 is necessary to cause lung toxicity ( green et al . inhibition of cyp2f2 also inhibited the cytotoxicity of 4-vp ( carlson 2002 ) , which is 10 times as toxic to the mouse lung as styrene and 5 times as toxic as so ( carlson et al . 2002 ) . because 4-vp can be metabolized by cyp2f2 into further ring - oxidized metabolites of styrene ( carlson et al . 2011 ) , this indicates that there is a subsequent metabolite of 4-vp that is responsible for lung cytotoxicity in mice . as mentioned above , the ring oxidation pathway of styrene metabolism is more predominant in mice than in rats or humans , and it has been shown that intraperitoneal administration of 4-vp induced cytotoxicity in the terminal bronchioles of mice , but not rats ( cruzan et al . the ntp profile for styrene states that the proposed mechanisms for the carcinogenicity of styrene include both genotoxic and non - genotoxic pathways . extensive data show , however , that a genotoxic mode of action for styrene is unlikely . although so can adduct to proteins and dna , low levels of so - dna adducts have been observed in vivo . increases in so - dna adducts have not been observed in mouse versus rat lung or mouse lung versus mouse liver after inhalation exposure to styrene ; thus , the increased incidence of lung tumors in mice is not accompanied by an increase in dna adducts ( boogaard et al . there have been some positive results from in vitro assays for chromosomal effects , but in vivo studies do not indicate increases in chromosomal aberrations or micronuclei . a small increase in sister chromatid exchanges ( sces ) has been observed in experimental animal studies of styrene , but in humans there has been no observed increase in sces in a dose - responsive manner . in a study by vodicka et al . ( 2004 ) , there was no clear association between styrene exposure and chromosomal aberrations , micronuclei , single strand breaks , and dna repair in styrene - exposed workers . although so is directly genotoxic in vitro , oral administration of so to mice and rats did not lead to systemic tumors , despite producing systemic concentrations of so that were equal to or higher than those resulting from inhalation exposures ( sarangapani et al . tumors were only observed at the site of contact , the forestomach ( conti et al . 1988 ; lijinsky 1986 ; ponomarkov et al . oral exposure to so also induced cell damage , repair , and increased proliferation , suggesting that the mechanism for tumor formation by so in the forestomach is attributable to the increased cell proliferation resulting from the cellular damage inducedby so . this type of non - genotoxic mechanism of action may also explain the increased lung tumor incidence in mice after chronic exposure to styrene . a non - genotoxic mode of action for styrene - induced lung tumors in mice is the most plausible mechanism for styrene carcinogenicity . increased tumor incidence caused by styrene exposure has only been observed in one species and at one tissue site . the tumors were mostly benign and were observed late in life , causing no early mortality . tumors were accompanied by organ toxicity and cell turnover , in the form of clara cell damage and proliferation . further support for a non - genotoxic mode of action comes from a 20-week study in which styrene exposure via intraperitoneal injection did not induce tumors of any type in mice ( brunnemann et al . in addition , target organ metabolism of styrene is necessary for the observed toxicity and tumor formation , as oral administration of the genotoxic primary metabolite , so , does not induce systemic tumors , and circulating levels of so are not associated with increased tumor incidence . the genotoxicity data for styrene are inconsistent , and dna adducts are observed at low levels after exposure and are not associated with tumor incidence . the mechanistic evidence that suggests a specific , non - genotoxic mode of action for styrene in the responding animals is of questionable applicability to other animals , other tissues , and to humans . styrene - induced cytotoxicity occurs in organs with high levels of the cyp2f family of enzymes . mouse lungs have a larger fraction of cyp2f2-containing clara cells than rat or human lungs , and the metabolism rates of styrene by the cyp2f family are higher in mice than in rats and are virtually non - existent in humans . in addition , styrene metabolites are removed more rapidly in rat and human tissues compared to mouse tissues ( green et al . consistent with these data , no styrene - induced toxicity , hyperplasia , or tumors have been observed in rat or human lungs . because the levels of cyp2f enzymes are higher in rat lungs compared to human lungs , the lack of lung toxicity or tumor formation in rats makes it very unlikely that a chemical that causes lung tumors by this mode of action in mice , but not rats , will cause human lung tumors . in accordance with the preceding mechanistic information , epidemiology data do not show an increased incidence of lung cancer , or any cancer type , in humans exposed to styrene . the ntp ( 2011 ) profile for styrene states that : although styrene disposition differs quantitatively among species , no qualitative differences between humans and experimental animals have been demonstrated that contradict the relevance of cancer studies in rodents for evaluation of human hazard . detection of styrene-7,8-oxide - dna adducts at base - pairing sites and chromosomal aberrations in lymphocytes of styrene - exposed workers supports the potential human cancer hazard from styrene through a genotoxic mode of action . there is no concordance among the human , rodent , and mode - of - action data on the effects of styrene . the styrene - induced lung toxicity and tumor formation observed in mice are species - specific , and the mechanistic data strongly suggest that these tumors result from the lung toxicity that depends upon the localized metabolism of styrene by mouse cyp2f2 . in humans , cyp2f1 does not appear to metabolize styrene . in addition , the metabolic pathway resulting in the formation of 4-vp , which may be the substrate for the cyp2f2-dependent cytotoxic metabolite in mouse lung , is a minor pathway in humans compared to mice . there is substantial evidence that the so mutagenicity , cited by the ntp ( 2011 ) profile as the basis for applying the mouse results to human risk projection , is not an important factor in vivo even in animals , is not consistent with the localization of mouse tumors , and in fact is not responsible for the mouse tumor response . the epidemiology data as a whole do not suggest that styrene exposure is associated with any specific cancer type in humans , either within or among studies . the ntp profile , however , interprets these data as suggesting that styrene exposure increases the incidence of lymphohematopoietic cancers , and possibly pancreatic and esophageal cancers , in humans ( ntp 2011 ) . even if one accepts this interpretation , there have been no corresponding responses observed in experimental animals , as no increased incidences of lymphohematopoietic , pancreatic , or esophageal tumors have been reported in styrene - exposed animals . the experimental animal data also indicate that orally - administered styrene does not induce tumors systemically ; although the data are unclear , in some studies with inhalation and oral exposures , an increased incidence of tumors was observed specifically in the mouse lung . the studies in which so - dna adducts and chromosomal aberrations were detected in styrene - exposed workers do not support a carcinogenic role for styrene . these studies are limited by their small size and lack of controlling for potential confounders . in addition , styrene - exposed rodents form similar dna adducts , with higher levels observed in rats than in mice , suggesting that these adducts are not associated with an increased incidence of tumors ( boogaard et al . furthermore , agents that are known or expected to cause lymphohematopoietic cancers in humans are believed to act through immune dysregulation and not through dna damage ( alexander et al . a genotoxic mode of action for styrene , either in animals or in humans , is not plausible . besides the genotoxicity data for styrene being inconsistent , styrene exposure has been associated with an increased tumor incidence in only one species and one site , mouse lung . the late onset and mostly benign lung tumors observed in mice were accompanied by lung cytotoxicity and cell proliferation and were dependent on lung - specific metabolism of styrene . these data suggest a non - genotoxic mechanism of action attributable to increased cell proliferation in the mouse lung resulting from the cellular damage induced upon styrene metabolism . taken together , the human , experimental animal , and mode - of - action data for the effects of styrene do not support the classification of styrene as a human carcinogen . the lack of correspondence of tumor incidence and tumor type among rodents , even within the same species , and humans indicates that there has been no particular type of cancer consistently observed among all available studies and renders the argument for the human carcinogenicity of styrene as implausible . the various data indicate that the only plausible mechanism for styrene - induced carcinogenesis is a non - genotoxic mode of action that is specific to the mouse lung . the ntp ( 2011 ) classification of styrene as reasonably anticipated to be ahuman carcinogen based on limited evidence of carcinogenicity in humans , sufficient evidence of carcinogenicity in animals , and supporting mechanistic data is not scientifically supported , given that the available data do not meet these criteria . because of this , styrene should not be listed in ntp 's report on carcinogens . the rationale for using rodent bioassay results as indicators of possible human carcinogenicity rests on the broad similarity among mammals in anatomy , physiology , and biochemistry ; the applicability of a rodent response as an indicator of potential human risk amounts to hypothesizing that , owing to this underlying commonality , the carcinogenic processes responsible for the animal results could also plausibly occur in humans . that is , one is hypothetically generalizing the phenomenon from the particular animal species showing the response to other mammals , including humans . for styrene , however , it is clear that the processes responsible for the tumorigenesis observed in mice do not occur in rats . it is not only that rats do not show a tumor impact of styrene inhalation ( the hypothesized generality of which across mammals is the basis for inferring its relevance to humans ) , but also that the specific mode of action the tissue - specific metabolic activation is not present . in short , moreover , there is no indication that the mode of action would be present in humans , either . to bring together experimental animal , human , and mode - of - action data into an overall weight - of - evidence conclusion about the potential for human carcinogenicity , one seeks to characterize the likelihood of a common thread that ties together the evidence from the different sources and proposes a biologically plausible line of reasoning as to why a potential hazard in humans is indicated . for styrene the mouse tumor responses are best interpreted as a species - specific phenomenon that does not apply to rats , so any sufficiency of animal evidence does not apply to all animals and to the degree that it does not , its applicability to humans is also questionable , because one would have to propose why , against all available evidence , humans should be like the mice and not like the rats in their response to styrene . there are no consistent responses among the human data of the kind that one would expect if there were true biological causation . the diversity of proposed tumor endpoints in human studies raises more questions than it answers why is it that styrene would affect some tumor responses in some studies , and other responses ( requiring other modes of action ) in other studies ? if there were truly a mode of action of sufficient generality and broadness to cause such a variety of tumor responses in humans , why is there no sign of it and why is there no indication of hematopoietic cancers in rats or in mice ? taken together , these data do not support the characterization of styrene as reasonably anticipated to be a human carcinogen and styrene should not be listed in ntp 's report on carcinogens .

styrene was listed as reasonably anticipated to be a human carcinogen in the twelfth edition of the national toxicology program 's report on carcinogens based on what we contend are erroneous findings of limited evidence of carcinogenicity in humans , sufficient evidence of carcinogenicity in experimental animals , and supporting mechanistic data . the epidemiology studies show no consistent increased incidence of , or mortality from , any type of cancer . in animal studies , increased incidence rates of mostly benign tumors have been observed only in certain strains of one species ( mice ) and at one tissue site ( lung ) . the lack of concordance of tumor incidence and tumor type among animals ( even within the same species ) and humans indicates that there has been no particular cancer consistently observed among all available studies . the only plausible mechanism for styrene - induced carcinogenesis a non - genotoxic mode of action that is specific to the mouse lung is not relevant to humans . as a whole , the evidence does not support the characterization of styrene as reasonably anticipated to be a human carcinogen , and styrene should not be listed in the report on carcinogens .

INTRODUCTION NTP CRITERIA FOR LIMITED OR SUFFICIENT EVIDENCE EPIDEMIOLOGY STUDIES SPECIES-SPECIFIC EFFECTS IN EXPERIMENTAL ANIMALS MODE-OF-ACTION DATA LACK OF CONCORDANCE AMONG HUMAN, EXPERIMENTAL ANIMAL, AND MODE-OF-ACTION DATA CONCLUSIONS

styrene was listed in the twelfth edition of the national toxicology program 's ( ntp 's ) report on carcinogens ( ntp 2011 ) . in its report , ntp ( 2011 ) states : styrene is reasonably anticipated to be a human carcinogen based on limited evidence of carcinogenicity from studies in humans , sufficient evidence of carcinogenicity from studies in experimental animals , and supporting data on mechanisms of carcinogenesis . as described in more detail below , the effects of styrene observed in experimental animals do not meet the standard of sufficient evidence in animals and the underlying mode of action is species - specific and not applicable to humans . in addition , the human data do not consistently show increased incidence of or mortality from cancer , and the epidemiology studies collectively do not support the standard of limited evidence . thus , based on ntp 's stated criteria , the evidence shows that styrene does not meet the standard for reasonably anticipated to be a human carcinogen and ntp ( 2011 ) states the following criteria for limited evidence of carcinogenicity in humans : there is limited evidence of carcinogenicity from studies in humans , which indicates that causal interpretation is credible , but that alternative explanations , such as chance , bias , or confounding factors , could not adequately be excluded . it is important to realize that limited evidence still requires a positive finding that a causal explanation is credible ; it is not simply applied when the data are inconsistent or inconclusive , and the mere presence of some positive evidence in some studies is not by itself grounds to conclude that a causal explanation is credible . instead , when results are mixed or inconsistent , an evaluation of all of the data must consider whether it is credible to hold that a truly causal relationship exists ( and the studies failing to show it do so because of chance , low statistical power , or because the true responses are somehow obscured by extraneous factors ) or whether it is more credible that there is no causal effect ( and the studies appearing to show an effect of exposure are in fact only showing chance findings or the effects of biases or confounding factors ) . that is , the limited evidence category does not simply consist of cases for which there are some positive and some negative results ; it is only when a case for a credible ( albeit unproven ) causative effect can be made that the limited evidence characterization should be applied . in our view , when all the human evidence is evaluated , and the low numbers of observed cases and the lack of consistent patterns in cancer outcomes within and across cohorts , combined with concerns about co - exposures and confounding are considered , one comes to the conclusion that a causal relationship between styrene exposure and human cancer is not credible , and the standards of limited evidence are not met . ntp ( 2011 ) also states the following criteria for sufficient evidence of carcinogenicity in experimental animals : there is an increased incidence of malignant and/or a combination of malignant and benign tumors ( 1 ) in multiple species or at multiple tissue sites , ( 2 ) by multiple routes of exposure , or ( 3 ) to an unusual degree with regard to incidence , site , or type of tumor , or age at onset . as described in more detail below , the data show that the ntp criteria for sufficient evidence in animals are not met for styrene . the ntp profile for styrene suggests that epidemiology studies of workers exposed to styrene show increased incidence of or mortality from lymphohematopoietic cancer and that some studies in the reinforced plastics industry provide suggestive evidence for increased incidence of or mortality due to pancreatic and esophageal tumors ( ntp 2011 ) . when considering the epidemiology evidence as a whole , however , there are no consistent associations between styrene exposure and any specific cancer type either within or among studies . according to the background document ( ntp 2008 ) , the major epidemiology studies of styrene focus on 10 cohorts from the reinforced plastics and composites ( rpc ) , styrene - butadiene latex rubber ( sbr ) , and styrene / polystyrene ( ps ) industries , an occupational cohort in finland reporting urinary concentrations of a styrene metabolite ( anttila et al . just as it is unlikely that these negative associations are reflective of a protective mechanism for styrene , the few positive associations are unlikely to reflect a causal association . there are no consistent associations seen across studies of the same cohort in the sbr industry , and co - exposure to 1,2-butadiene likely confounded results . although the ntp ( 2011 ) profile for styrene suggests increased risks for lymphohematopoietic cancers , each of these cancer types is unique , with an independent mode of action ( schottenfeld and fraumeni 2006 ) , and there were no consistent associations with any specific lymphohematopoietic cancer either within or across studies . taken together , the evidence does not support the profile 's suggestion that styrene exposure is associated with increased incidence of , or mortality from , lymphohematopoietic , pancreatic , or esophageal cancer and does not meet the ntp criteria for limited evidence of carcinogenicity in humans . there is inconsistency in the tumor incidence among different strains of mice , however , and each study that reports lung tumors in mice also suffers from limitations , as described below . in the only chronic inhalation study of styrene in mice , with exposures of 0 , 20 , 40 , 80 , or 160 ppm styrene vapor , increased incidence rates of bronchioalveolar adenomas ( benign tumors ) were observed in male and female cd-1 mice , but only at the end of the 24-month study period and with no dose response pattern ( cruzan et al . the historical control incidence rates of lung tumors for female cd-1 mice ranged from 04% for the laboratory , based on five different studies , and from 013.5% for the animal supplier ( charles river ) , based on nine different studies ( cruzan et al . the background document ( ntp 2008 ) examined vehicle controls from two studies in the same laboratory ( litton bionetics ) , as well as 14 studies from a nearby laboratory ( hazleton laboratories ) , and concluded that the tumor incidence in high dose males in the styrene study could be considered as outside the historical control range . other investigators have recommended that historical control comparisons should only use controls from the same testing laboratory because of the statistically significant inter - laboratory variability that has been observed in control mouse lung tumor incidence ( haseman et al . in other chronic oral gavage studies , an increased incidence of combined adenomas and carcinomas was observed in the lungs of male and female o20 mice treated with 1,350 mg / kg styrene in olive oil ( ponomarkov and tomatis 1978 ) . the increased incidence and early appearance of lung tumors could possibly indicate a carcinogenic effect for styrene in o20 mice . results from additional studies are needed before a final evaluation of the carcinogenicity of styrene in rodents can be made the same authors , using a similar study design , and a dose of 300 mg / kg styrene reported no increase in the incidence of tumors of any type in styrene - treated male and female c57b1 mice ( ponomarkov and tomatis 1978 ) . there were no increased tumor incidence rates reported in seven chronic rat studies of styrene that used various rat strains and exposure routes , including inhalation exposure in sprague - dawley ( sd ) rats ( cruzan et al . that is , there is a lack of generality of the tumorigenic response among rodent species . thus , an increased tumor incidence caused by styrene exposure has only been observed in one experimental animal species and at one tissue site . , the animal data for styrene do not meet the ntp criteria for sufficient evidence of carcinogenicity in experimental animals . neither lung toxicity nor lung tumors have been observed in humans exposed to styrene and , as described below , the proposed mode of action for styrene - induced lung tumors in mice is not applicable to humans , or even to other rodent species . in this section , the mode - of - action data that indicate the species - specific origin of this toxicity and how this toxicity is the likely cause of tumor formation in the mouse lung are described . these data include the results of studies examining differences in styrene metabolism across species and localized metabolism of styrene in the mouse lung , as well as genotoxicity data that strongly suggest a non - genotoxic mode of action for styrene - induced lung tumors in mice . lung toxicity has been reported as decreased cytoplasmic staining and increased replication of clara cells of the mouse bronchiolar epithelium , cell crowding in the terminal bronchioles , decreased eosinophilia of the mouse bronchiolar epithelium , and hyperplasia of clara cells in the terminal bronchioles ( cruzan et al . the differences in styrene - induced toxicity among mice , rats , and humans can be explained by differences in styrene metabolism . styrene metabolism occurs mainly in the liver and the lung , and results primarily in the formation of the weakly genotoxic metabolite , styrene-7,8-oxide ( so ) ( iarc 2002 ) . the ring oxidation and paa pathways play very small roles in styrene metabolism in humans and are used much more in the metabolism of styrene in mice than in rats , indicating that there are major species differences in the overall metabolism of styrene ( johanson et al . while cyp2e1 is the predominant enzyme for styrene metabolism in the liver , metabolism by cyp2f ( 2f1 in humans , 2f2 in mice , and 2f4 in rats ) is necessary for toxicity to occur in the lung ( hynes et al . there are significant species differences with respect to the activities and concentrations of the cyp2f enzymes in the lung . in mice and rats , the cyp2f enzymes readily metabolize styrene , whereas in humans , cyp2f1 does not appear to metabolize styrene at all ( green et al . in humans , clara cells are rare and can be found in small numbers in the distal bronchioles ( green 2000 ) . thus , the cells that metabolize styrene in the lung differ in their number , location , and specificity among mice , rats , and humans . this model also predicted that at a given airborne concentration of styrene , the level of total so in mouse terminal bronchioles is approximately 10 times higher than in rats and 100 times higher than in humans ( sarangapani et al . in addition , metabolism and cytotoxicity occur in the mouse lung after oral exposure to styrene ( green et al . 2001b ) , indicating that systemically absorbed concentrations of styrene are preferentially metabolized in the mouse lung . a recent study using cyp2f2 knockout mice demonstrated that both styrene and so toxicity in the mouse lung require cyp2f2 metabolism ( cruzan et al . inhibition of cyp2f2 also inhibited the cytotoxicity of 4-vp ( carlson 2002 ) , which is 10 times as toxic to the mouse lung as styrene and 5 times as toxic as so ( carlson et al . 2011 ) , this indicates that there is a subsequent metabolite of 4-vp that is responsible for lung cytotoxicity in mice . as mentioned above , the ring oxidation pathway of styrene metabolism is more predominant in mice than in rats or humans , and it has been shown that intraperitoneal administration of 4-vp induced cytotoxicity in the terminal bronchioles of mice , but not rats ( cruzan et al . the ntp profile for styrene states that the proposed mechanisms for the carcinogenicity of styrene include both genotoxic and non - genotoxic pathways . extensive data show , however , that a genotoxic mode of action for styrene is unlikely . increases in so - dna adducts have not been observed in mouse versus rat lung or mouse lung versus mouse liver after inhalation exposure to styrene ; thus , the increased incidence of lung tumors in mice is not accompanied by an increase in dna adducts ( boogaard et al . a small increase in sister chromatid exchanges ( sces ) has been observed in experimental animal studies of styrene , but in humans there has been no observed increase in sces in a dose - responsive manner . oral exposure to so also induced cell damage , repair , and increased proliferation , suggesting that the mechanism for tumor formation by so in the forestomach is attributable to the increased cell proliferation resulting from the cellular damage inducedby so . this type of non - genotoxic mechanism of action may also explain the increased lung tumor incidence in mice after chronic exposure to styrene . a non - genotoxic mode of action for styrene - induced lung tumors in mice is the most plausible mechanism for styrene carcinogenicity . increased tumor incidence caused by styrene exposure has only been observed in one species and at one tissue site . further support for a non - genotoxic mode of action comes from a 20-week study in which styrene exposure via intraperitoneal injection did not induce tumors of any type in mice ( brunnemann et al . in addition , target organ metabolism of styrene is necessary for the observed toxicity and tumor formation , as oral administration of the genotoxic primary metabolite , so , does not induce systemic tumors , and circulating levels of so are not associated with increased tumor incidence . the mechanistic evidence that suggests a specific , non - genotoxic mode of action for styrene in the responding animals is of questionable applicability to other animals , other tissues , and to humans . mouse lungs have a larger fraction of cyp2f2-containing clara cells than rat or human lungs , and the metabolism rates of styrene by the cyp2f family are higher in mice than in rats and are virtually non - existent in humans . consistent with these data , no styrene - induced toxicity , hyperplasia , or tumors have been observed in rat or human lungs . because the levels of cyp2f enzymes are higher in rat lungs compared to human lungs , the lack of lung toxicity or tumor formation in rats makes it very unlikely that a chemical that causes lung tumors by this mode of action in mice , but not rats , will cause human lung tumors . in accordance with the preceding mechanistic information , epidemiology data do not show an increased incidence of lung cancer , or any cancer type , in humans exposed to styrene . the ntp ( 2011 ) profile for styrene states that : although styrene disposition differs quantitatively among species , no qualitative differences between humans and experimental animals have been demonstrated that contradict the relevance of cancer studies in rodents for evaluation of human hazard . detection of styrene-7,8-oxide - dna adducts at base - pairing sites and chromosomal aberrations in lymphocytes of styrene - exposed workers supports the potential human cancer hazard from styrene through a genotoxic mode of action . the styrene - induced lung toxicity and tumor formation observed in mice are species - specific , and the mechanistic data strongly suggest that these tumors result from the lung toxicity that depends upon the localized metabolism of styrene by mouse cyp2f2 . in humans , cyp2f1 does not appear to metabolize styrene . in addition , the metabolic pathway resulting in the formation of 4-vp , which may be the substrate for the cyp2f2-dependent cytotoxic metabolite in mouse lung , is a minor pathway in humans compared to mice . there is substantial evidence that the so mutagenicity , cited by the ntp ( 2011 ) profile as the basis for applying the mouse results to human risk projection , is not an important factor in vivo even in animals , is not consistent with the localization of mouse tumors , and in fact is not responsible for the mouse tumor response . the epidemiology data as a whole do not suggest that styrene exposure is associated with any specific cancer type in humans , either within or among studies . even if one accepts this interpretation , there have been no corresponding responses observed in experimental animals , as no increased incidences of lymphohematopoietic , pancreatic , or esophageal tumors have been reported in styrene - exposed animals . the experimental animal data also indicate that orally - administered styrene does not induce tumors systemically ; although the data are unclear , in some studies with inhalation and oral exposures , an increased incidence of tumors was observed specifically in the mouse lung . the studies in which so - dna adducts and chromosomal aberrations were detected in styrene - exposed workers do not support a carcinogenic role for styrene . a genotoxic mode of action for styrene , either in animals or in humans , is not plausible . besides the genotoxicity data for styrene being inconsistent , styrene exposure has been associated with an increased tumor incidence in only one species and one site , mouse lung . these data suggest a non - genotoxic mechanism of action attributable to increased cell proliferation in the mouse lung resulting from the cellular damage induced upon styrene metabolism . taken together , the human , experimental animal , and mode - of - action data for the effects of styrene do not support the classification of styrene as a human carcinogen . the lack of correspondence of tumor incidence and tumor type among rodents , even within the same species , and humans indicates that there has been no particular type of cancer consistently observed among all available studies and renders the argument for the human carcinogenicity of styrene as implausible . the various data indicate that the only plausible mechanism for styrene - induced carcinogenesis is a non - genotoxic mode of action that is specific to the mouse lung . the ntp ( 2011 ) classification of styrene as reasonably anticipated to be ahuman carcinogen based on limited evidence of carcinogenicity in humans , sufficient evidence of carcinogenicity in animals , and supporting mechanistic data is not scientifically supported , given that the available data do not meet these criteria . because of this , styrene should not be listed in ntp 's report on carcinogens . it is not only that rats do not show a tumor impact of styrene inhalation ( the hypothesized generality of which across mammals is the basis for inferring its relevance to humans ) , but also that the specific mode of action the tissue - specific metabolic activation is not present . in short , moreover , there is no indication that the mode of action would be present in humans , either . for styrene the mouse tumor responses are best interpreted as a species - specific phenomenon that does not apply to rats , so any sufficiency of animal evidence does not apply to all animals and to the degree that it does not , its applicability to humans is also questionable , because one would have to propose why , against all available evidence , humans should be like the mice and not like the rats in their response to styrene . the diversity of proposed tumor endpoints in human studies raises more questions than it answers why is it that styrene would affect some tumor responses in some studies , and other responses ( requiring other modes of action ) in other studies ? if there were truly a mode of action of sufficient generality and broadness to cause such a variety of tumor responses in humans , why is there no sign of it and why is there no indication of hematopoietic cancers in rats or in mice ? taken together , these data do not support the characterization of styrene as reasonably anticipated to be a human carcinogen and styrene should not be listed in ntp 's report on carcinogens .

methotrexate ( mtx ) is the most common second - line therapeutic agent used to treat juvenile idiopathic arthritis ( jia ) worldwide . regardless of age or disease subtype , considerable interindividual variability in clinical response and adverse reactions exists with mtx , and thus far , there have been no predictive variables for outcomes in patients taking this medication . since the onset of clinical response may take months to manifest , the risk to benefit ratio early in treatment is altered , as there is risk for toxicity for several weeks to months before knowing if the medication has resulted in a clinical benefit . side effects may compromise efficacy due to patient noncompliance , clinician dose adjustment , or discontinuation even if the drug eventually is medically effective . medication dose alteration or discontinuation in the face of active disease is unacceptable when the alternatives for therapy in childhood arthritis are few and poorly studied in this population . by utilizing pharmacogenomic principles and a personalized therapeutic strategy , we hope to improve efficacy and prevent adverse drug reactions in children taking mtx to treat jia . when exploring the variability in response and toxicity to any medication used in children , a concept often overlooked is ontogeny . the effects of development can be applied at every level of drug disposition and response . these effects range from differences in gastric ph [ 2 , 3 ] and gastric emptying which may affect absorption , to changes in circulating plasma proteins with age that may affect drug distribution . developmental changes in phase i drug biotransformation and phase ii conjugating enzyme expression have the potential to alter drug metabolism , and developmental differences in glomerular filtration rates will affect drug excretion in children compared to adults . common drug biotransformation pathways are also shared with endogenous compounds involved in growth and development , such as testosterone , cortisol , and vitamin d3 , so it is not surprising that some of these pathways may be affected by the rapid growth and maturation of the pediatric patient , for example , during infancy and puberty . the developmental expression of these pathways at different rates or trajectories may also lead to variability in drug disposition and response . although pharmacogenetics appropriately strives to identify the correct dose of the correct drug for the correct person , the impact of development on an individual 's response to a drug must be taken into account . genotyping an individual for variations that affect function is an important step to understanding variability in outcomes ; however , knowing if and when that gene is expressed is a concept important to fully understanding genotype - phenotype relationships in children [ 9 , 10 ] . an approach to investigating hypotheses related to drug outcomes in children can be guided by the following questions . what gene products are quantitatively important in the disposition ( absorption , distribution , metabolism , and excretion ) of the drug in question ? for each gene product , what is the developmental trajectory for the acquisition of functional activity ? is allelic variation in the gene(s ) of interest associated with any functional consequences in vivo ? is there any evidence that allelic variation affects the developmental trajectory of the drug disposition phenotype ? what is the developmental context in which the gene(s ) of interest is / are operating ? this process is also relevant for genes / gene products involved in drug response . partnered with the understanding of genetic variation in an individual , appreciating the changes in gene expression throughout growth and development will allow us to manage the complexity of therapeutics in children . we strive to individualize therapy for children rather than extrapolate from adult experience , which traditionally has been the norm . juvenile idiopathic arthritis ( jia ) , formerly termed juvenile chronic arthritis ( jca ) or juvenile rheumatoid arthritis ( jra ) is one of the most common chronic diseases of childhood , and is an important cause of morbidity and disability in children . this disease is characterized by idiopathic peripheral arthritis with an immunoinflammatory pathogenesis , thought to be triggered by an external antigen . there is also speculation of a genetic predisposition for the disease [ 1215 ] . jia has a heterogeneous phenotypic expression , and includes several disease subtypes , whose classification continues to be revised and validated by clinicians worldwide . despite differences in disease expression between adults and children , like in many pediatric diseases , children are treated with generally the same armamentarium of drugs used to treat ra in adults . with the advent of disease modifying antirheumatic drugs ( dmards ) , the philosophy of treatment changed from simple pain control to prevention of erosions and long - term damage of the joints . methotrexate ( mtx ) , a folic acid antagonist , was approved by the food and drug administration for the treatment of ra in 1988 , and several uncontrolled descriptive studies suggested , and a randomized placebo - controlled double blind clinical trial demonstrated the effectiveness of mtx in children with jia [ 1621 ] . mtx has subsequently become the most common second - line therapeutic agent used to treat juvenile idiopathic arthritis ( jia ) worldwide . although the collective clinical experience with mtx has been vast , there are still unanswered questions about its mechanism of action , and considerable interindividual variability in clinical response and adverse reactions exists [ 2224 ] . thus far , there have been few predictors for efficacy or toxicity in pediatric patients taking this medication , and clinicians essentially dose by trial and error . factors that could contribute to this variability are extensive , and some are unique to the pediatric population . we would like to explore the potential sources of variability that may contribute to outcomes on mtx in jia . methotrexate , a folic acid analog and potent inhibitor of several enzymes within the folate pathway , has been used in low doses for the treatment of rheumatic disease over the last several decades . for rheumatic conditions , the dose range in pediatrics spans 10-fold , ranging from 0.1 mg / kg / dose to 1 mg / kg / dose , administered on a weekly basis . options include oral and subcutaneous administration , but intramuscular and intravenous administration are possible , although less practical in the outpatient setting . before being taken into the body , contributors to variability that can not be overlooked include patient compliance , differences in administered dose , and route of administration . children , who are a fraction of the weight of their adult counterparts , are dosed with the same absolute mtx dose , despite their smaller size . although attention has been brought to this phenomenon , there continues to be little understanding of why children appear to require , relative to body weight , higher doses of mtx or how these doses are tolerated . serum mtx concentrations have not been found useful to predict response or toxicity with little correlation with dose or outcome [ 2527 ] . it is known that mtx acts as a folate antagonist , entering the cells primarily through the reduced folate carrier ( rfc / slc19a1 ) . once intracellular , mtx is bioactivated to a polyglutamated form by folylpolyglutamyl synthase ( fpgs ) , which enhances the pharmacological activity and intracellular retention of mtx . in the ra and pediatric oncology literature , current evidence indicates that the enzymatic addition of glutamate residues to the mtx molecule in vivo ( polyglutamation / mtxglun ) is critical for pharmacologic activity by increasing the intracellular concentration of the drug and increasing its affinity for its therapeutic targets , thereby allowing more opportunity for its inhibitory effects to be exerted upon its target enzymes [ 2931 ] . the initial target of mtx to be identified was dihydrofolate reductase ( dhfr ) , which forms tetrahydrofolate , a precursor required for one carbon donation for synthesis of thymidylate , purines , methionine and serine , remethylation of homocysteine to form methionine , and provision of methyl donors for multiple methyltransferase enzymes . inhibition of thymidylate synthetase ( tyms ) , both directly and indirectly via depletion of tetrahydrofolate , leads to inhibition of pyrimidine biosynthesis with a resultant antiproliferative effect . the interruption of dna synthesis was thought to be the basis for rapidly dividing cell death in cancer cells . subsequently , the list of target genes has been extended to include amino - imidazole carboxamide ribonucleotide ( aicar ) transformylase ( gene name , atic ) , which inhibits de novo purine synthesis and promotes the accumulation of aicar ribotide , inhibiting adenosine deaminase and leading to a build up of adenosine , a potent anti - inflammatory agent [ 32 , 33 ] . adenosine 's effect is also mediated by adenosine a2 receptors ( adora2 ) , which are present on neutrophils , monocytes , lymphocytes and basophils , generally suppressing the immune function of these cells . gamma glutamyl hydrolase ( ggh ) , the enzyme responsible for glutamate removal from mtx , transforms mtx into a form that can be effluxed from the cell by the atp - binding cassette ( abc ) family of transporters . due to the rapid decline in serum drug concentrations , serum mtx concentrations are of limited utility in determining appropriate dosing or management of this medication [ 2527 ] . on the other hand , rbc folate concentrations are established during erythropoiesis and represent the average folate status over the preceding 120 days . by extension , mtx concentrations in rbcs represent a reasonable surrogate biomarker of average drug exposure over a similar period of time . in vitro studies have revealed that the cellular response to folate deprivation is associated with increased expression of fpgs and decreased expression of abcg2 , suggesting that the adaptive cellular response to low folate involves increased polyglutamation to promote the retention of folate . homozygosity for the variant allele of slc19a1 ( 80a / a genotype ) has been associated with increased concentrations of intracellular mtxglun compared to heterozygous or wt genotypes in ra patients . although the data are limited , these examples reveal that allelic variation in these genes resulting in increased or decreased activity may be associated with inter - individual variability in intracellular mtxglun . recent associations between mtxglun and clinical outcomes have also been reported in the adult rheumatology literature [ 33 , 36 , 37 ] . ( defined in adults as mtxglu3 or greater , parent mtx is mtxglu1 ) were associated with a number of improved response measures in ra . the relationship between mtxglun and side effects of the medication has not been established . in children , experience is limited to a single study reporting a total of 38 jia patients , and no relationship between intracellular total mtxglun concentrations and likelihood of response was apparent . as individual mtx polyglutamates differ with respect to their inhibitory effects on target enzymes and inhibition is modulated by folate polyglutamates , it is likely that multiple as yet unidentified factors contribute to variability in the relationship between mtxglun concentrations and efficacy and/or toxicity . in order to better identify factors that may contribute to the inconsistencies in response and toxicity to mtx , we sought to characterize the extent of variability of intracellular mtxglun concentrations in our jia patient population , and to investigate variables that may contribute to mtxglun variability . we have measured intracellular mtxglun concentrations in a cohort of 104 jia patients . in this cohort , total intracellular mtxglun ( mtxglutot , the sum of all individual mtxglun ) concentrations varied 40-fold with a mean of 85.4 48.8 concentrations of mtxglu1 - 7 were measured individually and as a percentage of each patient 's mtxglutot . mtxglu3 was the most prominent subtype identified , comprising 42% of mtxglutot , and was most highly correlated with mtxglutot ( r = 0.96 ) . higher concentrations of mtxglu1 + 2 were observed in patients receiving oral doses of mtx , whereas higher concentrations of mtxglu3 - 5 were observed in patients receiving subcutaneous doses of mtx ( p < these findings were also supported further by hierarchical clustering , which revealed distinct clusters of patients with higher proportions of mtxglu1 + 2 and a second cluster of patients in whom mtxglu35 predominated ( figure 2 ) . after controlling for mtx dose , subjects with higher proportions of mtxglu1 + 2 were more likely to be receiving oral mtx ( p < .0001 ) . those with higher proportions of mtxglu3 - 5 were more likely to be receiving subcutaneous mtx ( p = .0097 ) . in agreement with dolezalova and colleagues , we did not find a strong association of mtxglun concentrations ( total or long chain ) with mtx response ( unpublished data ) , but we are actively investigating associations with clinical outcomes such as gi toxicity and hepatic enzyme elevation . our experience demonstrates that mtxglun concentrations can be reliably measured in children and are extensively variable , yet the contributors to this variability are not fully explored . several studies have investigated the association of folate pathway pharmacogenetics and clinical outcomes with , at times , conflicting results . in genes associated with the cellular uptake and retention of mtx , there have been investigations studying allelic variations in influx transporters , ( rfc / slc19a1 ) , efflux transporters ( abcb1 and abcc2 ) , as well as enzymes responsible for glutamation and deglutamation , ( ggh and fpgs ) . previous investigations have found no association between snps in rfc / slc19a1 and clinical outcomes in ra patients [ 43 , 44 ] . alternatively , upregulation of the efflux transporter abcg2 protein expression has been associated with mtx resistance in cancer cells , but no associations with snps evaluated in fpgs and clinical effects of mtx have been observed . on the other hand , variations in ggh have shown conflicting associations with toxicity , however , a potential association with mtx response [ 33 , 44 ] . early work focused on genes directly involved in the methionine remethylation cycle , specifically methylenetetrahydrofolate reductase ( mthfr ) . certain polymorphisms in the mthfr gene have been associated with greater clinical improvement ( mthfr 1298aa and mthfr 677cc genotypes ) while mthfr 1298c and mthfr 677 t alleles have been associated with an increased risk for toxicity [ 43 , 46 , 47 ] . in a retrospective cohort study in jia , patients heterozygous for the mthfr 677c / t genotype also exhibited adverse effects more frequently than homozygous 677c / c genotype , strengthening the adult association . additionally , 5-methyltetrahydrofolate homocysteine methyltransferase ( mtr ) 2756gg was found to be overrepresented in pediatric osteosarcoma patients suffering gi toxicity after being treated with high dose mtx , and it has been associated with toxicity with low dose mtx in an adult ra cohort , supporting the potential for a similar effect in children with jia . genes within the adenosine pathway responsible for de novo purine synthesis have begun to receive attention from investigators following initial reports of the anti - inflammatory effects of the genes and enzymes within this pathway . favorable clinical response has been associated with polymorphisms in adenosine monophosphate deaminase ( ampd1 34 t allele ) , the atic 347cc genotype and inosine triphosphate pyrophosphatase ( itpa 94cc ) . in a recently reported jia cohort , presence of allelic variation in 2 atic snps ( rs12995526 and rs4673990 ) and itpa ( rs2295553 ) were associated with increased risk for lack of response . an increased risk of adverse effects was noted in atic 347 g allele carriers . further work investigating the adenosine receptor 2a gene ( adora2a ) revealed several polymorphisms associated with gi toxicity in an adult ra cohort . polymorphisms in this pathway carry the potential to effect outcomes of arthritis patients treated with mtx . however , available pediatric experience suggests that the ontogeny of genes in the purine biosynthesis and adenosine response pathways may warrant further investigation . evaluation of the pyrimidine pathway has focused on tyms , primarily a tandem repeat sequence in the 5-utr of the tyms gene with enhancing function as well as a 6-bp deletion sequence in the 3-utr . there have been data suggesting individuals homozygous for 2 tandem repeats had lower disease activity and better mtx response than patients with a third repeat . there have also been data suggesting there is an association with the side effect of alopecia . tyms genetic variants have been also used in combination with other genes including mthfr , atic , slc191a , and shmt to develop genomic indices , in an attempt to take into account the complexities of the cycle and gene - gene interactions that may contribute to understanding drug response and toxicity [ 33 , 38 , 55 ] . this concept makes more clinical sense than a single snp approach , as a system as important as the folate cycle is likely protected by redundancy and intricate feedback regulation such that the function of this critically important pathway is not subject to serious disruption by variation in single genes . thus far , there has been very little investigation into genetic variation within the folate pathway in relation to drug response in jia . being that this pathway is critical for growth and development by contributing to the synthesis of dna precursors and regulation of gene expression through methylation of cytosine and other methyltransferase reactions , it is likely that alterations in this pathway may have functional consequence , and if ontogeny plays a role , there may be more consequence during times of rapid growth and development . thus far , the ontogeny of the folate pathway during postnatal growth and development has not been investigated . investigating this pathway will require a better understanding of gene - gene interactions within the pathway , as well as the baseline folate status and the cell 's response to shifts in supply and demand during periods of active growth and development as well as perturbation by a drug such as mtx . with extensive 40-fold variability in intracellular mtxglun concentrations and distinct patterns of mtx polyglutamation highly associated with route of drug administration , one may consider absorption and cellular transport as key contributors to this observed variability . intestinal influx transporters include the solute carrier ( slc ) transporters , such as slc19a1 ( also known as the reduced folate carrier ( rfc ) ) and the proton coupled folate transporter / heme carrier protein-1 ( pcft ; slc46a1 ) and folate receptors ( fr , fr , and fr ) [ 56 , 57 ] . the importance of these transporters , specifically pcft has been exemplified by the autosomal recessive disorder hereditary folate malabsorption ( hfm ) where loss of function mutations in these families have resulted in impaired folate absorption leading to severe folate deficiency and impaired folate transport into the cns [ 5759 ] . further work has demonstrated that pcft also transports mtx , although less avidly than folate and has demonstrated transporter inhibition by anionic compounds , including sulfasalzine . in addition , proton - pump inhibitors also inhibit pcft transport function [ 59 , 60 ] , with the potential for clinically important consequences given that they often are coadministered with mtx in ra patients . in mice fed a low folate diet , expression of these transporters in the small intestine efflux transporters include the atp - binding cassette protein family of transporters ( abc transporters ) , which transport either back into the lumen ( abcg2/bcrp , abcc2/mrp-2 , abcc4/mrp-4 , and abcb1/mdr1 ) , or through the basolateral membrane into the blood ( abcc1/mrp-1 , abcc3/mrp-3 , abcc5/mpr-5 ) . the overlapping and compensatory functions of these efflux transporters make investigation of their function complex . gi toxicity has been reported in abcc1/mrp1 ( / ) knock out mice , found highly expressed in the small intestine . in pediatric all patients , the presence of at least one variant abcc2 - 24 c > t allele resulted in much higher mtx auc in female patients . additionally , in ra patients , increased mtx toxicity was noted in subjects carrying snps in the abcb1 and abcc2 genes [ 64 , 65 ] . variation in the function of intestinal transporters , such as pcft , may result in differences in mtx bioavailability , and may play a part in explaining why children , whose body surface area and weight are much less than adults , still require a similar mtx dose as adults to maintain an appropriate level of disease control . the combination and relation of both influx and efflux transporter function will likely need to be better elucidated before final associations with genotype and phenotype can be made . after being absorbed from the gut , mtx is further metabolized in the liver to its primary metabolite 7-oh - mtx , and influx and efflux transporters within the liver can also be contributors to interpatient variability in systemic availability and response to the drug , especially when considering the inherent risk for hepatotoxicity . the role of hepatic specific transporters such as organic anion transporter polypeptides ( oatp1b1/slco1b1 , oatp1b3/slco1b3 ) have received more attention with the reported influence of the slco1b1 * 5 haplotype ( c.51 t > oatp1b1 is located at the sinusoidal membrane of hepatocytes , and its transcript has been detected in hepatocytes . it has been shown to transport mtx in vitro , and in pediatric patients with all , variations in oatp1b1 were associated with clearance of mtx as well as gi toxicity . variations in the genotype of hepatic efflux transporters such as abcb1/mdr-1 and abcc2/mrp-2 may also play a role in drug response and toxicity . in abcc2-deficient rats , deficient mice , a dramatic increase in mtx and 7oh - mtx concentrations are found in the liver , as well as prolonged systemic exposure to both compounds after mtx administration compared to single gene knockout mice . there is currently a knowledge deficit regarding the ontogeny of these transporters . if dysfunction in efflux transporters such as abcc2 , abcc3 , and abcg2 leads to prolonged systemic exposure and elevated liver concentrations of mtx , perhaps more effective or efficient function of these transporters in children allow high doses to be tolerated and hepatic toxicity to be minimal . the complexity involved in predicting the cellular effects of mtx is only amplified by the multiple steps required to complete the journey to the cellular folate cycle , and recognizing that cell- and tissue - specific differences in cellular uptake and retention processes exist . the role of ontogeny adds a layer of complexity to an already intricate network of genes . very little is known about differences in gene expression with age within the network during normal growth and development , let alone after perturbation following administration of mtx . extensive variability in outcomes and intracellular mtxglun concentrations in our pediatric jia population , as well as the observation that children need and tolerate the same absolute doses of the drug routinely prescribed to adults , begs the question : what role does ontogeny play ? although mtx is widely used in clinical practice in a number of disease entities , at different doses and by different routes of administration there remains significant lack of understanding of its mechanisms of action and the factors that contribute to the variability in toxicity and response seen clinically . given the time lag between initiation of treatment and the first indication of patient response , this knowledge is essential to determine a priori the probability of beneficial therapeutic response and also take into consideration the probability of toxicity so that the best informed clinical decisions can be made . in addition to differences in drug administration , ( i.e. , dose , route , compliance ) factors that affect pharmacokinetics and pharamcodynamics such as genetic variation may explain individual differences in drug biotransformation . however , the pediatric population has an additional factor to consider , namely , the ontogeny of gene expression , which may invariably affect the relative expression of genes within the pathway as one carbon resources which are allocated to the different functions of the folate cycle ( purine and pyrimidine biosynthesis , homocysteine remethylation to methionine , one carbon donor for methyltransferases ) during periods of dynamic change in folate supply and demand . by taking into account not only the genes in question that may affect drug disposition , but also the developmental trajectory of genes involved in drug response , we may begin to better understand what makes children different , and identify and prevent unique adverse drug reactions in this population . future areas of study in this area may include investigating the developmental expression of tissue specific transporters , which may explaining the higher rate of subcutaneous administration and the higher doses of mtx used in children compared to adults . additionally , having a better understanding of how cellular folate concentrations and patterns change with age may also help explain the variation seen in mtx response . with newer techniques and a developmentally aware approach

although methotrexate is widely used in clinical practice there remains significant lack of understanding of its mechanisms of action and the factors that contribute to the variability in toxicity and response seen clinically . in addition to differences in drug administration , factors that affect pharmacokinetics and pharmacodynamics such as genetic variation may explain individual differences in drug biotransformation . however , the pediatric population has an additional factor to consider , namely the ontogeny of gene expression which may result in variation throughout growth and development . we review the current understanding of methotrexate biotransformation and the concept of ontogeny , with further discussion of how to implement a developmental pharmacogenomics approach in future studies .

1. Introduction 2. Developmental Pharmacogenetics 3. JIA Background 4. Cellular Effects of MTX 5. The Role of Methotrexate Polyglutamation 6. Methotrexate Pharmacogenetics and Clinical Outcomes 7. Systematc Approach to Address Methotrexate Disposition and Response in JIA 8. Conclusions

methotrexate ( mtx ) is the most common second - line therapeutic agent used to treat juvenile idiopathic arthritis ( jia ) worldwide . regardless of age or disease subtype , considerable interindividual variability in clinical response and adverse reactions exists with mtx , and thus far , there have been no predictive variables for outcomes in patients taking this medication . since the onset of clinical response may take months to manifest , the risk to benefit ratio early in treatment is altered , as there is risk for toxicity for several weeks to months before knowing if the medication has resulted in a clinical benefit . side effects may compromise efficacy due to patient noncompliance , clinician dose adjustment , or discontinuation even if the drug eventually is medically effective . when exploring the variability in response and toxicity to any medication used in children , a concept often overlooked is ontogeny . the effects of development can be applied at every level of drug disposition and response . these effects range from differences in gastric ph [ 2 , 3 ] and gastric emptying which may affect absorption , to changes in circulating plasma proteins with age that may affect drug distribution . developmental changes in phase i drug biotransformation and phase ii conjugating enzyme expression have the potential to alter drug metabolism , and developmental differences in glomerular filtration rates will affect drug excretion in children compared to adults . common drug biotransformation pathways are also shared with endogenous compounds involved in growth and development , such as testosterone , cortisol , and vitamin d3 , so it is not surprising that some of these pathways may be affected by the rapid growth and maturation of the pediatric patient , for example , during infancy and puberty . the developmental expression of these pathways at different rates or trajectories may also lead to variability in drug disposition and response . although pharmacogenetics appropriately strives to identify the correct dose of the correct drug for the correct person , the impact of development on an individual 's response to a drug must be taken into account . genotyping an individual for variations that affect function is an important step to understanding variability in outcomes ; however , knowing if and when that gene is expressed is a concept important to fully understanding genotype - phenotype relationships in children [ 9 , 10 ] . an approach to investigating hypotheses related to drug outcomes in children can be guided by the following questions . this process is also relevant for genes / gene products involved in drug response . partnered with the understanding of genetic variation in an individual , appreciating the changes in gene expression throughout growth and development will allow us to manage the complexity of therapeutics in children . juvenile idiopathic arthritis ( jia ) , formerly termed juvenile chronic arthritis ( jca ) or juvenile rheumatoid arthritis ( jra ) is one of the most common chronic diseases of childhood , and is an important cause of morbidity and disability in children . despite differences in disease expression between adults and children , like in many pediatric diseases , children are treated with generally the same armamentarium of drugs used to treat ra in adults . with the advent of disease modifying antirheumatic drugs ( dmards ) , the philosophy of treatment changed from simple pain control to prevention of erosions and long - term damage of the joints . methotrexate ( mtx ) , a folic acid antagonist , was approved by the food and drug administration for the treatment of ra in 1988 , and several uncontrolled descriptive studies suggested , and a randomized placebo - controlled double blind clinical trial demonstrated the effectiveness of mtx in children with jia [ 1621 ] . although the collective clinical experience with mtx has been vast , there are still unanswered questions about its mechanism of action , and considerable interindividual variability in clinical response and adverse reactions exists [ 2224 ] . thus far , there have been few predictors for efficacy or toxicity in pediatric patients taking this medication , and clinicians essentially dose by trial and error . factors that could contribute to this variability are extensive , and some are unique to the pediatric population . we would like to explore the potential sources of variability that may contribute to outcomes on mtx in jia . methotrexate , a folic acid analog and potent inhibitor of several enzymes within the folate pathway , has been used in low doses for the treatment of rheumatic disease over the last several decades . for rheumatic conditions , the dose range in pediatrics spans 10-fold , ranging from 0.1 mg / kg / dose to 1 mg / kg / dose , administered on a weekly basis . options include oral and subcutaneous administration , but intramuscular and intravenous administration are possible , although less practical in the outpatient setting . before being taken into the body , contributors to variability that can not be overlooked include patient compliance , differences in administered dose , and route of administration . children , who are a fraction of the weight of their adult counterparts , are dosed with the same absolute mtx dose , despite their smaller size . although attention has been brought to this phenomenon , there continues to be little understanding of why children appear to require , relative to body weight , higher doses of mtx or how these doses are tolerated . serum mtx concentrations have not been found useful to predict response or toxicity with little correlation with dose or outcome [ 2527 ] . once intracellular , mtx is bioactivated to a polyglutamated form by folylpolyglutamyl synthase ( fpgs ) , which enhances the pharmacological activity and intracellular retention of mtx . in the ra and pediatric oncology literature , current evidence indicates that the enzymatic addition of glutamate residues to the mtx molecule in vivo ( polyglutamation / mtxglun ) is critical for pharmacologic activity by increasing the intracellular concentration of the drug and increasing its affinity for its therapeutic targets , thereby allowing more opportunity for its inhibitory effects to be exerted upon its target enzymes [ 2931 ] . the initial target of mtx to be identified was dihydrofolate reductase ( dhfr ) , which forms tetrahydrofolate , a precursor required for one carbon donation for synthesis of thymidylate , purines , methionine and serine , remethylation of homocysteine to form methionine , and provision of methyl donors for multiple methyltransferase enzymes . subsequently , the list of target genes has been extended to include amino - imidazole carboxamide ribonucleotide ( aicar ) transformylase ( gene name , atic ) , which inhibits de novo purine synthesis and promotes the accumulation of aicar ribotide , inhibiting adenosine deaminase and leading to a build up of adenosine , a potent anti - inflammatory agent [ 32 , 33 ] . adenosine 's effect is also mediated by adenosine a2 receptors ( adora2 ) , which are present on neutrophils , monocytes , lymphocytes and basophils , generally suppressing the immune function of these cells . gamma glutamyl hydrolase ( ggh ) , the enzyme responsible for glutamate removal from mtx , transforms mtx into a form that can be effluxed from the cell by the atp - binding cassette ( abc ) family of transporters . due to the rapid decline in serum drug concentrations , serum mtx concentrations are of limited utility in determining appropriate dosing or management of this medication [ 2527 ] . on the other hand , rbc folate concentrations are established during erythropoiesis and represent the average folate status over the preceding 120 days . by extension , mtx concentrations in rbcs represent a reasonable surrogate biomarker of average drug exposure over a similar period of time . in vitro studies have revealed that the cellular response to folate deprivation is associated with increased expression of fpgs and decreased expression of abcg2 , suggesting that the adaptive cellular response to low folate involves increased polyglutamation to promote the retention of folate . although the data are limited , these examples reveal that allelic variation in these genes resulting in increased or decreased activity may be associated with inter - individual variability in intracellular mtxglun . the relationship between mtxglun and side effects of the medication has not been established . as individual mtx polyglutamates differ with respect to their inhibitory effects on target enzymes and inhibition is modulated by folate polyglutamates , it is likely that multiple as yet unidentified factors contribute to variability in the relationship between mtxglun concentrations and efficacy and/or toxicity . in order to better identify factors that may contribute to the inconsistencies in response and toxicity to mtx , we sought to characterize the extent of variability of intracellular mtxglun concentrations in our jia patient population , and to investigate variables that may contribute to mtxglun variability . we have measured intracellular mtxglun concentrations in a cohort of 104 jia patients . in this cohort , total intracellular mtxglun ( mtxglutot , the sum of all individual mtxglun ) concentrations varied 40-fold with a mean of 85.4 48.8 concentrations of mtxglu1 - 7 were measured individually and as a percentage of each patient 's mtxglutot . mtxglu3 was the most prominent subtype identified , comprising 42% of mtxglutot , and was most highly correlated with mtxglutot ( r = 0.96 ) . those with higher proportions of mtxglu3 - 5 were more likely to be receiving subcutaneous mtx ( p = .0097 ) . in agreement with dolezalova and colleagues , we did not find a strong association of mtxglun concentrations ( total or long chain ) with mtx response ( unpublished data ) , but we are actively investigating associations with clinical outcomes such as gi toxicity and hepatic enzyme elevation . several studies have investigated the association of folate pathway pharmacogenetics and clinical outcomes with , at times , conflicting results . previous investigations have found no association between snps in rfc / slc19a1 and clinical outcomes in ra patients [ 43 , 44 ] . on the other hand , variations in ggh have shown conflicting associations with toxicity , however , a potential association with mtx response [ 33 , 44 ] . early work focused on genes directly involved in the methionine remethylation cycle , specifically methylenetetrahydrofolate reductase ( mthfr ) . certain polymorphisms in the mthfr gene have been associated with greater clinical improvement ( mthfr 1298aa and mthfr 677cc genotypes ) while mthfr 1298c and mthfr 677 t alleles have been associated with an increased risk for toxicity [ 43 , 46 , 47 ] . in a retrospective cohort study in jia , patients heterozygous for the mthfr 677c / t genotype also exhibited adverse effects more frequently than homozygous 677c / c genotype , strengthening the adult association . genes within the adenosine pathway responsible for de novo purine synthesis have begun to receive attention from investigators following initial reports of the anti - inflammatory effects of the genes and enzymes within this pathway . favorable clinical response has been associated with polymorphisms in adenosine monophosphate deaminase ( ampd1 34 t allele ) , the atic 347cc genotype and inosine triphosphate pyrophosphatase ( itpa 94cc ) . in a recently reported jia cohort , presence of allelic variation in 2 atic snps ( rs12995526 and rs4673990 ) and itpa ( rs2295553 ) were associated with increased risk for lack of response . an increased risk of adverse effects was noted in atic 347 g allele carriers . however , available pediatric experience suggests that the ontogeny of genes in the purine biosynthesis and adenosine response pathways may warrant further investigation . evaluation of the pyrimidine pathway has focused on tyms , primarily a tandem repeat sequence in the 5-utr of the tyms gene with enhancing function as well as a 6-bp deletion sequence in the 3-utr . there have been data suggesting individuals homozygous for 2 tandem repeats had lower disease activity and better mtx response than patients with a third repeat . tyms genetic variants have been also used in combination with other genes including mthfr , atic , slc191a , and shmt to develop genomic indices , in an attempt to take into account the complexities of the cycle and gene - gene interactions that may contribute to understanding drug response and toxicity [ 33 , 38 , 55 ] . thus far , there has been very little investigation into genetic variation within the folate pathway in relation to drug response in jia . being that this pathway is critical for growth and development by contributing to the synthesis of dna precursors and regulation of gene expression through methylation of cytosine and other methyltransferase reactions , it is likely that alterations in this pathway may have functional consequence , and if ontogeny plays a role , there may be more consequence during times of rapid growth and development . thus far , the ontogeny of the folate pathway during postnatal growth and development has not been investigated . investigating this pathway will require a better understanding of gene - gene interactions within the pathway , as well as the baseline folate status and the cell 's response to shifts in supply and demand during periods of active growth and development as well as perturbation by a drug such as mtx . with extensive 40-fold variability in intracellular mtxglun concentrations and distinct patterns of mtx polyglutamation highly associated with route of drug administration , one may consider absorption and cellular transport as key contributors to this observed variability . intestinal influx transporters include the solute carrier ( slc ) transporters , such as slc19a1 ( also known as the reduced folate carrier ( rfc ) ) and the proton coupled folate transporter / heme carrier protein-1 ( pcft ; slc46a1 ) and folate receptors ( fr , fr , and fr ) [ 56 , 57 ] . in addition , proton - pump inhibitors also inhibit pcft transport function [ 59 , 60 ] , with the potential for clinically important consequences given that they often are coadministered with mtx in ra patients . the overlapping and compensatory functions of these efflux transporters make investigation of their function complex . gi toxicity has been reported in abcc1/mrp1 ( / ) knock out mice , found highly expressed in the small intestine . in pediatric all patients , the presence of at least one variant abcc2 - 24 c > t allele resulted in much higher mtx auc in female patients . additionally , in ra patients , increased mtx toxicity was noted in subjects carrying snps in the abcb1 and abcc2 genes [ 64 , 65 ] . variation in the function of intestinal transporters , such as pcft , may result in differences in mtx bioavailability , and may play a part in explaining why children , whose body surface area and weight are much less than adults , still require a similar mtx dose as adults to maintain an appropriate level of disease control . after being absorbed from the gut , mtx is further metabolized in the liver to its primary metabolite 7-oh - mtx , and influx and efflux transporters within the liver can also be contributors to interpatient variability in systemic availability and response to the drug , especially when considering the inherent risk for hepatotoxicity . the role of hepatic specific transporters such as organic anion transporter polypeptides ( oatp1b1/slco1b1 , oatp1b3/slco1b3 ) have received more attention with the reported influence of the slco1b1 * 5 haplotype ( c.51 t > oatp1b1 is located at the sinusoidal membrane of hepatocytes , and its transcript has been detected in hepatocytes . variations in the genotype of hepatic efflux transporters such as abcb1/mdr-1 and abcc2/mrp-2 may also play a role in drug response and toxicity . there is currently a knowledge deficit regarding the ontogeny of these transporters . if dysfunction in efflux transporters such as abcc2 , abcc3 , and abcg2 leads to prolonged systemic exposure and elevated liver concentrations of mtx , perhaps more effective or efficient function of these transporters in children allow high doses to be tolerated and hepatic toxicity to be minimal . the complexity involved in predicting the cellular effects of mtx is only amplified by the multiple steps required to complete the journey to the cellular folate cycle , and recognizing that cell- and tissue - specific differences in cellular uptake and retention processes exist . the role of ontogeny adds a layer of complexity to an already intricate network of genes . very little is known about differences in gene expression with age within the network during normal growth and development , let alone after perturbation following administration of mtx . extensive variability in outcomes and intracellular mtxglun concentrations in our pediatric jia population , as well as the observation that children need and tolerate the same absolute doses of the drug routinely prescribed to adults , begs the question : what role does ontogeny play ? although mtx is widely used in clinical practice in a number of disease entities , at different doses and by different routes of administration there remains significant lack of understanding of its mechanisms of action and the factors that contribute to the variability in toxicity and response seen clinically . given the time lag between initiation of treatment and the first indication of patient response , this knowledge is essential to determine a priori the probability of beneficial therapeutic response and also take into consideration the probability of toxicity so that the best informed clinical decisions can be made . in addition to differences in drug administration , ( i.e. , dose , route , compliance ) factors that affect pharmacokinetics and pharamcodynamics such as genetic variation may explain individual differences in drug biotransformation . however , the pediatric population has an additional factor to consider , namely , the ontogeny of gene expression , which may invariably affect the relative expression of genes within the pathway as one carbon resources which are allocated to the different functions of the folate cycle ( purine and pyrimidine biosynthesis , homocysteine remethylation to methionine , one carbon donor for methyltransferases ) during periods of dynamic change in folate supply and demand . by taking into account not only the genes in question that may affect drug disposition , but also the developmental trajectory of genes involved in drug response , we may begin to better understand what makes children different , and identify and prevent unique adverse drug reactions in this population . future areas of study in this area may include investigating the developmental expression of tissue specific transporters , which may explaining the higher rate of subcutaneous administration and the higher doses of mtx used in children compared to adults . additionally , having a better understanding of how cellular folate concentrations and patterns change with age may also help explain the variation seen in mtx response . with newer techniques and a developmentally aware approach

foreign body aspiration ( fba ) is a common and life - threatening emergency in children . the presentation varies from coughing , wheezing , recurrent pneumonia , and obstructive emphysema to respiratory distress . occasionally , fba could present with pneumomediastinum ( pm ) , subcutaneous emphysema ( sce ) , or pneumothorax ( pt ) . in the presence of an airway foreign body ( fb ) , persistence of expiratory outflow resistance and associated cough , crying , or vomiting leads to uninterrupted air leak through ruptured alveolus and thus progressive emphysema . besides , fb - associated pneumonitis could facilitate the alveolar rupture and precipitate the development of pm . the clinical presentation varies from chest pain , sce , dyspnea , hemodynamic instability to death . if the intramediastinum pressure rises abruptly or the decompression by air dissecting into subcutaneous tissue is insufficient to relieve the tension , the mediastinal parietal pleura may tear , resulting in concomitant pt , which would compromise the cardiopulmonary reserve . early diagnosis and proper intervention of pm secondary to fba are critical in preventing life - threatening complications . however , given the infrequent nature and the lack of treatment guide , there was no consensus on the treatment and the management is often based on personal experience . while chest drainage is recommended if pm is present with fba , simple bronchoscopy is performed in some patients with good outcomes . a simple and practical assessment is needed to differentiate those patients who simply need bronchoscopy from those who need the preceding air evacuation . in this study , we retrospectively reviewed our experience in the treatment of pediatric pm secondary to fba from january 2010 to december 2015 . the aims of this retrospective study were : ( 1 ) to introduce a fast and practical assessment system based on the degree of dyspnea and ( 2 ) to define the proper management according to the clinical severity . patients with pm secondary to fba treated in beijing children 's hospital between january 2010 and december 2015 were included . the inclusion criteria included the presence of tracheobronchial fbs identified by rigid bronchoscopy and the presence of pm recognized by chest radiograph or chest computed tomography ( ct ) at admission . exclusion criteria included asthma , chest wounds and injuries , infection by gas - producing germs , and pm developed during or after bronchoscopies . the study protocol was reviewed and approved by the ethics committee of beijing children 's hospital . data were collected including demographic data , fbs characteristics , clinical presentation , precipitating factors , anteroposterior and lateral neck and chest films , interventions , hospital admission ( regular ward versus pediatric intensive care unit [ picu ] ) , length of hospital stay , time to resolution of pm , clinical outcomes , and recurrence . for all patients , a detailed history , careful physical examinations , neck and chest x - rays ( anteroposterior and lateral ) ct was performed when there was no choking episode or when other associated diseases were suspected . follow - up chest radiographs were undertaken every 35 days to confirm the resolution of pm based on clinical status . all patients were clinically assessed based on a comprehensive medical history , physical examination , and radiographic findings . the clinical severity was stratified according to the degree of respiratory distress as follows : ( 1 ) grade i was defined as mild to moderate dyspnea without anxiety or restlessness . the child had normal intake of food and drink and appeared interested in playing ; ( 2 ) grade ii was defined as severe dyspnea with anxiety and restlessness . there was refusal of food and drink and no interest in playing ; ( 3 ) grade iii was defined as exhausted child with slowing down of respiratory rate and heart rate . when dealing with clinically stable patients in grade i dyspnea , emergent rigid bronchoscopy was performed under general anesthesia . for some small fbs in the distal bronchi which were difficult for the rigid bronchoscopy , flexible bronchoscopy was performed . for the clinically fragile patients in grade ii dyspnea , skin cutting preceded emergent rigid bronchoscopy . for exhausted children of grade iii , emergent skin cutting and chest drainage were performed followed by cardiopulmonary resuscitation . all bronchoscopies were performed under general anesthesia in the operating room , except for the exhausted patients for whom tropical anesthesia was applied to save time . air evacuation included skin cutting , which was bilateral infraclavicular 3 cm incisions , and chest drainage , which was performed as described by cerfolio . conservative management , which consisted of reassurance , sedation , oxygen , antibiotics , and analgesics when needed , was applied on all patients perioperatively . valsalva maneuver and other activities , which could increase the pulmonary pressure , were prohibited for 2 weeks . descriptive data were expressed as frequency ( percentage ) , median ( range ) , or mean standard deviation ( sd ) . kruskal - wallis rank sum test ( for continuous nonnormal variable ) and chi - square test ( for unordered categorical variable ) were used to analyze differences among the subgroups . patients with pm secondary to fba treated in beijing children 's hospital between january 2010 and december 2015 were included . the inclusion criteria included the presence of tracheobronchial fbs identified by rigid bronchoscopy and the presence of pm recognized by chest radiograph or chest computed tomography ( ct ) at admission . exclusion criteria included asthma , chest wounds and injuries , infection by gas - producing germs , and pm developed during or after bronchoscopies . the study protocol was reviewed and approved by the ethics committee of beijing children 's hospital . data were collected including demographic data , fbs characteristics , clinical presentation , precipitating factors , anteroposterior and lateral neck and chest films , interventions , hospital admission ( regular ward versus pediatric intensive care unit [ picu ] ) , length of hospital stay , time to resolution of pm , clinical outcomes , and recurrence . for all patients , a detailed history , careful physical examinations , neck and chest x - rays ( anteroposterior and lateral ) were conducted routinely . ct was performed when there was no choking episode or when other associated diseases were suspected . follow - up chest radiographs were undertaken every 35 days to confirm the resolution of pm based on clinical status . all patients were clinically assessed based on a comprehensive medical history , physical examination , and radiographic findings . the clinical severity was stratified according to the degree of respiratory distress as follows : ( 1 ) grade i was defined as mild to moderate dyspnea without anxiety or restlessness . the child had normal intake of food and drink and appeared interested in playing ; ( 2 ) grade ii was defined as severe dyspnea with anxiety and restlessness . there was refusal of food and drink and no interest in playing ; ( 3 ) grade iii was defined as exhausted child with slowing down of respiratory rate and heart rate . when dealing with clinically stable patients in grade i dyspnea , emergent rigid bronchoscopy was performed under general anesthesia . for some small fbs in the distal bronchi which were difficult for the rigid bronchoscopy , flexible bronchoscopy was performed . for the clinically fragile patients in grade ii dyspnea , skin cutting preceded emergent rigid bronchoscopy . for exhausted children of grade iii , emergent skin cutting and chest drainage were performed followed by cardiopulmonary resuscitation . all bronchoscopies were performed under general anesthesia in the operating room , except for the exhausted patients for whom tropical anesthesia was applied to save time . air evacuation included skin cutting , which was bilateral infraclavicular 3 cm incisions , and chest drainage , which was performed as described by cerfolio . conservative management , which consisted of reassurance , sedation , oxygen , antibiotics , and analgesics when needed , was applied on all patients perioperatively . all patients were discharged when the condition stabilized . valsalva maneuver and other activities , which could increase the pulmonary pressure , were prohibited for 2 weeks . descriptive data were expressed as frequency ( percentage ) , median ( range ) , or mean standard deviation ( sd ) . kruskal - wallis rank sum test ( for continuous nonnormal variable ) and chi - square test ( for unordered categorical variable ) were used to analyze differences among the subgroups . a total of 2643 patients with fba were identified between january 2010 and december 2015 in beijing children 's hospital . of all patients , pm occurred in 39 patients ( 1.5% ) , including 28 boys and 11 girls . the characteristics of patients and fbs and the clinical presentations and management are listed in table 1 . demographics , clinical characteristics , and management in pm secondary to fba ( n = 39 ) the data are shown as n ( % ) , median ( range ) , or mean sd . fbs : foreign bodies ; fba : foreign body aspiration ; pm : pneumomediastinum ; sce : subcutaneous emphysema ; pt : pneumothorax ; sd : standard deviation . among the 39 patients , 37 patients ( 94.9% ) had wheezing as the main symptom . the most common site of fbs was right main bronchus in 20 patients ( 51.3% ) , followed by left main bronchus in 17 patients ( 43.6% ) , both bronchi in one patient ( 2.6% ) , and subglottis in one patient ( 2.6% ) . there was sce in 27 patients ( 69.2% ) , which was the most common comorbid condition on radiograph [ table 1 ] . thirty - nine patients were assessed and categorized into three grades according to the degree of dyspnea . thirty - one patients fell into grade i ; six into grade ii ; and two into grade iii . the management among the three subgroups were different ( = 35.858 , p = 0.000 ) . skin cutting was applied on eight patients while chest drainage and intubation on three children . of all patients , two patients died while 37 got uneventful recovery with a mean time of 5.5 1.9 days for the pm resolution . the median lengths of hospital stay were 5.7 days ( range 120 days ) for grade i , 9.5 days ( range 517 days ) for grade ii , and 5.0 days ( range 19 days ) for grade iii . however , the median lengths of hospital stay among the three subgroups had no statistical difference ( h = 4.415 , p = 0.110 ) . radiographic findings , management and clinical outcomes for patients with different grades of dyspnea ( n = 39 ) * chi - square values ; h values . pm : pneumomediastinum ; sce : subcutaneous emphysema ; pt : pneumothorax ; sd : standard deviation . rigid bronchoscopies were performed on 26 patients ( 83.9% ) while flexible bronchoscopies on six patients ( 19.4% ) . in one case , a small peanut particle was removed by a flexible bronchoscopy from the distal right bronchus after the failure of a rigid bronchoscopy . after a short period of observation in hospital , they all recovered uneventfully and their pm resolved spontaneously with a mean time of 6.7 1.5 days . one patient , a 13-month - old male child , got cyanosis and swelling over neck and face after an inhalation of a peanut for 5 h. although a skin cutting was performed immediately for decompression , he got acute progressive sce with tachycardia and unstable blood pressure immediately after an episode of violent crying . given the breath sound disappeared on the right side , a pleural aspiration was performed immediately and the right - sided pt was confirmed . thereafter , a chest drainage and intubation were put immediately . on the rigid bronchoscopy , a chest radiograph on the 7 day revealed good lung expansion with resolution of pm , and he was discharged . during follow - up , they had been intubated outside our hospital and got skin cutting and chest drainage immediately on admission . thereafter a 22-month - old male child was admitted to the emergency department of another hospital for respiratory arrest . the patient had suffered from a choking episode for 3.5 h until he reached a physician . since there was a swollen neck and face with anxiety and restlessness , intubation and artificial ventilation were performed , and the child was transferred to our hospital in an ambulance . on admission , there were coma , cyanosis , progression of sce , and hemodynamic instability . the skin cutting and chest drainage were performed once the diffuse pm , sce , and bilateral pt were confirmed on a bedside radiograph [ figure 1 ] . an emergent rigid bronchoscopy under tropical anesthesia revealed peanut particles obstructing both the bronchi completely , which were removed successfully . the clinical status deteriorated and he died of progressive and irreversible hypoxia and hypoxic encephalopathy 9 days later . chest radiograph showing mediastinal air , massive subcutaneous emphysema , and bilateral pneumothorax compressing both lungs to 50% . another 39-month - old female child was admitted to another hospital with swollen over neck and face after inhalation of a bean 9 h ago . after an episode of violent crying 1 h later , the swelling progressed suddenly with cyanosis and anxiety . after immediate intubation and cardiopulmonary resuscitation , she was transferred to our hospital for further treatment . there were coma and massive swelling over the neck , face , and chest . an emergent bedside radiograph revealed diffuse pm and right - sided pt [ figure 2 ] . given the desaturation and hemodynamic instability , an intercostal chest tube and skin cutting were put to drain the air . on subsequent rigid bronchoscopy , chest radiograph showing mediastinal air and right - sided pneumothorax with the right lung compressed to approximately 30% . a total of 2643 patients with fba were identified between january 2010 and december 2015 in beijing children 's hospital . of all patients , pm occurred in 39 patients ( 1.5% ) , including 28 boys and 11 girls . the characteristics of patients and fbs and the clinical presentations and management are listed in table 1 . demographics , clinical characteristics , and management in pm secondary to fba ( n = 39 ) the data are shown as n ( % ) , median ( range ) , or mean sd . fbs : foreign bodies ; fba : foreign body aspiration ; pm : pneumomediastinum ; sce : subcutaneous emphysema ; pt : pneumothorax ; sd : standard deviation . among the 39 patients , 37 patients ( 94.9% ) had wheezing as the main symptom . peanuts ( in 23 patients , 59.0% ) remained the most common aspirated fb . the most common site of fbs was right main bronchus in 20 patients ( 51.3% ) , followed by left main bronchus in 17 patients ( 43.6% ) , both bronchi in one patient ( 2.6% ) , and subglottis in one patient ( 2.6% ) . there was sce in 27 patients ( 69.2% ) , which was the most common comorbid condition on radiograph [ table 1 ] . thirty - nine patients were assessed and categorized into three grades according to the degree of dyspnea . thirty - one patients fell into grade i ; six into grade ii ; and two into grade iii . the management among the three subgroups were different ( = 35.858 , p = 0.000 ) . skin cutting was applied on eight patients while chest drainage and intubation on three children . of all patients , two patients died while 37 got uneventful recovery with a mean time of 5.5 1.9 days for the pm resolution . the median lengths of hospital stay were 5.7 days ( range 120 days ) for grade i , 9.5 days ( range 517 days ) for grade ii , and 5.0 days ( range 19 days ) for grade iii . however , the median lengths of hospital stay among the three subgroups had no statistical difference ( h = 4.415 , p = 0.110 ) . radiographic findings , management and clinical outcomes for patients with different grades of dyspnea ( n = 39 ) * chi - square values ; h values . pm : pneumomediastinum ; sce : subcutaneous emphysema ; pt : pneumothorax ; sd : standard deviation . rigid bronchoscopies were performed on 26 patients ( 83.9% ) while flexible bronchoscopies on six patients ( 19.4% ) . in one case , a small peanut particle was removed by a flexible bronchoscopy from the distal right bronchus after the failure of a rigid bronchoscopy . after a short period of observation in hospital , they all recovered uneventfully and their pm resolved spontaneously with a mean time of 6.7 1.5 days . one patient , a 13-month - old male child , got cyanosis and swelling over neck and face after an inhalation of a peanut for 5 h. although a skin cutting was performed immediately for decompression , he got acute progressive sce with tachycardia and unstable blood pressure immediately after an episode of violent crying . given the breath sound disappeared on the right side , a pleural aspiration was performed immediately and the right - sided pt was confirmed . thereafter , a chest drainage and intubation were put immediately . on the rigid bronchoscopy , a chest radiograph on the 7 day revealed good lung expansion with resolution of pm , and he was discharged . during follow - up , the child was thriving well without any respiratory complaint . they had been intubated outside our hospital and got skin cutting and chest drainage immediately on admission . thereafter a 22-month - old male child was admitted to the emergency department of another hospital for respiratory arrest . the patient had suffered from a choking episode for 3.5 h until he reached a physician . since there was a swollen neck and face with anxiety and restlessness , intubation and artificial ventilation were performed , and the child was transferred to our hospital in an ambulance . on admission , there were coma , cyanosis , progression of sce , and hemodynamic instability . the skin cutting and chest drainage were performed once the diffuse pm , sce , and bilateral pt were confirmed on a bedside radiograph [ figure 1 ] . an emergent rigid bronchoscopy under tropical anesthesia revealed peanut particles obstructing both the bronchi completely , which were removed successfully . the clinical status deteriorated and he died of progressive and irreversible hypoxia and hypoxic encephalopathy 9 days later . chest radiograph showing mediastinal air , massive subcutaneous emphysema , and bilateral pneumothorax compressing both lungs to 50% . another 39-month - old female child was admitted to another hospital with swollen over neck and face after inhalation of a bean 9 h ago . after an episode of violent crying 1 h later , the swelling progressed suddenly with cyanosis and anxiety . after immediate intubation and cardiopulmonary resuscitation , she was transferred to our hospital for further treatment . there were coma and massive swelling over the neck , face , and chest . an emergent bedside radiograph revealed diffuse pm and right - sided pt [ figure 2 ] . given the desaturation and hemodynamic instability , an intercostal chest tube and skin cutting were put to drain the air . on subsequent rigid bronchoscopy , chest radiograph showing mediastinal air and right - sided pneumothorax with the right lung compressed to approximately 30% . the incidence in our study was 1.5% ( 39/2643 ) , which was similar to previous reports in the usa and israel . in this study , we characterized the presentation and management for this clinical entity in the past 6 years . a quick assessment system based on the degree of dyspnea was applied to evaluate the clinical severity , and management was performed accordingly on all patients . to the best of our knowledge , we aimed to help guide pediatric physicians in their assessment and management with this rare clinical entity . the clinical presentation of pm secondary to fba varies from chest pain , sce , dyspnea , hemodynamic instability to death . in this study , the most common presentations were wheezing , coughing , reduced breath sounds , and swelling over face and neck , which was similar to those with spontaneous pm . even though the majority of patients were generally well or with minimal symptoms , life - threatening complications should be kept in mind , including tension pm , tension pt , and increased pressure in the pulmonary interstitial . in our study , there were six patients in severe dyspnea and two in respiratory failure . the diagnosis of pm resulting from fba could be made from a comprehensive history , clinical signs , and radiographic studies . however , since some complications are life - threatening , the first and most important thing in practice is to identify clinical severity . the treatment principle for pm secondary to fba is early bronchoscopic fbs removals and supportive perioperative care . we believe rigid bronchoscopy and flexible bronchoscopy are both effective for the removal of airway fbs in experienced hands . skin cutting and chest drainage , which could be accomplished quickly at the patient 's bedside under local anesthesia , have already been reported as effective and safe procedures for air decompression for massive sce and pm and large pt , respectively . it is noted that for children with pm secondary to fba , a team of experienced emergency physicians , otolaryngologists , thoracic surgeons , and anesthetists should be available immediately to proceed with air evacuation , cardiopulmonary resuscitation , bronchoscopy , or tracheotomy in case of any accident . most importantly , the management must be decided cautiously on a case - by - case basis depending on the status of the child . in clinical practice , a simple and rapid assessment is needed to differentiate those patients who need bronchoscopy simply from patients who need both air evacuation and bronchoscopy . however , there are no well - defined criteria that could grade the severity of pm to date . since pm - associated compression of the airway and cardiac venous return can lead to dyspnea , desaturation , and consequent hemodynamic instability , we believe the degree of dyspnea could be used for stratification . it is simple and practical with initial careful observation . in this study , all patients were assessed on admission and patients in different degree of dyspnea were treated differently . ( grade i ) , an early bronchoscopy under general anesthesia is recommended with careful preparations . in our series after the removal of airway fbs , pms resolved spontaneously and all patients were discharged within a few days . it is noted that high - frequency jet ventilation is not advocated to prevent the potential aggravation of air trapping . in a generally well child , we recommend that an early bronchoscopy and a short period of observation are sufficient to promise a good outcome . for children in severe dyspnea ( grade ii ) , air evacuation for decompression should be the first step while intubation and cardiovascular resuscitation should be available if condition deteriorates . after the decompression , rigid bronchoscopy should be performed as soon as possible to remove the fbs . in our series , there were six children in severe dyspnea with diffuse pm , sce , and/or small pt . they all received immediate skin cutting for decompression , followed by rigid bronchoscopy . with subsequent supportive treatment , pms resolved spontaneously with a mean time of 6.7 days and they all got full recovery . the two deceased cases highlighted early air evacuation . both the children fell into grade ii dyspnea on their initial visits to the doctors . however , pm and pt were not recognized and no efforts were made to withdraw air from the mediastinum or thoracic cavity , a simple and fast procedure which might have saved their lives . besides , the intubation and artificial ventilation might have aggravated the air trapping and the mediastinal compression . similarly , a child was reported to die from possible tension pm secondary to fba due to lack of air evacuation . this reminds us that for patients in severe dyspnea with suspected pm and/or pt , immediate air evacuation , a simple and fast procedure , should come first for decompression . pm could progress at any time with activities which could increase the alveoli pressure . in our study , there was a 13-month - old male child who fell into grade ii on admission . however , pm progressed suddenly after an episode of violent crying . therefore , if there is sudden onset of dyspnea in a previously stable patient , such complications as tension pm or large pt should be suspected and air evacuation should come first to save life . supportive care for pm secondary to fba includes bed rest , oxygen therapy , analgesics , treatment of comorbidities , and avoidance of valsalva maneuver . in light of the benign and self - limiting nature of pediatric pm , we recommend a short period of observation in hospital and avoidance of valsalva maneuvers for approximately 2 weeks . besides , the hospital stay should depend on patient 's clinical condition instead of the presence of sce or the radiologic presence of pm . second , the volume of pt in some children was assessed on supine chest x - rays instead of upright chest x - rays because of severe dyspnea or young ages . in addition , time to resolution was based on chest x - rays , and thus patients who were discharged may have a longer time to their next chest x - rays when compared with admitted patients . moreover , the hospital stay was gradually reduced along the study from 14 days in severe patients to few hours in stable children with our experience increasing although 48-h medical supervision seems necessary for stable children . despite this , we felt the data are valid to help guide pediatric physicians in their assessment and management with this rare clinical entity . in conclusion , pm is a rare presentation of fba . an assessment system based on the degree of dyspnea is critical to help make intervention options and anticipate the outcomes . for patients with mild and moderate dyspnea , simple bronchoscopic removal of airway fbs , an effective and less invasive procedure , is recommended . for patients with severe dyspnea , immediate air evacuation followed by bronchoscopy , a simple and fast procedure to improve the cardiopulmonary reserve , could promise a good outcome . this work was supported by the grants from beijing municipal administration of hospital clinical medicine development of special funding support ( no . this work was supported by the grants from beijing municipal administration of hospital clinical medicine development of special funding support ( no . z131100006813028 ) , and beijing municipal science and technology project ( no . d131100005313014 ) .

background : pneumomediastinum ( pm ) secondary to foreign body aspiration ( fba ) is rare in children . although it is mainly benign , some cases may be fatal . due to the rare nature of this clinical entity , proper assessment and management have been poorly studied so far . here , we characterized the presentation and management of this clinical entity and provided an evaluation system for the management.methods:we retrospectively reviewed children with pm secondary to fba , who were treated in beijing children 's hospital from january 2010 to december 2015 . all patients were stratified according to the degree of dyspnea on admission , and interventions were given accordingly . bronchoscopic removals of airway foreign bodies ( fbs ) were performed on all patients . for patients in acute respiratory distress , emergent air evacuation and/or resuscitations were performed first . admission data , interventions , and clinical outcomes were recorded.results:a total of 39 patients were included in this study . the clinical severity was divided into three grades ( grades i , ii , and iii ) according to the degree of dyspnea . thirty - one patients were in grade i dyspnea , and they simply underwent bronchoscopic fbs removals . pm resolved spontaneously and all patients recovered uneventfully . six patients were in grade ii dyspnea , and emergent drainage preceded rigid bronchoscopy . they all recovered uneventfully under close observation . two exhausted patients were in grade iii dyspnea . they died from large pm and bilateral pneumothorax , respectively , despite of aggressive interventions in our hospital.conclusions:pm secondary to fba could be life - threatening in some patients . the degree of dyspnea should be evaluated immediately , and patients in different dyspnea should be treated accordingly . for patients in grade i dyspnea , simple bronchoscopic fbs removals could promise a good outcome . for patients in grade ii dyspnea , emergent air evacuation and/or resuscitation should precede a bronchoscopy before the children become exhausted .

I M Study setting and population Clinical variables Diagnosis measures Assessment of stratification Intervention procedures Statistical analysis R Patient characteristics Clinical features Assessment and management Deceased cases in Grade III dyspnea D Financial support and sponsorship Conflicts of interest

foreign body aspiration ( fba ) is a common and life - threatening emergency in children . the presentation varies from coughing , wheezing , recurrent pneumonia , and obstructive emphysema to respiratory distress . occasionally , fba could present with pneumomediastinum ( pm ) , subcutaneous emphysema ( sce ) , or pneumothorax ( pt ) . in the presence of an airway foreign body ( fb ) , persistence of expiratory outflow resistance and associated cough , crying , or vomiting leads to uninterrupted air leak through ruptured alveolus and thus progressive emphysema . the clinical presentation varies from chest pain , sce , dyspnea , hemodynamic instability to death . early diagnosis and proper intervention of pm secondary to fba are critical in preventing life - threatening complications . however , given the infrequent nature and the lack of treatment guide , there was no consensus on the treatment and the management is often based on personal experience . while chest drainage is recommended if pm is present with fba , simple bronchoscopy is performed in some patients with good outcomes . in this study , we retrospectively reviewed our experience in the treatment of pediatric pm secondary to fba from january 2010 to december 2015 . the aims of this retrospective study were : ( 1 ) to introduce a fast and practical assessment system based on the degree of dyspnea and ( 2 ) to define the proper management according to the clinical severity . patients with pm secondary to fba treated in beijing children 's hospital between january 2010 and december 2015 were included . the inclusion criteria included the presence of tracheobronchial fbs identified by rigid bronchoscopy and the presence of pm recognized by chest radiograph or chest computed tomography ( ct ) at admission . exclusion criteria included asthma , chest wounds and injuries , infection by gas - producing germs , and pm developed during or after bronchoscopies . the study protocol was reviewed and approved by the ethics committee of beijing children 's hospital . data were collected including demographic data , fbs characteristics , clinical presentation , precipitating factors , anteroposterior and lateral neck and chest films , interventions , hospital admission ( regular ward versus pediatric intensive care unit [ picu ] ) , length of hospital stay , time to resolution of pm , clinical outcomes , and recurrence . for all patients , a detailed history , careful physical examinations , neck and chest x - rays ( anteroposterior and lateral ) ct was performed when there was no choking episode or when other associated diseases were suspected . all patients were clinically assessed based on a comprehensive medical history , physical examination , and radiographic findings . the clinical severity was stratified according to the degree of respiratory distress as follows : ( 1 ) grade i was defined as mild to moderate dyspnea without anxiety or restlessness . the child had normal intake of food and drink and appeared interested in playing ; ( 2 ) grade ii was defined as severe dyspnea with anxiety and restlessness . there was refusal of food and drink and no interest in playing ; ( 3 ) grade iii was defined as exhausted child with slowing down of respiratory rate and heart rate . when dealing with clinically stable patients in grade i dyspnea , emergent rigid bronchoscopy was performed under general anesthesia . for some small fbs in the distal bronchi which were difficult for the rigid bronchoscopy , flexible bronchoscopy was performed . for the clinically fragile patients in grade ii dyspnea , skin cutting preceded emergent rigid bronchoscopy . for exhausted children of grade iii , emergent skin cutting and chest drainage were performed followed by cardiopulmonary resuscitation . all bronchoscopies were performed under general anesthesia in the operating room , except for the exhausted patients for whom tropical anesthesia was applied to save time . air evacuation included skin cutting , which was bilateral infraclavicular 3 cm incisions , and chest drainage , which was performed as described by cerfolio . conservative management , which consisted of reassurance , sedation , oxygen , antibiotics , and analgesics when needed , was applied on all patients perioperatively . patients with pm secondary to fba treated in beijing children 's hospital between january 2010 and december 2015 were included . the inclusion criteria included the presence of tracheobronchial fbs identified by rigid bronchoscopy and the presence of pm recognized by chest radiograph or chest computed tomography ( ct ) at admission . the study protocol was reviewed and approved by the ethics committee of beijing children 's hospital . data were collected including demographic data , fbs characteristics , clinical presentation , precipitating factors , anteroposterior and lateral neck and chest films , interventions , hospital admission ( regular ward versus pediatric intensive care unit [ picu ] ) , length of hospital stay , time to resolution of pm , clinical outcomes , and recurrence . for all patients , a detailed history , careful physical examinations , neck and chest x - rays ( anteroposterior and lateral ) were conducted routinely . all patients were clinically assessed based on a comprehensive medical history , physical examination , and radiographic findings . the clinical severity was stratified according to the degree of respiratory distress as follows : ( 1 ) grade i was defined as mild to moderate dyspnea without anxiety or restlessness . the child had normal intake of food and drink and appeared interested in playing ; ( 2 ) grade ii was defined as severe dyspnea with anxiety and restlessness . there was refusal of food and drink and no interest in playing ; ( 3 ) grade iii was defined as exhausted child with slowing down of respiratory rate and heart rate . when dealing with clinically stable patients in grade i dyspnea , emergent rigid bronchoscopy was performed under general anesthesia . for some small fbs in the distal bronchi which were difficult for the rigid bronchoscopy , flexible bronchoscopy was performed . for the clinically fragile patients in grade ii dyspnea , skin cutting preceded emergent rigid bronchoscopy . for exhausted children of grade iii , emergent skin cutting and chest drainage were performed followed by cardiopulmonary resuscitation . all bronchoscopies were performed under general anesthesia in the operating room , except for the exhausted patients for whom tropical anesthesia was applied to save time . air evacuation included skin cutting , which was bilateral infraclavicular 3 cm incisions , and chest drainage , which was performed as described by cerfolio . conservative management , which consisted of reassurance , sedation , oxygen , antibiotics , and analgesics when needed , was applied on all patients perioperatively . all patients were discharged when the condition stabilized . a total of 2643 patients with fba were identified between january 2010 and december 2015 in beijing children 's hospital . of all patients , pm occurred in 39 patients ( 1.5% ) , including 28 boys and 11 girls . the characteristics of patients and fbs and the clinical presentations and management are listed in table 1 . demographics , clinical characteristics , and management in pm secondary to fba ( n = 39 ) the data are shown as n ( % ) , median ( range ) , or mean sd . fbs : foreign bodies ; fba : foreign body aspiration ; pm : pneumomediastinum ; sce : subcutaneous emphysema ; pt : pneumothorax ; sd : standard deviation . among the 39 patients , 37 patients ( 94.9% ) had wheezing as the main symptom . the most common site of fbs was right main bronchus in 20 patients ( 51.3% ) , followed by left main bronchus in 17 patients ( 43.6% ) , both bronchi in one patient ( 2.6% ) , and subglottis in one patient ( 2.6% ) . thirty - nine patients were assessed and categorized into three grades according to the degree of dyspnea . thirty - one patients fell into grade i ; six into grade ii ; and two into grade iii . the management among the three subgroups were different ( = 35.858 , p = 0.000 ) . of all patients , two patients died while 37 got uneventful recovery with a mean time of 5.5 1.9 days for the pm resolution . the median lengths of hospital stay were 5.7 days ( range 120 days ) for grade i , 9.5 days ( range 517 days ) for grade ii , and 5.0 days ( range 19 days ) for grade iii . radiographic findings , management and clinical outcomes for patients with different grades of dyspnea ( n = 39 ) * chi - square values ; h values . pm : pneumomediastinum ; sce : subcutaneous emphysema ; pt : pneumothorax ; sd : standard deviation . rigid bronchoscopies were performed on 26 patients ( 83.9% ) while flexible bronchoscopies on six patients ( 19.4% ) . after a short period of observation in hospital , they all recovered uneventfully and their pm resolved spontaneously with a mean time of 6.7 1.5 days . on the rigid bronchoscopy , a chest radiograph on the 7 day revealed good lung expansion with resolution of pm , and he was discharged . during follow - up , they had been intubated outside our hospital and got skin cutting and chest drainage immediately on admission . thereafter a 22-month - old male child was admitted to the emergency department of another hospital for respiratory arrest . since there was a swollen neck and face with anxiety and restlessness , intubation and artificial ventilation were performed , and the child was transferred to our hospital in an ambulance . on admission , there were coma , cyanosis , progression of sce , and hemodynamic instability . the skin cutting and chest drainage were performed once the diffuse pm , sce , and bilateral pt were confirmed on a bedside radiograph [ figure 1 ] . the clinical status deteriorated and he died of progressive and irreversible hypoxia and hypoxic encephalopathy 9 days later . chest radiograph showing mediastinal air , massive subcutaneous emphysema , and bilateral pneumothorax compressing both lungs to 50% . after immediate intubation and cardiopulmonary resuscitation , she was transferred to our hospital for further treatment . an emergent bedside radiograph revealed diffuse pm and right - sided pt [ figure 2 ] . on subsequent rigid bronchoscopy , chest radiograph showing mediastinal air and right - sided pneumothorax with the right lung compressed to approximately 30% . a total of 2643 patients with fba were identified between january 2010 and december 2015 in beijing children 's hospital . of all patients , pm occurred in 39 patients ( 1.5% ) , including 28 boys and 11 girls . the characteristics of patients and fbs and the clinical presentations and management are listed in table 1 . demographics , clinical characteristics , and management in pm secondary to fba ( n = 39 ) the data are shown as n ( % ) , median ( range ) , or mean sd . fbs : foreign bodies ; fba : foreign body aspiration ; pm : pneumomediastinum ; sce : subcutaneous emphysema ; pt : pneumothorax ; sd : standard deviation . the most common site of fbs was right main bronchus in 20 patients ( 51.3% ) , followed by left main bronchus in 17 patients ( 43.6% ) , both bronchi in one patient ( 2.6% ) , and subglottis in one patient ( 2.6% ) . thirty - nine patients were assessed and categorized into three grades according to the degree of dyspnea . thirty - one patients fell into grade i ; six into grade ii ; and two into grade iii . of all patients , two patients died while 37 got uneventful recovery with a mean time of 5.5 1.9 days for the pm resolution . the median lengths of hospital stay were 5.7 days ( range 120 days ) for grade i , 9.5 days ( range 517 days ) for grade ii , and 5.0 days ( range 19 days ) for grade iii . radiographic findings , management and clinical outcomes for patients with different grades of dyspnea ( n = 39 ) * chi - square values ; h values . rigid bronchoscopies were performed on 26 patients ( 83.9% ) while flexible bronchoscopies on six patients ( 19.4% ) . in one case , a small peanut particle was removed by a flexible bronchoscopy from the distal right bronchus after the failure of a rigid bronchoscopy . after a short period of observation in hospital , they all recovered uneventfully and their pm resolved spontaneously with a mean time of 6.7 1.5 days . on the rigid bronchoscopy , a chest radiograph on the 7 day revealed good lung expansion with resolution of pm , and he was discharged . they had been intubated outside our hospital and got skin cutting and chest drainage immediately on admission . thereafter a 22-month - old male child was admitted to the emergency department of another hospital for respiratory arrest . since there was a swollen neck and face with anxiety and restlessness , intubation and artificial ventilation were performed , and the child was transferred to our hospital in an ambulance . on admission , there were coma , cyanosis , progression of sce , and hemodynamic instability . the skin cutting and chest drainage were performed once the diffuse pm , sce , and bilateral pt were confirmed on a bedside radiograph [ figure 1 ] . the clinical status deteriorated and he died of progressive and irreversible hypoxia and hypoxic encephalopathy 9 days later . chest radiograph showing mediastinal air , massive subcutaneous emphysema , and bilateral pneumothorax compressing both lungs to 50% . after immediate intubation and cardiopulmonary resuscitation , she was transferred to our hospital for further treatment . the incidence in our study was 1.5% ( 39/2643 ) , which was similar to previous reports in the usa and israel . in this study , we characterized the presentation and management for this clinical entity in the past 6 years . a quick assessment system based on the degree of dyspnea was applied to evaluate the clinical severity , and management was performed accordingly on all patients . to the best of our knowledge , we aimed to help guide pediatric physicians in their assessment and management with this rare clinical entity . the clinical presentation of pm secondary to fba varies from chest pain , sce , dyspnea , hemodynamic instability to death . in this study , the most common presentations were wheezing , coughing , reduced breath sounds , and swelling over face and neck , which was similar to those with spontaneous pm . even though the majority of patients were generally well or with minimal symptoms , life - threatening complications should be kept in mind , including tension pm , tension pt , and increased pressure in the pulmonary interstitial . in our study , there were six patients in severe dyspnea and two in respiratory failure . the diagnosis of pm resulting from fba could be made from a comprehensive history , clinical signs , and radiographic studies . however , since some complications are life - threatening , the first and most important thing in practice is to identify clinical severity . the treatment principle for pm secondary to fba is early bronchoscopic fbs removals and supportive perioperative care . we believe rigid bronchoscopy and flexible bronchoscopy are both effective for the removal of airway fbs in experienced hands . skin cutting and chest drainage , which could be accomplished quickly at the patient 's bedside under local anesthesia , have already been reported as effective and safe procedures for air decompression for massive sce and pm and large pt , respectively . it is noted that for children with pm secondary to fba , a team of experienced emergency physicians , otolaryngologists , thoracic surgeons , and anesthetists should be available immediately to proceed with air evacuation , cardiopulmonary resuscitation , bronchoscopy , or tracheotomy in case of any accident . most importantly , the management must be decided cautiously on a case - by - case basis depending on the status of the child . since pm - associated compression of the airway and cardiac venous return can lead to dyspnea , desaturation , and consequent hemodynamic instability , we believe the degree of dyspnea could be used for stratification . it is simple and practical with initial careful observation . in this study , all patients were assessed on admission and patients in different degree of dyspnea were treated differently . ( grade i ) , an early bronchoscopy under general anesthesia is recommended with careful preparations . in our series after the removal of airway fbs , pms resolved spontaneously and all patients were discharged within a few days . in a generally well child , we recommend that an early bronchoscopy and a short period of observation are sufficient to promise a good outcome . for children in severe dyspnea ( grade ii ) , air evacuation for decompression should be the first step while intubation and cardiovascular resuscitation should be available if condition deteriorates . after the decompression , rigid bronchoscopy should be performed as soon as possible to remove the fbs . they all received immediate skin cutting for decompression , followed by rigid bronchoscopy . with subsequent supportive treatment , pms resolved spontaneously with a mean time of 6.7 days and they all got full recovery . the two deceased cases highlighted early air evacuation . both the children fell into grade ii dyspnea on their initial visits to the doctors . however , pm and pt were not recognized and no efforts were made to withdraw air from the mediastinum or thoracic cavity , a simple and fast procedure which might have saved their lives . similarly , a child was reported to die from possible tension pm secondary to fba due to lack of air evacuation . this reminds us that for patients in severe dyspnea with suspected pm and/or pt , immediate air evacuation , a simple and fast procedure , should come first for decompression . in our study , there was a 13-month - old male child who fell into grade ii on admission . therefore , if there is sudden onset of dyspnea in a previously stable patient , such complications as tension pm or large pt should be suspected and air evacuation should come first to save life . supportive care for pm secondary to fba includes bed rest , oxygen therapy , analgesics , treatment of comorbidities , and avoidance of valsalva maneuver . in light of the benign and self - limiting nature of pediatric pm , we recommend a short period of observation in hospital and avoidance of valsalva maneuvers for approximately 2 weeks . in addition , time to resolution was based on chest x - rays , and thus patients who were discharged may have a longer time to their next chest x - rays when compared with admitted patients . despite this , we felt the data are valid to help guide pediatric physicians in their assessment and management with this rare clinical entity . an assessment system based on the degree of dyspnea is critical to help make intervention options and anticipate the outcomes . for patients with mild and moderate dyspnea , simple bronchoscopic removal of airway fbs , an effective and less invasive procedure , is recommended . for patients with severe dyspnea , immediate air evacuation followed by bronchoscopy , a simple and fast procedure to improve the cardiopulmonary reserve , could promise a good outcome . z131100006813028 ) , and beijing municipal science and technology project ( no .

the past 10 years have seen a dramatic increase in the number of structures of ribosomal particles determined by cryo - electron microscopy ( cryo - em ) and x - ray crystallography . from a crystallographic viewpoint , structures of ribosomal particles are now available from three different bacterial organisms [ escherichia coli 70s ( 1 ) , thermus thermophilus 30s ( 2,3 ) and 70s ( 4,5 ) , as well as deinococcus radiodurans 50s ( 6 ) ] , one archaeon [ haloarcula marismortui 50s ] and two eukaryotes [ tetrahymena thermophila 40s ( 7 ) and saccharomyces cerevisiae 80s ( 8) ] . similarly , cryo - em has been used to determine structures of a wide - range of bacterial ribosomes , predominantly from e. coli ( 9 ) , but also t. thermophilus ( 10 ) , organellar ribosomes [ spinach chloroplast 70s ( 11 ) and mammalian mitochondrial 55s ( 12 ) ] as well as cytoplasmic ribosomes from both higher eukaryotes , including mammals [ e.g. human 40s ( 13 ) , rat 80s ( 14 ) , rabbit 80s ( 15 ) and canine 80s ( 16 ) ] and plants [ triticum aestivum 80s ( 17 ) ] , and lower eukaryotes , such as yeast ( 18 ) and other fungi ( 19 ) . to date , there are approximately 130 cryo - em maps of ribosomal particles deposited in the electron microscopy data bank ( emdb ; http://www.ebi.ac.uk/pdbe/emdb/ ) and more than 300 pdb entries for atomic coordinates and models of ribosomal particles deposited in the protein data bank ( pdb ; http://www.pdb.org/pdb ) . their deposition has followed the rapid exponential increase in the overall number of cryo - em maps and models deposited in the emdb and pdb , respectively ( 20,21 ) . indeed , the new emdatabank ( http://emdatabank.org ) has already more than 100 entries for emdb maps of ribosomal particles linked with associated pdb models ( 21 ) . both cryo - em and x - ray crystallography have been successfully utilized to determine structures of ribosomal particles in complex with various ligands , ranging from mrna and trna substrates to protein factors and antibiotics [ reviewed by ( 22 ) ] . at the time of writing , approximately 70 cryo - em maps of ribosomes in complex with trnas and 100 in complex with translation factors were deposited in the emdb , whereas the pdb contained more than 100 atomic coordinates of ribosomal particles associated with trnas , approximately 150 with antibiotics and approximately 100 with protein factors ( or domains thereof ) . the huge wealth of structural information available provides insight into ribosome function by enabling multiple different types of comparisons to be made , for example , between ( i ) ribosomes of different kingdoms , which provides evolutionary insight into the conserved and kingdom - specific regions of the ribosomes ( 23,24 ) , ( ii ) different conformational states of the same ribosomal particle , which can indicate conformational dynamics , for example , the rotational movement of the small subunit with respect to the large subunit ( 18,25,26 ) , ( iii ) ribosomes of different species interacting with the same ligand , which is particularly relevant when comparing the binding of antibiotics to antibiotic - susceptible bacteria or resistant archaea ( 27 ) , ( iv ) different but closely related ligands bound to the same ribosomal particle , which is exemplified by the plethora of substrate and intermediate mimics used to decipher the mechanism of peptide - bond formation ( 28 ) , ( v ) different functional states of the ribosome with the same ligand bound , such as the differing degrees of subunit rotation , head swiveling and l1 stalk movement seen when comparing trnas in pre- , intermediate or post - translocational states ribosomes ( 29,30 ) and ( vi ) different functional states of the ribosome induced upon ligand binding , for example subunit rotation upon ef - g binding ( 25 ) or remodeling of the small subunit upon hcv ires binding ( 13 ) . however , because the maps and atomic structures deposited in the emdb and pdb have no common coordinate system , it is usually necessary for the researcher to realign the maps and models of interest to one another . unfortunately , for the non - expert , this is not always trivial , particular when working with em density maps . therefore , we have developed a database of aligned ribosomal complexes , the darc site , where users can freely access and download aligned pdbs and cryo - em density maps . thus , the downloaded pdb and map ( brix ) files can be opened in common and freely available viewing programs , such as pymol ( www.pymol.org ) , chimera ( 31 ) , vmd ( 32 ) and directly compared . the darc site continuously monitors the release of new structures into the public domain by the emdb and pdb and imports maps and pdbs of entries that contain ribosomes or ribosomal subunits . currently , this encompasses cryo - em maps of bacterial 30s , 50s or 70s particles , archaeal 50s subunits , organellar 70s as well as eukaryotic 40s , 60s and 80s particles . the darc site does not yet include cryo - em maps of bacterial or eukaryotic assembly intermediates . the darc site comprises atomic coordinates from the pdb related to crystal structures of the bacterial 30s , 50s and 70s , the archaeal 50s and eukaryotic 40s and 80s . in addition , the darc site contains atomic coordinates for ribosomal structures built into cryo - em maps , however , does not include all structures of fragments of ribosomal rna alone or in complex with ligands due to ambiguity with alignment . similarly , the darc site does not include some incomplete models that contain only partial fragments of the small or large subunit rrnas , but does however include atomic coordinates with chains containing only backbone atoms , such as phosphates for rrna or c for ribosomal proteins . the darc site uses a common coordinate system based on the cryo - em maps ( emdb-1067 ) and associated molecular models ( 40s , pdb-1s1h and 60s , pdb-1s1i ) of the yeast 80s ribosome ( 18 ) . cryo - em maps of ribosomal particles were parsed from the emdb and the pixel and box size was adjusted to the reference map using spider ( 33 ) . the readjusted maps were then manually aligned to the emdb-1067 reference map in chimera ( 31 ) . subsequently an automatic fitting algorithm ( fit - in - map ) was applied that maximized the cross - correlation coefficient between the parsed and reference maps using a series of alternating rotation and translation steps until convergence is attained . the coordinate system of the aligned map was subsequently resampled to the reference coordinate system using the chimera command vop. atomic coordinates of ribosomes obtained from the pdb were aligned to the reference model for the yeast 80s ribosome , based either on the small or large subunit rrna , i.e. models based on crystal structures or cryo - em reconstructions of the small subunits used the small subunit rrna to align to the 18s rrna ( chain a ) of pdb-1s1h , whereas models based on crystal structures or cryo - em reconstructions of the large subunits used the large subunit rrna to align to the 25s rrna ( chain 3 ) of pdb-1s1i . in contrast , models for the small and large subunit from crystal structures or cryo - em reconstructions of 70s or 80s ribosomes were both aligned to the 25s rrna ( chain 3 ) of pdb-1s1i on the basis of the large subunit rrna . this is necessary to retain the same relative orientation of the small and large subunit within the 70s or 80s ribosomes in the re - aligned positions . since the atomic coordinates for the small and large subunit of a 70s or 80s ribosome are in separate pdb files due to their size , the alignment was done in two steps . firstly , the large subunit was aligned as described above , and then the large subunit alignment matrix was applied to the small subunit . alignment of the rrna was performed by using the matchmaker function in chimera ( 31 ) , which performs an initial pairwise alignment of the two rrna sequences via the needleman - wunsch - algorithm and the nucleic substitution matrix , and then employs an iterative fit algorithm that removes aligned pairs until the root mean square deviation ( rmsd ) of the fitted pairs is < 2 . the darc site has a user - friendly relational interface where users can either enter the known emdb or pdb accession number ( i d ) into the search panel or use the drop - down menu to restrict the search to source database or i d ( emdb or pdb ) , title , author , abstract or pudmed i d , as well as method ( cryo or x - ray ) , organism ( chloroplast , coli , human , mitochondria , thermophilus , rabbit , marismortui ) , ligand ( ef - g , ef2 , srp , trna , antibiotic ) , classification ( bacteria , archaeon , eukaryote ) or particle type ( 30s , 50s , 70s , 40s , 60s , 80s ) ( figure 1 ) . search results are displayed as a list alphabetically arranged on the basis of the pdb or emdb i d and the associated aligned darc structures can be directly downloaded from the right - hand column by clicking the aligned file link . figure 1.sample darc output . alternatively , the user can access further information on individual files by clicking the pdb or emdb accession number link . each entry has a header with the title and a link to the original emdb ( or pdb file ) is provided together with a link to the darc aligned file , either as brix file for maps or pdb files for models . for emdb entries having an associated pdb entry , additional links to the aligned files are provided below the title , for example , emdb-1858 is associated with pdb entries 3j00 and 3j01 . an automatically generated image of the relevant structure is presented on the right - hand side and additional details are presented on the left , including classification ( e.g. bacteria ) , particle ( 70s ) , organism ( e.g. e. coli ) , ligand name(s ) ( e.g. secyeg ) , method ( cryo - em or x - ray ) , publication authors ( frauenfeld et al . ) , publication year ( 2011 ) and pubmed i d ( with link to pubmed abstract ) . downloaded darc - aligned map and pdb files can be opened in common viewers such as pymol , chimera ( 31 ) or vmd ( 32 ) and directly compared . figure 2a c illustrates direct comparison of darc aligned cryo - em maps for a bacterial e. coli 70s ribosome ( emdb-1657 ) ( 34 ) with a eukaryotic t. aestivum 80s ribosome ( emdb-1780 ) ( 24,35 ) . alignment of cryo - em maps and associated pdbs of the protein conducting channel ( pcc ) ( 36 ) and the signal recognition particle ( srp ) ( 17 ) bound to the t. aestivum 80s ribosome reveals an overlap in their binding site at the tunnel exit site ( figure 2d f ) . similarly , darc - aligned pdbs comparing the antibiotic paromomycin [ paro ; pdb-1ibk , ( 37 ) ] and kasugamycin [ ksg ; pdb-2hhh , ( 38 ) ] bound to bacterial t. thermophilus 30s subunit reveal the different location of these drugs on the interface side of the particle ( figure 2g figure 2.example comparisons of darc - aligned emdb cryo - em maps and pdb atomic coordinates . ( a c ) comparison of the cryo - em map of the bacterial e. coli 70s ribosome ( emdb-1657 ) with the eukaryotic t. aestivum 80s ribosome ( emdb-1780 ) . ( d f ) overview ( above ) and zoom ( below ) of the cryo - em map and associated pdbs of the t. aestivum 80s ribosome bound with either the protein - conducting channel ( pcc ) ( emdb-1652/pdb-2wwb ) or the signal recognition particle ( srp ) ( emdb-1264/pdb-1ry1 ) . ( g i ) comparison of the structures of the 30s subunit in complex with the antibiotic paromomycin ( paro ) ( pdb-1ibk ) or kasugamycin ( ksg ) ( pdb-2hhh ) . example comparisons of darc - aligned emdb cryo - em maps and pdb atomic coordinates . ( a c ) comparison of the cryo - em map of the bacterial e. coli 70s ribosome ( emdb-1657 ) with the eukaryotic t. aestivum 80s ribosome ( emdb-1780 ) . ( d f ) overview ( above ) and zoom ( below ) of the cryo - em map and associated pdbs of the t. aestivum 80s ribosome bound with either the protein - conducting channel ( pcc ) ( emdb-1652/pdb-2wwb ) or the signal recognition particle ( srp ) ( emdb-1264/pdb-1ry1 ) . ( g i ) comparison of the structures of the 30s subunit in complex with the antibiotic paromomycin ( paro ) ( pdb-1ibk ) or kasugamycin ( ksg ) ( pdb-2hhh ) . the darc site continuously monitors the release of new structures into the public domain by the emdb and pdb and imports maps and pdbs of entries that contain ribosomes or ribosomal subunits . currently , this encompasses cryo - em maps of bacterial 30s , 50s or 70s particles , archaeal 50s subunits , organellar 70s as well as eukaryotic 40s , 60s and 80s particles . the darc site does not yet include cryo - em maps of bacterial or eukaryotic assembly intermediates . the darc site comprises atomic coordinates from the pdb related to crystal structures of the bacterial 30s , 50s and 70s , the archaeal 50s and eukaryotic 40s and 80s . in addition , the darc site contains atomic coordinates for ribosomal structures built into cryo - em maps , however , does not include all structures of fragments of ribosomal rna alone or in complex with ligands due to ambiguity with alignment . similarly , the darc site does not include some incomplete models that contain only partial fragments of the small or large subunit rrnas , but does however include atomic coordinates with chains containing only backbone atoms , such as phosphates for rrna or c for ribosomal proteins . the darc site uses a common coordinate system based on the cryo - em maps ( emdb-1067 ) and associated molecular models ( 40s , pdb-1s1h and 60s , pdb-1s1i ) of the yeast 80s ribosome ( 18 ) . cryo - em maps of ribosomal particles were parsed from the emdb and the pixel and box size was adjusted to the reference map using spider ( 33 ) . the readjusted maps were then manually aligned to the emdb-1067 reference map in chimera ( 31 ) . subsequently an automatic fitting algorithm ( fit - in - map ) was applied that maximized the cross - correlation coefficient between the parsed and reference maps using a series of alternating rotation and translation steps until convergence is attained . the coordinate system of the aligned map was subsequently resampled to the reference coordinate system using the chimera command vop. atomic coordinates of ribosomes obtained from the pdb were aligned to the reference model for the yeast 80s ribosome , based either on the small or large subunit rrna , i.e. models based on crystal structures or cryo - em reconstructions of the small subunits used the small subunit rrna to align to the 18s rrna ( chain a ) of pdb-1s1h , whereas models based on crystal structures or cryo - em reconstructions of the large subunits used the large subunit rrna to align to the 25s rrna ( chain 3 ) of pdb-1s1i . in contrast , models for the small and large subunit from crystal structures or cryo - em reconstructions of 70s or 80s ribosomes were both aligned to the 25s rrna ( chain 3 ) of pdb-1s1i on the basis of the large subunit rrna . this is necessary to retain the same relative orientation of the small and large subunit within the 70s or 80s ribosomes in the re - aligned positions . since the atomic coordinates for the small and large subunit of a 70s or 80s ribosome are in separate pdb files due to their size , the alignment was done in two steps . firstly , the large subunit was aligned as described above , and then the large subunit alignment matrix was applied to the small subunit . alignment of the rrna was performed by using the matchmaker function in chimera ( 31 ) , which performs an initial pairwise alignment of the two rrna sequences via the needleman - wunsch - algorithm and the nucleic substitution matrix , and then employs an iterative fit algorithm that removes aligned pairs until the root mean square deviation ( rmsd ) of the fitted pairs is < 2 . the darc site has a user - friendly relational interface where users can either enter the known emdb or pdb accession number ( i d ) into the search panel or use the drop - down menu to restrict the search to source database or i d ( emdb or pdb ) , title , author , abstract or pudmed i d , as well as method ( cryo or x - ray ) , organism ( chloroplast , coli , human , mitochondria , thermophilus , rabbit , marismortui ) , ligand ( ef - g , ef2 , srp , trna , antibiotic ) , classification ( bacteria , archaeon , eukaryote ) or particle type ( 30s , 50s , 70s , 40s , 60s , 80s ) ( figure 1 ) . search results are displayed as a list alphabetically arranged on the basis of the pdb or emdb i d and the associated aligned darc structures can be directly downloaded from the right - hand column by clicking the aligned file link . alternatively , the user can access further information on individual files by clicking the pdb or emdb accession number link . each entry has a header with the title and a link to the original emdb ( or pdb file ) is provided together with a link to the darc aligned file , either as brix file for maps or pdb files for models . for emdb entries having an associated pdb entry , additional links to the aligned files are provided below the title , for example , emdb-1858 is associated with pdb entries 3j00 and 3j01 . an automatically generated image of the relevant structure is presented on the right - hand side and additional details are presented on the left , including classification ( e.g. bacteria ) , particle ( 70s ) , organism ( e.g. e. coli ) , ligand name(s ) ( e.g. secyeg ) , method ( cryo - em or x - ray ) , publication authors ( frauenfeld et al . ) , publication year ( 2011 ) and pubmed i d ( with link to pubmed abstract ) . downloaded darc - aligned map and pdb files can be opened in common viewers such as pymol , chimera ( 31 ) or vmd ( 32 ) and directly compared . figure 2a c illustrates direct comparison of darc aligned cryo - em maps for a bacterial e. coli 70s ribosome ( emdb-1657 ) ( 34 ) with a eukaryotic t. aestivum 80s ribosome ( emdb-1780 ) ( 24,35 ) . alignment of cryo - em maps and associated pdbs of the protein conducting channel ( pcc ) ( 36 ) and the signal recognition particle ( srp ) ( 17 ) bound to the t. aestivum 80s ribosome reveals an overlap in their binding site at the tunnel exit site ( figure 2d f ) . similarly , darc - aligned pdbs comparing the antibiotic paromomycin [ paro ; pdb-1ibk , ( 37 ) ] and kasugamycin [ ksg ; pdb-2hhh , ( 38 ) ] bound to bacterial t. thermophilus 30s subunit reveal the different location of these drugs on the interface side of the particle ( figure 2g figure 2.example comparisons of darc - aligned emdb cryo - em maps and pdb atomic coordinates . ( a c ) comparison of the cryo - em map of the bacterial e. coli 70s ribosome ( emdb-1657 ) with the eukaryotic t. aestivum 80s ribosome ( emdb-1780 ) . ( d f ) overview ( above ) and zoom ( below ) of the cryo - em map and associated pdbs of the t. aestivum 80s ribosome bound with either the protein - conducting channel ( pcc ) ( emdb-1652/pdb-2wwb ) or the signal recognition particle ( srp ) ( emdb-1264/pdb-1ry1 ) . ( g i ) comparison of the structures of the 30s subunit in complex with the antibiotic paromomycin ( paro ) ( pdb-1ibk ) or kasugamycin ( ksg ) ( pdb-2hhh ) . example comparisons of darc - aligned emdb cryo - em maps and pdb atomic coordinates . ( a c ) comparison of the cryo - em map of the bacterial e. coli 70s ribosome ( emdb-1657 ) with the eukaryotic t. aestivum 80s ribosome ( emdb-1780 ) . ( d f ) overview ( above ) and zoom ( below ) of the cryo - em map and associated pdbs of the t. aestivum 80s ribosome bound with either the protein - conducting channel ( pcc ) ( emdb-1652/pdb-2wwb ) or the signal recognition particle ( srp ) ( emdb-1264/pdb-1ry1 ) . ( g i ) comparison of the structures of the 30s subunit in complex with the antibiotic paromomycin ( paro ) ( pdb-1ibk ) or kasugamycin ( ksg ) ( pdb-2hhh ) . given the large number of crystal and nmr structures of ribosomal ligands determined in the non - bound state , one future perspective would be to incorporate these structures aligned to the ribosomal - bound form of the ligand . this would provide users with the ability to easily compare conformational changes that indeed occur in numerous ligands upon ribosome binding . moreover , the database could be expanded progressively to include alignments of other large macromolecular complexes , that are subject to intense structural studies such as rna polymerases ( 39 ) or aaa+ atpases ( 40 ) . database name : the darc sitemain resource url : http://darcsite.genzentrum.lmu.de/darc/contact information ( e - mail ; postal mail ) : curator : hauser@lmb.uni-muenchen.de ; gene center , feodor - lynenstr . 25 , 81377 munich , germanydate resource established ( year ) : 2011conditions of use ( free , or type of license ) : freescope : data types captured , curation policy , standards used : pdb and brix filesstandards : mis , data formats , terminologies : pdb and brix filestaxonomic coverage : all speciesdata accessibility / output options : download from urldata release frequency : updated monthlyversioning period and access to historical files : yearly , nonedocumentation available : not necessaryuser support options : not necessarydata submission policy : data parsed from pdb and emdbrelevant publications : noneresource 's wikipedia url : nonetools available : none database name : the darc site main resource url : http://darcsite.genzentrum.lmu.de/darc/ contact information ( e - mail ; postal mail ) : curator : hauser@lmb.uni-muenchen.de ; gene center , feodor - lynenstr . 25 , 81377 munich , germany date resource established ( year ) : 2011 conditions of use ( free , or type of license ) : free scope : data types captured , curation policy , standards used : pdb and brix files standards : mis , data formats , terminologies : pdb and brix files taxonomic coverage : all species data accessibility / output options : download from url data release frequency : updated monthly versioning period and access to historical files : yearly , none documentation available : not necessary user support options : not necessary data submission policy : data parsed from pdb and emdb relevant publications : none resource 's wikipedia url : none tools available : none deutsche forschungsgemeinschaft sfb594 and sfb646 ( to r.b . ) and wi3285/1 - 1 ( to d.n.w . ) .

the ribosome is a highly dynamic machine responsible for protein synthesis within the cell . cryo - electron microscopy ( cryo - em ) and x - ray crystallography structures of ribosomal particles , alone and in complex with diverse ligands ( protein factors , rnas and small molecules ) , have revealed the dynamic nature of the ribosome and provided much needed insight into translation and its regulation . in the past years , there has been exponential growth in the deposition of cryo - em maps into the electron microscopy data bank ( emdb ) as well as atomic structures into the protein data bank ( pdb ) . unfortunately , the deposited ribosomal particles usually have distinct orientations with respect to one another , which complicate the comparison of the available structures . to simplify this , we have developed a database of aligned ribosomal complexes , the darc site ( http://darcsite.genzentrum.lmu.de/darc/ ) , which houses the available cryo - em maps and atomic coordinates of ribosomal particles from the emdb and pdb aligned within a common coordinate system . an easy - to - use , searchable interface allows users to access and download > 130 cryo - em maps and > 300 atomic models in the format of brix and pdb files , respectively . the aligned coordinate system substantially simplifies direct visualization of conformational changes in the ribosome , such as subunit rotation and head - swiveling , as well as direct comparison of bound ligands , such as antibiotics or translation factors .

INTRODUCTION RESULTS Database content Structural alignments Querying and retrieval from the DARC site Visualization of examples of DARC aligned maps and PDBs FUTURE PERSPECTIVES FUNDING

the past 10 years have seen a dramatic increase in the number of structures of ribosomal particles determined by cryo - electron microscopy ( cryo - em ) and x - ray crystallography . from a crystallographic viewpoint , structures of ribosomal particles are now available from three different bacterial organisms [ escherichia coli 70s ( 1 ) , thermus thermophilus 30s ( 2,3 ) and 70s ( 4,5 ) , as well as deinococcus radiodurans 50s ( 6 ) ] , one archaeon [ haloarcula marismortui 50s ] and two eukaryotes [ tetrahymena thermophila 40s ( 7 ) and saccharomyces cerevisiae 80s ( 8) ] . similarly , cryo - em has been used to determine structures of a wide - range of bacterial ribosomes , predominantly from e. coli ( 9 ) , but also t. thermophilus ( 10 ) , organellar ribosomes [ spinach chloroplast 70s ( 11 ) and mammalian mitochondrial 55s ( 12 ) ] as well as cytoplasmic ribosomes from both higher eukaryotes , including mammals [ e.g. human 40s ( 13 ) , rat 80s ( 14 ) , rabbit 80s ( 15 ) and canine 80s ( 16 ) ] and plants [ triticum aestivum 80s ( 17 ) ] , and lower eukaryotes , such as yeast ( 18 ) and other fungi ( 19 ) . to date , there are approximately 130 cryo - em maps of ribosomal particles deposited in the electron microscopy data bank ( emdb ; http://www.ebi.ac.uk/pdbe/emdb/ ) and more than 300 pdb entries for atomic coordinates and models of ribosomal particles deposited in the protein data bank ( pdb ; http://www.pdb.org/pdb ) . their deposition has followed the rapid exponential increase in the overall number of cryo - em maps and models deposited in the emdb and pdb , respectively ( 20,21 ) . both cryo - em and x - ray crystallography have been successfully utilized to determine structures of ribosomal particles in complex with various ligands , ranging from mrna and trna substrates to protein factors and antibiotics [ reviewed by ( 22 ) ] . at the time of writing , approximately 70 cryo - em maps of ribosomes in complex with trnas and 100 in complex with translation factors were deposited in the emdb , whereas the pdb contained more than 100 atomic coordinates of ribosomal particles associated with trnas , approximately 150 with antibiotics and approximately 100 with protein factors ( or domains thereof ) . the huge wealth of structural information available provides insight into ribosome function by enabling multiple different types of comparisons to be made , for example , between ( i ) ribosomes of different kingdoms , which provides evolutionary insight into the conserved and kingdom - specific regions of the ribosomes ( 23,24 ) , ( ii ) different conformational states of the same ribosomal particle , which can indicate conformational dynamics , for example , the rotational movement of the small subunit with respect to the large subunit ( 18,25,26 ) , ( iii ) ribosomes of different species interacting with the same ligand , which is particularly relevant when comparing the binding of antibiotics to antibiotic - susceptible bacteria or resistant archaea ( 27 ) , ( iv ) different but closely related ligands bound to the same ribosomal particle , which is exemplified by the plethora of substrate and intermediate mimics used to decipher the mechanism of peptide - bond formation ( 28 ) , ( v ) different functional states of the ribosome with the same ligand bound , such as the differing degrees of subunit rotation , head swiveling and l1 stalk movement seen when comparing trnas in pre- , intermediate or post - translocational states ribosomes ( 29,30 ) and ( vi ) different functional states of the ribosome induced upon ligand binding , for example subunit rotation upon ef - g binding ( 25 ) or remodeling of the small subunit upon hcv ires binding ( 13 ) . however , because the maps and atomic structures deposited in the emdb and pdb have no common coordinate system , it is usually necessary for the researcher to realign the maps and models of interest to one another . therefore , we have developed a database of aligned ribosomal complexes , the darc site , where users can freely access and download aligned pdbs and cryo - em density maps . thus , the downloaded pdb and map ( brix ) files can be opened in common and freely available viewing programs , such as pymol ( www.pymol.org ) , chimera ( 31 ) , vmd ( 32 ) and directly compared . the darc site continuously monitors the release of new structures into the public domain by the emdb and pdb and imports maps and pdbs of entries that contain ribosomes or ribosomal subunits . currently , this encompasses cryo - em maps of bacterial 30s , 50s or 70s particles , archaeal 50s subunits , organellar 70s as well as eukaryotic 40s , 60s and 80s particles . the darc site does not yet include cryo - em maps of bacterial or eukaryotic assembly intermediates . the darc site comprises atomic coordinates from the pdb related to crystal structures of the bacterial 30s , 50s and 70s , the archaeal 50s and eukaryotic 40s and 80s . in addition , the darc site contains atomic coordinates for ribosomal structures built into cryo - em maps , however , does not include all structures of fragments of ribosomal rna alone or in complex with ligands due to ambiguity with alignment . similarly , the darc site does not include some incomplete models that contain only partial fragments of the small or large subunit rrnas , but does however include atomic coordinates with chains containing only backbone atoms , such as phosphates for rrna or c for ribosomal proteins . the darc site uses a common coordinate system based on the cryo - em maps ( emdb-1067 ) and associated molecular models ( 40s , pdb-1s1h and 60s , pdb-1s1i ) of the yeast 80s ribosome ( 18 ) . cryo - em maps of ribosomal particles were parsed from the emdb and the pixel and box size was adjusted to the reference map using spider ( 33 ) . the coordinate system of the aligned map was subsequently resampled to the reference coordinate system using the chimera command vop. atomic coordinates of ribosomes obtained from the pdb were aligned to the reference model for the yeast 80s ribosome , based either on the small or large subunit rrna , i.e. since the atomic coordinates for the small and large subunit of a 70s or 80s ribosome are in separate pdb files due to their size , the alignment was done in two steps . the darc site has a user - friendly relational interface where users can either enter the known emdb or pdb accession number ( i d ) into the search panel or use the drop - down menu to restrict the search to source database or i d ( emdb or pdb ) , title , author , abstract or pudmed i d , as well as method ( cryo or x - ray ) , organism ( chloroplast , coli , human , mitochondria , thermophilus , rabbit , marismortui ) , ligand ( ef - g , ef2 , srp , trna , antibiotic ) , classification ( bacteria , archaeon , eukaryote ) or particle type ( 30s , 50s , 70s , 40s , 60s , 80s ) ( figure 1 ) . secyeg ) , method ( cryo - em or x - ray ) , publication authors ( frauenfeld et al . ) downloaded darc - aligned map and pdb files can be opened in common viewers such as pymol , chimera ( 31 ) or vmd ( 32 ) and directly compared . figure 2a c illustrates direct comparison of darc aligned cryo - em maps for a bacterial e. coli 70s ribosome ( emdb-1657 ) ( 34 ) with a eukaryotic t. aestivum 80s ribosome ( emdb-1780 ) ( 24,35 ) . alignment of cryo - em maps and associated pdbs of the protein conducting channel ( pcc ) ( 36 ) and the signal recognition particle ( srp ) ( 17 ) bound to the t. aestivum 80s ribosome reveals an overlap in their binding site at the tunnel exit site ( figure 2d f ) . similarly , darc - aligned pdbs comparing the antibiotic paromomycin [ paro ; pdb-1ibk , ( 37 ) ] and kasugamycin [ ksg ; pdb-2hhh , ( 38 ) ] bound to bacterial t. thermophilus 30s subunit reveal the different location of these drugs on the interface side of the particle ( figure 2g figure 2.example comparisons of darc - aligned emdb cryo - em maps and pdb atomic coordinates . ( a c ) comparison of the cryo - em map of the bacterial e. coli 70s ribosome ( emdb-1657 ) with the eukaryotic t. aestivum 80s ribosome ( emdb-1780 ) . ( d f ) overview ( above ) and zoom ( below ) of the cryo - em map and associated pdbs of the t. aestivum 80s ribosome bound with either the protein - conducting channel ( pcc ) ( emdb-1652/pdb-2wwb ) or the signal recognition particle ( srp ) ( emdb-1264/pdb-1ry1 ) . ( g i ) comparison of the structures of the 30s subunit in complex with the antibiotic paromomycin ( paro ) ( pdb-1ibk ) or kasugamycin ( ksg ) ( pdb-2hhh ) . example comparisons of darc - aligned emdb cryo - em maps and pdb atomic coordinates . ( a c ) comparison of the cryo - em map of the bacterial e. coli 70s ribosome ( emdb-1657 ) with the eukaryotic t. aestivum 80s ribosome ( emdb-1780 ) . ( d f ) overview ( above ) and zoom ( below ) of the cryo - em map and associated pdbs of the t. aestivum 80s ribosome bound with either the protein - conducting channel ( pcc ) ( emdb-1652/pdb-2wwb ) or the signal recognition particle ( srp ) ( emdb-1264/pdb-1ry1 ) . ( g i ) comparison of the structures of the 30s subunit in complex with the antibiotic paromomycin ( paro ) ( pdb-1ibk ) or kasugamycin ( ksg ) ( pdb-2hhh ) . the darc site continuously monitors the release of new structures into the public domain by the emdb and pdb and imports maps and pdbs of entries that contain ribosomes or ribosomal subunits . currently , this encompasses cryo - em maps of bacterial 30s , 50s or 70s particles , archaeal 50s subunits , organellar 70s as well as eukaryotic 40s , 60s and 80s particles . the darc site does not yet include cryo - em maps of bacterial or eukaryotic assembly intermediates . the darc site comprises atomic coordinates from the pdb related to crystal structures of the bacterial 30s , 50s and 70s , the archaeal 50s and eukaryotic 40s and 80s . in addition , the darc site contains atomic coordinates for ribosomal structures built into cryo - em maps , however , does not include all structures of fragments of ribosomal rna alone or in complex with ligands due to ambiguity with alignment . similarly , the darc site does not include some incomplete models that contain only partial fragments of the small or large subunit rrnas , but does however include atomic coordinates with chains containing only backbone atoms , such as phosphates for rrna or c for ribosomal proteins . the darc site uses a common coordinate system based on the cryo - em maps ( emdb-1067 ) and associated molecular models ( 40s , pdb-1s1h and 60s , pdb-1s1i ) of the yeast 80s ribosome ( 18 ) . cryo - em maps of ribosomal particles were parsed from the emdb and the pixel and box size was adjusted to the reference map using spider ( 33 ) . the coordinate system of the aligned map was subsequently resampled to the reference coordinate system using the chimera command vop. atomic coordinates of ribosomes obtained from the pdb were aligned to the reference model for the yeast 80s ribosome , based either on the small or large subunit rrna , i.e. models based on crystal structures or cryo - em reconstructions of the small subunits used the small subunit rrna to align to the 18s rrna ( chain a ) of pdb-1s1h , whereas models based on crystal structures or cryo - em reconstructions of the large subunits used the large subunit rrna to align to the 25s rrna ( chain 3 ) of pdb-1s1i . this is necessary to retain the same relative orientation of the small and large subunit within the 70s or 80s ribosomes in the re - aligned positions . the darc site has a user - friendly relational interface where users can either enter the known emdb or pdb accession number ( i d ) into the search panel or use the drop - down menu to restrict the search to source database or i d ( emdb or pdb ) , title , author , abstract or pudmed i d , as well as method ( cryo or x - ray ) , organism ( chloroplast , coli , human , mitochondria , thermophilus , rabbit , marismortui ) , ligand ( ef - g , ef2 , srp , trna , antibiotic ) , classification ( bacteria , archaeon , eukaryote ) or particle type ( 30s , 50s , 70s , 40s , 60s , 80s ) ( figure 1 ) . secyeg ) , method ( cryo - em or x - ray ) , publication authors ( frauenfeld et al . ) downloaded darc - aligned map and pdb files can be opened in common viewers such as pymol , chimera ( 31 ) or vmd ( 32 ) and directly compared . figure 2a c illustrates direct comparison of darc aligned cryo - em maps for a bacterial e. coli 70s ribosome ( emdb-1657 ) ( 34 ) with a eukaryotic t. aestivum 80s ribosome ( emdb-1780 ) ( 24,35 ) . alignment of cryo - em maps and associated pdbs of the protein conducting channel ( pcc ) ( 36 ) and the signal recognition particle ( srp ) ( 17 ) bound to the t. aestivum 80s ribosome reveals an overlap in their binding site at the tunnel exit site ( figure 2d f ) . similarly , darc - aligned pdbs comparing the antibiotic paromomycin [ paro ; pdb-1ibk , ( 37 ) ] and kasugamycin [ ksg ; pdb-2hhh , ( 38 ) ] bound to bacterial t. thermophilus 30s subunit reveal the different location of these drugs on the interface side of the particle ( figure 2g figure 2.example comparisons of darc - aligned emdb cryo - em maps and pdb atomic coordinates . ( a c ) comparison of the cryo - em map of the bacterial e. coli 70s ribosome ( emdb-1657 ) with the eukaryotic t. aestivum 80s ribosome ( emdb-1780 ) . ( d f ) overview ( above ) and zoom ( below ) of the cryo - em map and associated pdbs of the t. aestivum 80s ribosome bound with either the protein - conducting channel ( pcc ) ( emdb-1652/pdb-2wwb ) or the signal recognition particle ( srp ) ( emdb-1264/pdb-1ry1 ) . ( g i ) comparison of the structures of the 30s subunit in complex with the antibiotic paromomycin ( paro ) ( pdb-1ibk ) or kasugamycin ( ksg ) ( pdb-2hhh ) . example comparisons of darc - aligned emdb cryo - em maps and pdb atomic coordinates . ( a c ) comparison of the cryo - em map of the bacterial e. coli 70s ribosome ( emdb-1657 ) with the eukaryotic t. aestivum 80s ribosome ( emdb-1780 ) . ( d f ) overview ( above ) and zoom ( below ) of the cryo - em map and associated pdbs of the t. aestivum 80s ribosome bound with either the protein - conducting channel ( pcc ) ( emdb-1652/pdb-2wwb ) or the signal recognition particle ( srp ) ( emdb-1264/pdb-1ry1 ) . ( g i ) comparison of the structures of the 30s subunit in complex with the antibiotic paromomycin ( paro ) ( pdb-1ibk ) or kasugamycin ( ksg ) ( pdb-2hhh ) . given the large number of crystal and nmr structures of ribosomal ligands determined in the non - bound state , one future perspective would be to incorporate these structures aligned to the ribosomal - bound form of the ligand . 25 , 81377 munich , germanydate resource established ( year ) : 2011conditions of use ( free , or type of license ) : freescope : data types captured , curation policy , standards used : pdb and brix filesstandards : mis , data formats , terminologies : pdb and brix filestaxonomic coverage : all speciesdata accessibility / output options : download from urldata release frequency : updated monthlyversioning period and access to historical files : yearly , nonedocumentation available : not necessaryuser support options : not necessarydata submission policy : data parsed from pdb and emdbrelevant publications : noneresource 's wikipedia url : nonetools available : none database name : the darc site main resource url : http://darcsite.genzentrum.lmu.de/darc/ contact information ( e - mail ; postal mail ) : curator : hauser@lmb.uni-muenchen.de ; gene center , feodor - lynenstr .

in 2014 , there were an estimated 2.6 million children younger than 15 years living with hiv and 190,000 children became infected . hiv - infected children and adolescents have unique social and psychological issues that could affect their adherence to antiretroviral treatment ( art ) and health outcomes . one particular issue that may influence pediatric outcomes is knowledge of their own hiv status . timely and supportive disclosure may improve treatment adherence , retention in care , psychological adjustment , family relationships , and morbidity and mortality in hiv - infected children and adolescents . however , disclosing an hiv - positive status to a child remains a global challenge . in high hiv prevalence settings , most perinatally hiv - infected children and adolescents are unaware of their diagnosis , including those who attend regular clinic visits and take art . there are several barriers to pediatric hiv disclosure , including caregiver fears and lack of health care worker ( hcw ) knowledge and tools for disclosure . caregivers are reluctant to disclose because of potential to experience hiv stigma , guilt regarding transmission , uncertainty in how to disclose , and fears of negative child reactions or questions the child may ask . in addition , high - volume pediatric hiv clinics often lack systematized processes or standardized materials for disclosure , making disclosure a challenging task for overburdened hcws . interventions that address caregiver fears , as well as provide more training and standardized materials to hcws , may help to improve disclosure rates and experiences and improve child outcomes . to date , limited peer - reviewed literature describes disclosure interventions and their associated outcomes . after a rapid expansion in art access for children , hcws in namibia noted that they were unprepared for dealing with complex issues associated with telling an hiv - infected child their diagnosis . to address these concerns , the namibian ministry of health and social services ( mohss ) hcws who were providing pediatric hiv services , and the international education and training center for health ( i - tech ) , collaboratively and iteratively developed a pediatric hiv disclosure intervention . in 2010 , we have previously published evaluation data describing how the intervention improved hcw and caregiver 's confidence and communication skills for pediatric disclosure . in this retrospective study , we evaluated the impact of the intervention on child knowledge of their hiv status , adherence to art , and viral suppression , using the most complete routine service delivery data available . the evaluation was conducted at 4 high - volume hiv clinics in namibia : onandjokwe , oshakati , engela , and katutura . evaluation sites were selected based on the timing of intervention roll - out and pediatric hiv patient volumes . briefly , the disclosure intervention is intended to be used with children aged 618 years . the centerpiece of the intervention is a 5-chapter cartoon book which uses empowering language and metaphors of body soldiers being strengthened by medicine [ antiretroviral medications ( arvs ) ] and keeping the bad guys ( hiv virus ) asleep . the further the child progresses in reading the book , the more information about his or her disease and the role of medications is revealed . it is not until chapter 5 that the words hiv or a portion of the book is read , or reread , at each visit by a hcw until the caregiver and child are ready to read chapter 5 in which full disclosure occurs . the chapters are read in a highly interactive manner with each one taking approximately 510 minutes to complete the first time it is read . a disclosure form is attached to the patient care booklet on which the hcw notes how far in the disclosure book the child has gone at each visit and why the child thinks they are taking medicine . a readiness assessment form helps hcws assess the child 's and family 's readiness to engage in the full disclosure process . there is variation in how the intervention is implemented at each site because of site - specific contexts . for example , in facilities where children are unaccompanied by caregivers at their clinic visit , book chapters 14 are frequently used in group education settings for children . although the intervention is implemented in all sites , the completeness of routine documentation associated with the intervention varies widely . given that the disclosure intervention had been implemented nationally by the namibian mohss as part of routinely offered pediatric hiv treatment services , the university of washington institutional review board determined that the evaluation of this program was not human subjects research . data for this evaluation was abstracted between september and december of 2013 from routinely collected programmatic data . data sources included patient charts and 3 national electronic databases : ( 1 ) the national institute of pathology database that contains all hiv viral load ( vl ) test results performed in the country , ( 2 ) the electronic patient management system ( epms ) that stores general contact and demographic information on all children enrolled in hiv care , and ( 3 ) the electronic dispensing tool ( edt ) which contains prescription and medication information for all hiv - infected children receiving medications . initial participant lists for each of the 4 target clinics were generated by searching the epms database and identifying all children with birth dates within the appropriate date range ( age 715 years at the time of data abstraction ) who had been on art for at least one year . data abstractors pulled patient charts and verified and abstracted demographic data for all children identified through the epms database . we abstracted data for 2 components of evaluation : ( 1 ) a disclosure process evaluation to determine disclosure outcomes and changes in medication knowledge and ( 2 ) a clinical outcome evaluation to assess the impact of partial and full disclosure on cd4 count , vl , and adherence to art . children were included in the disclosure process evaluation if they had at least one hiv vl in the national institute of pathology database within the previous 6 months and documentation of initiating the disclosure intervention at least 13 months before the date of abstraction . of the children included in the disclosure process evaluation , the clinical outcome analysis was limited to children enrolled in the intervention during 2011 who had preintervention and postintervention initiation vl , cd4 , and/or adherence data . the 2011 enrollment cutoff was selected so that children had at least 2 years of follow - up postintervention initiation at the time of data abstraction ( fig . detailed flow chart of inclusion and exclusion criteria for participants included in disclosure evaluations . dashed line boxes include potential populations while solid - lined boxes describe actual populations included in the evaluation . adherence scores were calculated using pill pick - up and dispensing information found in edt and the following formula provided by the mohss : ppc = previous pill count pc = current pill count qd = quantity dispensed d = days since last visit cnppd = number of pills per day children in namibia begin receiving tablets at ages 34 years or earlier . a select few reported visits in the edt database included syrups for enrolled children , and those visits were removed before adherence analysis . for children documented as initiating the disclosure intervention , disclosure status was classified as full or partial . children who specifically mentioned that they took medication for hiv or had the full disclosure box checked and a corresponding date listed that was at or before enrollment in the intervention program were considered fully disclosed at baseline . children participating in the program were asked if they knew why they took their medicine at each visit , and responses were recorded . children characterized as having reached full disclosure during the intervention period had the full disclosure box checked and an appropriate date listed , had a record of reading the intervention booklet chapter where hiv is named , or a recorded response to the question why do you take your medicine ? that included the word hiv . data abstracted from patient charts and electronic medical record databases were analyzed using intercooled stata version 13.0 ( college station , tx ) . correlates of partial vs. full disclosure were determined using univariate logistic regression , and variables statistically significant ( p 0.05 ) in univariate analyses were included in a multivariate logistic regression model . variables were assessed for collinearity before being included in the multivariate model , and only one variable from collinear groupings was selected to be included in the multivariate model . for the subset of children enrolled in the disclosure program during 2011 , paired t - tests and mcnemar tests were used to compare mean differences in adherence scores , cd4 counts , cd4 percent , and log vls or the proportion of children virally suppressed or considered adherent before versus after enrollment into the intervention . we evaluated virologic success using 2 categories of clinical significance : 100 copies per milliliter and 1000 copies per milliliter . these categories reflect the who threshold for virologic suppression ( 1000 copies / ml ) and good viral suppression ( 100 copies / ml ) . children were considered adherent if they had a mean adherence percentage at or above 80% during the time period described . the evaluation was conducted at 4 high - volume hiv clinics in namibia : onandjokwe , oshakati , engela , and katutura . evaluation sites were selected based on the timing of intervention roll - out and pediatric hiv patient volumes . briefly , the disclosure intervention is intended to be used with children aged 618 years . the centerpiece of the intervention is a 5-chapter cartoon book which uses empowering language and metaphors of body soldiers being strengthened by medicine [ antiretroviral medications ( arvs ) ] and keeping the bad guys ( hiv virus ) asleep . the further the child progresses in reading the book , the more information about his or her disease and the role of medications is revealed . it is not until chapter 5 that the words hiv or a portion of the book is read , or reread , at each visit by a hcw until the caregiver and child are ready to read chapter 5 in which full disclosure occurs . the chapters are read in a highly interactive manner with each one taking approximately 510 minutes to complete the first time it is read . a disclosure form is attached to the patient care booklet on which the hcw notes how far in the disclosure book the child has gone at each visit and why the child thinks they are taking medicine . a readiness assessment form helps hcws assess the child 's and family 's readiness to engage in the full disclosure process . there is variation in how the intervention is implemented at each site because of site - specific contexts . for example , in facilities where children are unaccompanied by caregivers at their clinic visit , book chapters 14 are frequently used in group education settings for children . although the intervention is implemented in all sites , the completeness of routine documentation associated with the intervention varies widely . the namibian mohss ethics review committee reviewed and approved the study . given that the disclosure intervention had been implemented nationally by the namibian mohss as part of routinely offered pediatric hiv treatment services , the university of washington institutional review board determined that the evaluation of this program was not human subjects research . data for this evaluation was abstracted between september and december of 2013 from routinely collected programmatic data . data sources included patient charts and 3 national electronic databases : ( 1 ) the national institute of pathology database that contains all hiv viral load ( vl ) test results performed in the country , ( 2 ) the electronic patient management system ( epms ) that stores general contact and demographic information on all children enrolled in hiv care , and ( 3 ) the electronic dispensing tool ( edt ) which contains prescription and medication information for all hiv - infected children receiving medications . initial participant lists for each of the 4 target clinics were generated by searching the epms database and identifying all children with birth dates within the appropriate date range ( age 715 years at the time of data abstraction ) who had been on art for at least one year . data abstractors pulled patient charts and verified and abstracted demographic data for all children identified through the epms database . we abstracted data for 2 components of evaluation : ( 1 ) a disclosure process evaluation to determine disclosure outcomes and changes in medication knowledge and ( 2 ) a clinical outcome evaluation to assess the impact of partial and full disclosure on cd4 count , vl , and adherence to art . children were included in the disclosure process evaluation if they had at least one hiv vl in the national institute of pathology database within the previous 6 months and documentation of initiating the disclosure intervention at least 13 months before the date of abstraction . of the children included in the disclosure process evaluation , children included in the clinical outcomes evaluation met additional inclusion criteria . the clinical outcome analysis was limited to children enrolled in the intervention during 2011 who had preintervention and postintervention initiation vl , cd4 , and/or adherence data . the 2011 enrollment cutoff was selected so that children had at least 2 years of follow - up postintervention initiation at the time of data abstraction ( fig . detailed flow chart of inclusion and exclusion criteria for participants included in disclosure evaluations . dashed line boxes include potential populations while solid - lined boxes describe actual populations included in the evaluation . adherence scores were calculated using pill pick - up and dispensing information found in edt and the following formula provided by the mohss : ppc = previous pill count pc = current pill count qd = quantity dispensed d = days since last visit cnppd = number of pills per day children in namibia begin receiving tablets at ages 34 years or earlier . a select few reported visits in the edt database included syrups for enrolled children , and those visits were removed before adherence analysis . for children documented as initiating the disclosure intervention , children who specifically mentioned that they took medication for hiv or had the full disclosure box checked and a corresponding date listed that was at or before enrollment in the intervention program were considered fully disclosed at baseline . children participating in the program were asked if they knew why they took their medicine at each visit , and responses were recorded . children characterized as having reached full disclosure during the intervention period had the full disclosure box checked and an appropriate date listed , had a record of reading the intervention booklet chapter where hiv is named , or a recorded response to the question why do you take your medicine ? that included the word hiv . data abstracted from patient charts and electronic medical record databases were analyzed using intercooled stata version 13.0 ( college station , tx ) . correlates of partial vs. full disclosure were determined using univariate logistic regression , and variables statistically significant ( p 0.05 ) in univariate analyses were included in a multivariate logistic regression model . variables were assessed for collinearity before being included in the multivariate model , and only one variable from collinear groupings was selected to be included in the multivariate model . for the subset of children enrolled in the disclosure program during 2011 , paired t - tests and mcnemar tests were used to compare mean differences in adherence scores , cd4 counts , cd4 percent , and log vls or the proportion of children virally suppressed or considered adherent before versus after enrollment into the intervention . we evaluated virologic success using 2 categories of clinical significance : 100 copies per milliliter and 1000 copies per milliliter . these categories reflect the who threshold for virologic suppression ( 1000 copies / ml ) and good viral suppression ( 100 copies / ml ) . children were considered adherent if they had a mean adherence percentage at or above 80% during the time period described . of the 1466 children screened for inclusion in the evaluation , only 314 satisfied all inclusion criteria ( fig . the median age of children was 12 years , and approximately half ( 47% ) were female ( table 1 ) . more than 50% of the children had been on art for more than 6 years . only 64% of the study participants had a cd4 count or percent recorded within 1 year before data abstraction . almost half ( 46% ) of children had vls at or below 100 copies per milliliter , and an additional 18% were suppressed at or below 1000 copies per milliliter . population description at the time of data abstraction , the median time of enrollment in the intervention was just below 3 years ( table 1 ) . most children ( 89% ) had more than 1 visit recorded in the disclosure tracking form . for children with multiple entries , the median number of entries tracking responses to the question was 5 , and the average time between entries was 4.6 months . at enrollment , only 34 children ( 11% ) knew their hiv status . during their time enrolled in the program , there was documented full disclosure to 120 ( 43% ) children . when stratified by age , only 20% of children aged 710 years were fully disclosed during the course of the intervention compared with 57% of children aged 1115 years ( p < 0.001 ) . however , among those who reached full disclosure , the average time to full disclosure was not significantly different between younger and older children ( 29 vs. 31 months , respectively ; p = 0.40 ) . just below half ( 48% ) of the children who were disclosed after enrollment into the intervention had a record of reading chapter 5 of the intervention booklet , suggesting that many caregivers may have decided to disclose to their children outside the clinic setting , which is one of the options discussed with individual caregivers as part of the intervention . children who reached full disclosure during the course of the intervention were similar to children who remained only partially disclosed with respect to sex and cd4 count measurements ( table 2 ) . children who reached full disclosure were almost 1.5 years older at enrollment and at the time of data abstraction ( p < 0.001 for both ) and had been in hiv care [ odds ratio ( or ) : 1.33 , p < 0.001 ] and on art for longer ( or : 1.36 , p < 0.001 ) . more children from the clinic at katutura were fully disclosed compared with those from the other 3 clinics . whether evaluated continuously or as clinical cutoffs below 100 copies per milliliter or 1000 copies per milliliter , children with lower vls were more likely to have been fully disclosed during the course of the intervention . children enrolled in the intervention longer ( or : 1.08 , p < 0.001 ) and who had more intervention visits ( or : 1.26 , p < correlates of disclosure at their first visit , more than half ( 61% ) of children had no knowledge or incorrect knowledge of why they take their medications ( fig . this included responses in which the child reported that they did not know why they took medicines or reported taking medication for another ailment such as cough or malaria . initially , only 10% of children used hiv - specific terms to describe why they take medications , and 16% used basic health and wellness descriptions . by the last visit recorded before data abstraction , the number of children who had no knowledge or incorrect knowledge of fwhy they take their medicine dropped to 18% . of 153 children who did not know why they took medications initially , 42% became fully disclosed and used hiv - specific language , while 34% adopted language specific to the disclosure program . changes in children 's responses to the question why do you take your medicine ? from enrollment to the time of data abstraction among 278 children with more than 1 entry in the disclosure tracking form . the 92 children included in the preanalysis or postanalysis of vl and adherence measures had been on arvs for at least 18 months at the time of enrollment into the intervention and had at least one vl or adherence measurement during 2 periods of assessment time : ( 1 ) 12 months before enrollment in the intervention and ( 2 ) 012 months after intervention enrollment or 1224 months after intervention enrollment . although we found no significant difference between pre - enrollment and postenrollment vl measurements 012 months after enrollment ( p = 0.896 ) , we did find that vl measurements significantly decreased from pre - enrollment measurements by 0.5 log10 copies per milliliter ( p = 0.004 ) by 1224 months after enrollment into the disclosure program . we also observed a decrease in vl measurements between 012 months after enrollment and 1224 months after enrollment ( p = 0.053 ) . when evaluating sustained viral suppression at or below 1000 copies per milliliter during the 12-month period before and after enrollment , we saw no effect of enrollment in the intervention on virologic failure . although not statistically significant , we observed slightly improved viral suppression at or below 100 copies per milliliter when comparing viral suppression before enrollment to 1224 months after enrollment ( p = 0.103 ) . by 12 months after intervention , 18 ( 30% ) children had reached full disclosure , and 8 more children reached full disclosure by 24 months . we saw no statistically significant association between full disclosure status at 12 months and mean vl values or proportion of children virally suppressed during the 1224-month period after intervention enrollment . 83 of these children were on first line regimens and 6 were on second line regimens . when evaluating calculated adherence percentage measurements before and after enrollment , we observed a significant adherence percentage increase over pre - enrollment measurements by 8% ( p < 0.001 ) by 012 months after enrollment into the disclosure program and by 10% ( p < 0.001 ) by 1224 months after enrollment ( fig . 3 ) . there was no significant difference between adherence percentages 012 months and 1224 months after enrollment . when evaluating adherence measures categorically ( mean adherence 80% ) , we also observed a significant increase in the proportion of children adherent to medications during the 12 months ( p < 0.001 ) and 24 months ( p < 0.001 ) after enrollment . by 12 months and 24 months after intervention , 22 and 31 children , respectively , we did not observe statistically significant differences between full disclosure status and adherence measurements between the 12 months before enrollment and 12 months or 24 months after enrollment into the disclosure program . panel b , percent of children adherent ( defined as mean adherence 80% ) or with sustained viral suppression ( defined as all vl measurements 1000 copies / ml or 100 copies / ml ) during the period specified . panel c , paired t - tests and mcnemar tests comparing adherence and vl measurements during the periods specified for all children with data recorded during the 2 periods specified . of the 1466 children screened for inclusion in the evaluation , only 314 satisfied all inclusion criteria ( fig . the median age of children was 12 years , and approximately half ( 47% ) were female ( table 1 ) . more than 50% of the children had been on art for more than 6 years . only 64% of the study participants had a cd4 count or percent recorded within 1 year before data abstraction . almost half ( 46% ) of children had vls at or below 100 copies per milliliter , and an additional 18% were suppressed at or below 1000 copies per milliliter . at the time of data abstraction , the median time of enrollment in the intervention was just below 3 years ( table 1 ) . most children ( 89% ) had more than 1 visit recorded in the disclosure tracking form . for children with multiple entries , the median number of entries tracking responses to the question was 5 , and the average time between entries was 4.6 months . at enrollment , enrolled in the program , there was documented full disclosure to 120 ( 43% ) children . when stratified by age , only 20% of children aged 710 years were fully disclosed during the course of the intervention compared with 57% of children aged 1115 years ( p < 0.001 ) . however , among those who reached full disclosure , the average time to full disclosure was not significantly different between younger and older children ( 29 vs. 31 months , respectively ; p = 0.40 ) . just below half ( 48% ) of the children who were disclosed after enrollment into the intervention had a record of reading chapter 5 of the intervention booklet , suggesting that many caregivers may have decided to disclose to their children outside the clinic setting , which is one of the options discussed with individual caregivers as part of the intervention children who reached full disclosure during the course of the intervention were similar to children who remained only partially disclosed with respect to sex and cd4 count measurements ( table 2 ) . children who reached full disclosure were almost 1.5 years older at enrollment and at the time of data abstraction ( p < 0.001 for both ) and had been in hiv care [ odds ratio ( or ) : 1.33 , p < 0.001 ] and on art for longer ( or : 1.36 , p < 0.001 ) . more children from the clinic at katutura were fully disclosed compared with those from the other 3 clinics . whether evaluated continuously or as clinical cutoffs below 100 copies per milliliter or 1000 copies per milliliter , children with lower vls were more likely to have been fully disclosed during the course of the intervention . children enrolled in the intervention longer ( or : 1.08 , p < 0.001 ) and who had more intervention visits ( or : 1.26 , p < at their first visit , more than half ( 61% ) of children had no knowledge or incorrect knowledge of why they take their medications ( fig . this included responses in which the child reported that they did not know why they took medicines or reported taking medication for another ailment such as cough or malaria . initially , only 10% of children used hiv - specific terms to describe why they take medications , and 16% used basic health and wellness descriptions . by the last visit recorded before data abstraction , the number of children who had no knowledge or incorrect knowledge of fwhy they take their medicine dropped to 18% . of 153 children who did not know why they took medications initially , 42% became fully disclosed and used hiv - specific language , while 34% adopted language specific to the disclosure program . changes in children 's responses to the question why do you take your medicine ? from enrollment to the time of data abstraction among 278 children with more than 1 entry in the disclosure tracking form . the 92 children included in the preanalysis or postanalysis of vl and adherence measures had been on arvs for at least 18 months at the time of enrollment into the intervention and had at least one vl or adherence measurement during 2 periods of assessment time : ( 1 ) 12 months before enrollment in the intervention and ( 2 ) 012 months after intervention enrollment or 1224 months after intervention enrollment . although we found no significant difference between pre - enrollment and postenrollment vl measurements 012 months after enrollment ( p = 0.896 ) , we did find that vl measurements significantly decreased from pre - enrollment measurements by 0.5 log10 copies per milliliter ( p = 0.004 ) by 1224 months after enrollment into the disclosure program . we also observed a decrease in vl measurements between 012 months after enrollment and 1224 months after enrollment ( p = 0.053 ) . when evaluating sustained viral suppression at or below 1000 copies per milliliter during the 12-month period before and after enrollment , we saw no effect of enrollment in the intervention on virologic failure . although not statistically significant , we observed slightly improved viral suppression at or below 100 copies per milliliter when comparing viral suppression before enrollment to 1224 months after enrollment ( p = 0.103 ) . by 12 months after intervention , 18 ( 30% ) children had reached full disclosure , and 8 more children reached full disclosure by 24 months . we saw no statistically significant association between full disclosure status at 12 months and mean vl values or proportion of children virally suppressed during the 1224-month period after intervention enrollment . 83 of these children were on first line regimens and 6 were on second line regimens . when evaluating calculated adherence percentage measurements before and after enrollment , we observed a significant adherence percentage increase over pre - enrollment measurements by 8% ( p < 0.001 ) by 012 months after enrollment into the disclosure program and by 10% ( p < 0.001 ) by 1224 months after enrollment ( fig . there was no significant difference between adherence percentages 012 months and 1224 months after enrollment . when evaluating adherence measures categorically ( mean adherence 80% ) , we also observed a significant increase in the proportion of children adherent to medications during the 12 months ( p < 0.001 ) and 24 months ( p < we did not observe statistically significant differences between full disclosure status and adherence measurements between the 12 months before enrollment and 12 months or 24 months after enrollment into the disclosure program . panel a , mean summary of adherence or vl measurements during the period specified . includes only children who have measurements collected during all 3 periods . panel b , percent of children adherent ( defined as mean adherence 80% ) or with sustained viral suppression ( defined as all vl measurements 1000 copies / ml or 100 copies / ml ) during the period specified . panel c , paired t - tests and mcnemar tests comparing adherence and vl measurements during the periods specified for all children with data recorded during the 2 periods specified . this evaluation provides additional support to previously published qualitative results indicating that this hiv disclosure intervention was beneficial to pediatric patients , their primary caregivers , and health providers . previous publications cited caregiver and hcw descriptions of how the disclosure intervention improved their care of hiv - infected children and reports of children 's improved adherence to care and treatment . the analysis presented here contributes clinical outcome , quantitative data describing statistically significant improvements in adherence measurements before and after enrollment into the intervention . children exhibited better adherence by 12 and 24 months after the initial exposure to the intervention . children also showed improved vl measurements between pre - enrollment and postenrollment into the intervention , although these changes were not statistically significant when evaluated at a threshold of 1000 copies per milliliter or 100 copies per milliliter , measuring virologic success . interestingly , when evaluating continuous measures of vl and adherence , we saw improved adherence measurements preceding improvements in vl . although nonintervention studies have also shown that disclosure of hiv status is associated with improved adherence to art regimens among children and adolescents , our study is the first to evaluate changes in adherence longitudinally before and after the introduction of a disclosure intervention . in evaluating knowledge of why they take their medicine over time , we found that there was a dramatic decrease in the number of children who did not know why they took their medicines . our data , captured from routine tracking of pediatric patient knowledge which is a component of the intervention , depict the evolution of patient thinking about adherence and can directly relate it to the intervention 's communication and education strategy , thus unpacking the black box of programmatic interventions . after exposure to the intervention , most children changed responses and either related their partial understanding to terminology described in the disclosure book ( to keep bad guys asleep and/or soldiers strong ) or had documented full disclosure . we found that almost half of the children enrolled in the program had reached full disclosure by the time of data abstraction . this is almost 4.5 times the number of children who knew their status at enrollment . similarly to other studies , we saw that younger children were fully disclosed less frequently than older children . guidelines and current literature suggest that disclosure should be a guided step - by - step process , progressing from partial to full disclosure depending on caregiver readiness and child 's maturity . however , this is the first study of a disclosure intervention implemented at scale ( nationally ) to provide a description of the length and steps in that process . our study found that the average length of time from partial to full disclosure was almost 2.5 years . interestingly , our study did not find that the time to full disclosure differed by age . rather , the time required to reach full disclosure may instead reflect the frequency that children attend clinic visits , the need to overcome caregiver barriers to disclosure , and variable child readiness for full disclosure , regardless of age . the data on which the findings presented in this article are based were drawn from routinely collected patient information , based on health care service delivery as routinely implemented in busy practice settings . thus , we were limited on the variables we were able to evaluate , the time when variables were collected , and the number of participants we were able to include . unfortunately , no data on who performed disclosure was collected , and we were unable to assess the location where disclosure happened . however , despite these limitations , the results of this study are promising and demonstrate that disclosure can impact clinical outcomes and improve hiv knowledge in children and adolescents . analysis of each of 3 key variables indicates a consistent picture of a disclosure intervention that facilitates the disclosure process in such a way as to improve adherence and decrease vl . the fact that the intervention was successful in nonresearch settings suggests that while some specific intervention content adaptation would be necessary for different cultural contexts , major adaptations for real world implementation would not . there is an urgent need to develop interventions to assist hcws with the challenging but crucial process of hiv disclosure to children and adolescents . throughout sub - saharan africa the namibia hiv disclosure intervention seems to have improved disclosure rates , child knowledge of why they take their medicine , vl , and adherence measurements for children enrolled in the disclosure program . the namibian disclosure intervention may provide a helpful example of what could be adapted and used in other settings .

objectives : using routinely collected data , we evaluated a nationally implemented intervention to assist health care workers and caregivers with hiv disclosure to children . we assessed the impact of the intervention on child 's knowledge and health outcomes.methods:data were abstracted from national databases and patient charts for hiv - infected children aged 715 years attending 4 high - volume hiv clinics in namibia . disclosure rates , time to disclosure , and hiv knowledge in 314 children participating in the intervention were analyzed . logistic regression was used to identify correlates of partial vs. full disclosure . paired t - tests and mcnemar tests were used to compare adherence and viral load ( vl ) before versus after intervention enrollment.results:among children who participated in the disclosure intervention , 11% knew their hiv status at enrollment and an additional 38% reached full disclosure after enrollment . the average time to full disclosure was 2.5 years ( interquartile range : 1.23 years ) . children who achieved full disclosure were more likely to be older , have lower vls , and have been enrolled in the intervention longer . among children who reported incorrect knowledge regarding why they take their medicine , 83% showed improved knowledge after the intervention , defined as knowledge of hiv status or adopting intervention - specific language . on comparing 012 months before vs. 1224 months after enrollment in the intervention , vl decreased by 0.5 log10 copies per milliliter ( n = 42 , p = 0.004 ) , whereas mean adherence scores increased by 10% ( n = 88 , p value < 0.001).conclusions : this hiv disclosure intervention demonstrated improved viral suppression , adherence , and hiv knowledge and should be considered for translation to other settings .

INTRODUCTION METHODS Intervention Design and Evaluation Sites Ethical Considerations Data Collection Data Analysis RESULTS Cohort Characteristics Disclosure Process and HIV Knowledge Correlates of Full Disclosure Knowledge Changes During the Intervention Clinical Outcomes DISCUSSION CONCLUSIONS

hiv - infected children and adolescents have unique social and psychological issues that could affect their adherence to antiretroviral treatment ( art ) and health outcomes . timely and supportive disclosure may improve treatment adherence , retention in care , psychological adjustment , family relationships , and morbidity and mortality in hiv - infected children and adolescents . there are several barriers to pediatric hiv disclosure , including caregiver fears and lack of health care worker ( hcw ) knowledge and tools for disclosure . in addition , high - volume pediatric hiv clinics often lack systematized processes or standardized materials for disclosure , making disclosure a challenging task for overburdened hcws . to address these concerns , the namibian ministry of health and social services ( mohss ) hcws who were providing pediatric hiv services , and the international education and training center for health ( i - tech ) , collaboratively and iteratively developed a pediatric hiv disclosure intervention . in this retrospective study , we evaluated the impact of the intervention on child knowledge of their hiv status , adherence to art , and viral suppression , using the most complete routine service delivery data available . the evaluation was conducted at 4 high - volume hiv clinics in namibia : onandjokwe , oshakati , engela , and katutura . briefly , the disclosure intervention is intended to be used with children aged 618 years . data sources included patient charts and 3 national electronic databases : ( 1 ) the national institute of pathology database that contains all hiv viral load ( vl ) test results performed in the country , ( 2 ) the electronic patient management system ( epms ) that stores general contact and demographic information on all children enrolled in hiv care , and ( 3 ) the electronic dispensing tool ( edt ) which contains prescription and medication information for all hiv - infected children receiving medications . we abstracted data for 2 components of evaluation : ( 1 ) a disclosure process evaluation to determine disclosure outcomes and changes in medication knowledge and ( 2 ) a clinical outcome evaluation to assess the impact of partial and full disclosure on cd4 count , vl , and adherence to art . children were included in the disclosure process evaluation if they had at least one hiv vl in the national institute of pathology database within the previous 6 months and documentation of initiating the disclosure intervention at least 13 months before the date of abstraction . of the children included in the disclosure process evaluation , the clinical outcome analysis was limited to children enrolled in the intervention during 2011 who had preintervention and postintervention initiation vl , cd4 , and/or adherence data . children who specifically mentioned that they took medication for hiv or had the full disclosure box checked and a corresponding date listed that was at or before enrollment in the intervention program were considered fully disclosed at baseline . children participating in the program were asked if they knew why they took their medicine at each visit , and responses were recorded . children characterized as having reached full disclosure during the intervention period had the full disclosure box checked and an appropriate date listed , had a record of reading the intervention booklet chapter where hiv is named , or a recorded response to the question why do you take your medicine ? correlates of partial vs. full disclosure were determined using univariate logistic regression , and variables statistically significant ( p 0.05 ) in univariate analyses were included in a multivariate logistic regression model . for the subset of children enrolled in the disclosure program during 2011 , paired t - tests and mcnemar tests were used to compare mean differences in adherence scores , cd4 counts , cd4 percent , and log vls or the proportion of children virally suppressed or considered adherent before versus after enrollment into the intervention . we evaluated virologic success using 2 categories of clinical significance : 100 copies per milliliter and 1000 copies per milliliter . the evaluation was conducted at 4 high - volume hiv clinics in namibia : onandjokwe , oshakati , engela , and katutura . briefly , the disclosure intervention is intended to be used with children aged 618 years . data sources included patient charts and 3 national electronic databases : ( 1 ) the national institute of pathology database that contains all hiv viral load ( vl ) test results performed in the country , ( 2 ) the electronic patient management system ( epms ) that stores general contact and demographic information on all children enrolled in hiv care , and ( 3 ) the electronic dispensing tool ( edt ) which contains prescription and medication information for all hiv - infected children receiving medications . we abstracted data for 2 components of evaluation : ( 1 ) a disclosure process evaluation to determine disclosure outcomes and changes in medication knowledge and ( 2 ) a clinical outcome evaluation to assess the impact of partial and full disclosure on cd4 count , vl , and adherence to art . children were included in the disclosure process evaluation if they had at least one hiv vl in the national institute of pathology database within the previous 6 months and documentation of initiating the disclosure intervention at least 13 months before the date of abstraction . the clinical outcome analysis was limited to children enrolled in the intervention during 2011 who had preintervention and postintervention initiation vl , cd4 , and/or adherence data . for children documented as initiating the disclosure intervention , children who specifically mentioned that they took medication for hiv or had the full disclosure box checked and a corresponding date listed that was at or before enrollment in the intervention program were considered fully disclosed at baseline . children participating in the program were asked if they knew why they took their medicine at each visit , and responses were recorded . children characterized as having reached full disclosure during the intervention period had the full disclosure box checked and an appropriate date listed , had a record of reading the intervention booklet chapter where hiv is named , or a recorded response to the question why do you take your medicine ? data abstracted from patient charts and electronic medical record databases were analyzed using intercooled stata version 13.0 ( college station , tx ) . correlates of partial vs. full disclosure were determined using univariate logistic regression , and variables statistically significant ( p 0.05 ) in univariate analyses were included in a multivariate logistic regression model . variables were assessed for collinearity before being included in the multivariate model , and only one variable from collinear groupings was selected to be included in the multivariate model . for the subset of children enrolled in the disclosure program during 2011 , paired t - tests and mcnemar tests were used to compare mean differences in adherence scores , cd4 counts , cd4 percent , and log vls or the proportion of children virally suppressed or considered adherent before versus after enrollment into the intervention . almost half ( 46% ) of children had vls at or below 100 copies per milliliter , and an additional 18% were suppressed at or below 1000 copies per milliliter . population description at the time of data abstraction , the median time of enrollment in the intervention was just below 3 years ( table 1 ) . at enrollment , only 34 children ( 11% ) knew their hiv status . during their time enrolled in the program , there was documented full disclosure to 120 ( 43% ) children . when stratified by age , only 20% of children aged 710 years were fully disclosed during the course of the intervention compared with 57% of children aged 1115 years ( p < 0.001 ) . however , among those who reached full disclosure , the average time to full disclosure was not significantly different between younger and older children ( 29 vs. 31 months , respectively ; p = 0.40 ) . just below half ( 48% ) of the children who were disclosed after enrollment into the intervention had a record of reading chapter 5 of the intervention booklet , suggesting that many caregivers may have decided to disclose to their children outside the clinic setting , which is one of the options discussed with individual caregivers as part of the intervention . children who reached full disclosure during the course of the intervention were similar to children who remained only partially disclosed with respect to sex and cd4 count measurements ( table 2 ) . children who reached full disclosure were almost 1.5 years older at enrollment and at the time of data abstraction ( p < 0.001 for both ) and had been in hiv care [ odds ratio ( or ) : 1.33 , p < 0.001 ] and on art for longer ( or : 1.36 , p < 0.001 ) . whether evaluated continuously or as clinical cutoffs below 100 copies per milliliter or 1000 copies per milliliter , children with lower vls were more likely to have been fully disclosed during the course of the intervention . children enrolled in the intervention longer ( or : 1.08 , p < 0.001 ) and who had more intervention visits ( or : 1.26 , p < correlates of disclosure at their first visit , more than half ( 61% ) of children had no knowledge or incorrect knowledge of why they take their medications ( fig . initially , only 10% of children used hiv - specific terms to describe why they take medications , and 16% used basic health and wellness descriptions . by the last visit recorded before data abstraction , the number of children who had no knowledge or incorrect knowledge of fwhy they take their medicine dropped to 18% . of 153 children who did not know why they took medications initially , 42% became fully disclosed and used hiv - specific language , while 34% adopted language specific to the disclosure program . the 92 children included in the preanalysis or postanalysis of vl and adherence measures had been on arvs for at least 18 months at the time of enrollment into the intervention and had at least one vl or adherence measurement during 2 periods of assessment time : ( 1 ) 12 months before enrollment in the intervention and ( 2 ) 012 months after intervention enrollment or 1224 months after intervention enrollment . although we found no significant difference between pre - enrollment and postenrollment vl measurements 012 months after enrollment ( p = 0.896 ) , we did find that vl measurements significantly decreased from pre - enrollment measurements by 0.5 log10 copies per milliliter ( p = 0.004 ) by 1224 months after enrollment into the disclosure program . we also observed a decrease in vl measurements between 012 months after enrollment and 1224 months after enrollment ( p = 0.053 ) . when evaluating sustained viral suppression at or below 1000 copies per milliliter during the 12-month period before and after enrollment , we saw no effect of enrollment in the intervention on virologic failure . although not statistically significant , we observed slightly improved viral suppression at or below 100 copies per milliliter when comparing viral suppression before enrollment to 1224 months after enrollment ( p = 0.103 ) . by 12 months after intervention , 18 ( 30% ) children had reached full disclosure , and 8 more children reached full disclosure by 24 months . we saw no statistically significant association between full disclosure status at 12 months and mean vl values or proportion of children virally suppressed during the 1224-month period after intervention enrollment . when evaluating calculated adherence percentage measurements before and after enrollment , we observed a significant adherence percentage increase over pre - enrollment measurements by 8% ( p < 0.001 ) by 012 months after enrollment into the disclosure program and by 10% ( p < 0.001 ) by 1224 months after enrollment ( fig . there was no significant difference between adherence percentages 012 months and 1224 months after enrollment . when evaluating adherence measures categorically ( mean adherence 80% ) , we also observed a significant increase in the proportion of children adherent to medications during the 12 months ( p < 0.001 ) and 24 months ( p < 0.001 ) after enrollment . by 12 months and 24 months after intervention , 22 and 31 children , respectively , we did not observe statistically significant differences between full disclosure status and adherence measurements between the 12 months before enrollment and 12 months or 24 months after enrollment into the disclosure program . panel c , paired t - tests and mcnemar tests comparing adherence and vl measurements during the periods specified for all children with data recorded during the 2 periods specified . almost half ( 46% ) of children had vls at or below 100 copies per milliliter , and an additional 18% were suppressed at or below 1000 copies per milliliter . at the time of data abstraction , the median time of enrollment in the intervention was just below 3 years ( table 1 ) . at enrollment , enrolled in the program , there was documented full disclosure to 120 ( 43% ) children . when stratified by age , only 20% of children aged 710 years were fully disclosed during the course of the intervention compared with 57% of children aged 1115 years ( p < 0.001 ) . however , among those who reached full disclosure , the average time to full disclosure was not significantly different between younger and older children ( 29 vs. 31 months , respectively ; p = 0.40 ) . just below half ( 48% ) of the children who were disclosed after enrollment into the intervention had a record of reading chapter 5 of the intervention booklet , suggesting that many caregivers may have decided to disclose to their children outside the clinic setting , which is one of the options discussed with individual caregivers as part of the intervention children who reached full disclosure during the course of the intervention were similar to children who remained only partially disclosed with respect to sex and cd4 count measurements ( table 2 ) . children who reached full disclosure were almost 1.5 years older at enrollment and at the time of data abstraction ( p < 0.001 for both ) and had been in hiv care [ odds ratio ( or ) : 1.33 , p < 0.001 ] and on art for longer ( or : 1.36 , p < 0.001 ) . whether evaluated continuously or as clinical cutoffs below 100 copies per milliliter or 1000 copies per milliliter , children with lower vls were more likely to have been fully disclosed during the course of the intervention . children enrolled in the intervention longer ( or : 1.08 , p < 0.001 ) and who had more intervention visits ( or : 1.26 , p < at their first visit , more than half ( 61% ) of children had no knowledge or incorrect knowledge of why they take their medications ( fig . initially , only 10% of children used hiv - specific terms to describe why they take medications , and 16% used basic health and wellness descriptions . by the last visit recorded before data abstraction , the number of children who had no knowledge or incorrect knowledge of fwhy they take their medicine dropped to 18% . of 153 children who did not know why they took medications initially , 42% became fully disclosed and used hiv - specific language , while 34% adopted language specific to the disclosure program . the 92 children included in the preanalysis or postanalysis of vl and adherence measures had been on arvs for at least 18 months at the time of enrollment into the intervention and had at least one vl or adherence measurement during 2 periods of assessment time : ( 1 ) 12 months before enrollment in the intervention and ( 2 ) 012 months after intervention enrollment or 1224 months after intervention enrollment . although we found no significant difference between pre - enrollment and postenrollment vl measurements 012 months after enrollment ( p = 0.896 ) , we did find that vl measurements significantly decreased from pre - enrollment measurements by 0.5 log10 copies per milliliter ( p = 0.004 ) by 1224 months after enrollment into the disclosure program . we also observed a decrease in vl measurements between 012 months after enrollment and 1224 months after enrollment ( p = 0.053 ) . when evaluating sustained viral suppression at or below 1000 copies per milliliter during the 12-month period before and after enrollment , we saw no effect of enrollment in the intervention on virologic failure . although not statistically significant , we observed slightly improved viral suppression at or below 100 copies per milliliter when comparing viral suppression before enrollment to 1224 months after enrollment ( p = 0.103 ) . by 12 months after intervention , 18 ( 30% ) children had reached full disclosure , and 8 more children reached full disclosure by 24 months . we saw no statistically significant association between full disclosure status at 12 months and mean vl values or proportion of children virally suppressed during the 1224-month period after intervention enrollment . when evaluating calculated adherence percentage measurements before and after enrollment , we observed a significant adherence percentage increase over pre - enrollment measurements by 8% ( p < 0.001 ) by 012 months after enrollment into the disclosure program and by 10% ( p < 0.001 ) by 1224 months after enrollment ( fig . there was no significant difference between adherence percentages 012 months and 1224 months after enrollment . when evaluating adherence measures categorically ( mean adherence 80% ) , we also observed a significant increase in the proportion of children adherent to medications during the 12 months ( p < 0.001 ) and 24 months ( p < we did not observe statistically significant differences between full disclosure status and adherence measurements between the 12 months before enrollment and 12 months or 24 months after enrollment into the disclosure program . panel c , paired t - tests and mcnemar tests comparing adherence and vl measurements during the periods specified for all children with data recorded during the 2 periods specified . this evaluation provides additional support to previously published qualitative results indicating that this hiv disclosure intervention was beneficial to pediatric patients , their primary caregivers , and health providers . previous publications cited caregiver and hcw descriptions of how the disclosure intervention improved their care of hiv - infected children and reports of children 's improved adherence to care and treatment . children also showed improved vl measurements between pre - enrollment and postenrollment into the intervention , although these changes were not statistically significant when evaluated at a threshold of 1000 copies per milliliter or 100 copies per milliliter , measuring virologic success . although nonintervention studies have also shown that disclosure of hiv status is associated with improved adherence to art regimens among children and adolescents , our study is the first to evaluate changes in adherence longitudinally before and after the introduction of a disclosure intervention . in evaluating knowledge of why they take their medicine over time , we found that there was a dramatic decrease in the number of children who did not know why they took their medicines . our data , captured from routine tracking of pediatric patient knowledge which is a component of the intervention , depict the evolution of patient thinking about adherence and can directly relate it to the intervention 's communication and education strategy , thus unpacking the black box of programmatic interventions . after exposure to the intervention , most children changed responses and either related their partial understanding to terminology described in the disclosure book ( to keep bad guys asleep and/or soldiers strong ) or had documented full disclosure . we found that almost half of the children enrolled in the program had reached full disclosure by the time of data abstraction . this is almost 4.5 times the number of children who knew their status at enrollment . guidelines and current literature suggest that disclosure should be a guided step - by - step process , progressing from partial to full disclosure depending on caregiver readiness and child 's maturity . our study found that the average length of time from partial to full disclosure was almost 2.5 years . interestingly , our study did not find that the time to full disclosure differed by age . rather , the time required to reach full disclosure may instead reflect the frequency that children attend clinic visits , the need to overcome caregiver barriers to disclosure , and variable child readiness for full disclosure , regardless of age . there is an urgent need to develop interventions to assist hcws with the challenging but crucial process of hiv disclosure to children and adolescents . throughout sub - saharan africa the namibia hiv disclosure intervention seems to have improved disclosure rates , child knowledge of why they take their medicine , vl , and adherence measurements for children enrolled in the disclosure program .

the discovery of mirror neurons that fire when an animal performs a goal - directed action or sees others perform the action gave rise to both monkey and human studies on the neural correlates of imitation ( rizzolatti and craighero , 2004 ; iacoboni and dapretto , 2006 ) . while the circuitry for motor imitation is described as encompassing the posterior inferior gyrus ( including area f5 ) , and adjacent ventral premotor cortex , as well as a posterior area located in the rostral part of the inferior parietal lobule , mirror neurons are also thought to have importance in language development . according to iacoboni and dapretto ( 2006 ) this evolutionary argument is based on the homology between area f5 of the macaque brain and brodman area 44 in the posterior inferior frontal gyrus of the human brain , an area linked with language . iacoboni and dapretto ( 2006 ) also point out while trans - cranial magnetic stimulation ( tms ) has demonstrated bilaterality for the human mirror system ( mns ) , the question is raised how a relatively bilateral system for action observation and imitation contributes to a left - lateralized system for language . the authors describe a further tms study which investigated motor facilitation in the two hemispheres , while listening to acoustic sounds . the authors point out that as mirror neurons also respond to the sound of an action , the motor system of listeners should be facilitated when listening to the sounds produced by actions , but such facilitation only occurred in the left hemisphere . this suggested that the left hemisphere of the human brain has a multimodal ( visual , auditory ) mns , whereas the right hemisphere has only a visual mns . in humans , the shift from a purely visual to a multimodal mns , could have determined functional changes that could have facilitated language and a left - lateralization of language functions arbib ( 2010 ) has proposed a mirror system hypothesis ( msh ) of language evolution . he suggests that the mechanisms which support language in the human evolved atop a basic mechanism not originally related to communication , namely the mirror system for grasping . in the macaque monkey brain an area f5 just in front of the primary motor cortex ( thought to be homologous with f1 in humans ) contains neurons , active during manual and orofacial actions . according to arbib , a subset of these neurons ( mirror neurons ) , are also active when the monkey observes actions such as a precision pinch or a power grasp by a monkey or a human . similarly , a mirror system for grasping has been shown in the human brain . most significantly frontal activation was found in or near broca s area , a region which in most humans lies in the left hemisphere and traditionally is associated with speech production . arbib hypothesized seven stages in the evolution of languages , with the first three stages s1 ( grasping ) , s2 ( grasping shared with the common ancestor of human and monkey ) , s3 ( a simple imitation system for grasping shared with the common ancestor ) thought to be pre - hominid , and the next three s4 ( a complex imitation system for grasping ) , s5 ( protosign , a manual - based communication system , breaking through the fixed repertoire of primate vocalizations to yield an open repertoire ) , s6 ( protospeech , the ability of control mechanisms evolved for protosign coming to control the vocal apparatus with increasing flexibility , thought to distinguish the hominid line from that of the great apes ) , while the final stage s7 is that of language ( arbib and rizzolatti , 1997 ) . according to arbib , msh is simply the assertion that the mechanisms which get us to the role of broca s area in language depend in a crucial way on the mechanisms established in stage 2 , namely mirror mechanisms . while the mirror system for grasping is described as evolving to support protosign and protospeech in humans ( arbib and rizzolatti , 1997 ; rizzolatti and arbib , 1998 ; iacoboni and dapretto , 2006 ; arbib , 2010 ) . arbib and bota ( 2010 ) distinguish between the neural representation of the sign ( as distinct from symbol ) , which inherits mirror properties linking the production of vocal , manual , and/or facial gestures for a word , on one hand and the phonological loop and working memory systems on the other ( baddeley , 2003 ) . broca s area must be linked into prefrontal cortical ( pfc ) planning ( and its administration by the basal ganglia ) to assemble verb - argument and more complex hierarchical structures , finding the words , and binding them correctly . " by implication , the mirror aspect of language suggests an early feed - forward pantomime stage , while the development of grammar implies a sequential recursive capacity based on pfc developments ( arbib and bota , 2010 ) . fitch ( 2005 ) has pointed out that the weakest link in the arbib mns model is the crucial link from protosign to protospeech , " specifically his elison between two distinct forms of imitation : vocal and manual . fitch suggests that the co - evolution of vocal and manual gesture may have been more closely tied to music and dance than pantomime and linguistic communication . by this hypothesis , the crucial first step in human evolution from our last common ancestor with chimpanzees was the development of vocal imitation , similar in form and function to that independently evolved in many other vertebrate lineages ( including cetaceans , pinnipeds , and multiple avian lineages ) this hypothetical musical protolanguage preceded any truly linguistic system , capable of communicating particulate propositional meanings while dogs , birds , and apes can learn to map between meanings and words presented in isolation , the ability to extract words from arbitrary complex contexts and to recombine them in equally complex novel contexts is unattested in any non - human animal ( fitch , 2005 ) . leap by the age of three , without tutelage , feedback , or specific scaffolding , in contrast with skills such as alphabetic writing . in terms of deacon s ( 1997 ) classification above , mirror mechanisms should originally be classified as iconic , but according to arbib and bota ( 2010 ) , they provide a platform on which symbolic language representation may be built . where mirror systems reproduce a motor or vocal action in the absence of a concurrent stimulus , a further analogy with early language development is provided by the investigation of birdsong . for example , aronov et al . ( 2008 ) point out that babbling is an early behavior produced by juveniles of vocal mammals and birds . while much of the brain of birds from spinal cord to midbrain reflects an organization common to most vertebrates , the higher brain regions including the forebrain are different . however , there are large nuclear masses , which resemble the mammalian basal ganglia ( striatum ) , and a laminated isocortex ( gray matter ) , which is separated from the underlying basal ganglia by a band of myelinated axons ( white matter ) . like humans , birdsong and language both consist of ordered strings of sounds , separated by brief silent intervals . song syllables are usually grouped together to form phrases or motifs ( aronov et al . , 2008 ) . in zebra finches , babbling ( called subsong ) occurs roughly from 30 to 45 days post - hatch ( dph ) . plastic song follows , with the gradual appearance of distinctive identifiable , but variable , vocal elements ( syllables ) . according to the authors , plastic - song is by 90 dph gradually transformed into highly complex , stereotyped , motifs , or sequences of syllables that constitute adult song . the premotor circuit for adult song production is believed to consist of the high vocal center ( hvc ) , robust nucleus of the archipallium ( ra ) , and brainstem motor nuclei . this motor pathway is crucial for generating stereotyped , learned vocalizations , and exhibits firing , that is precisely time - locked to the song output ( aronov et al . , 2008 ) . ( 2008 ) another circuit , the anterior forebrain pathway ( afp ) is homologous to the basal ganglia thalamo - cortical loops in mammals , and projects to ra through a forebrain nucleus , lateral magnocellular nucleus of the nidopallium ( lman ) . although lman is not required for singing in adult birds , it is necessary for normal song learning in juveniles , and plays a role in producing song variability in adult and juvenile birds aronov and colleagues eliminated the hvc bilaterally ( important in adult singing ) in nine subsong ( 3344 dph ) , and in three additional birds in which they left the hvc intact , but specifically eliminated its projection to ra . also 12 older birds in the plastic - song stage ( 4573 dph ) and five adults also sang after hvc elimination , but lost structure and stereotypy and reverted to subsong - like vocalizations . in addition , when hvc was pharmacologically inactivated this reversion was fast and reversible , suggesting an immediate rather than long - term circuit change . the investigators posited three possibilities in relation to subsong : it is entirely produced by the midbrain or brainstem ; it is driven by circuitry intrinsic to ra , even in the absence of hvc and lman ; and third it is driven by or requires inputs from lman or ra . the investigators found that ra lesions entirely blocked singing in juvenile birds ( n = 5 , 3573 dph ) , indicating that subsong - like vocalizations required descending inputs from forebrain . similarly , song production was abolished by lesions of the hvc and subsequent inactivation of lman ( n = 5 , 5175 dph ) , indicating that ra circuitry without its afferent paths was not sufficient to generate singing ( aronov et al . , 2008 ) . the above authors concluded that lman and possibly other components of the afp constitute an essential premotor circuit for the production of early babbling . at the same time , the classical premotor nucleus hvc was not necessary for the generation of subsong . they proposed two premotor pathways in the songbird function , to produce vocalizations at different stages of development . in young juveniles , the afp generates poorly structured subsong , whereas in adult birds , the classical hvc - motor pathway generates highly stereotypic motor sequences . these pathways interact in the intermediate song stage to generate structured but variable vocalizations , upon which vocal learning operates . the transfer of functional dominance from one pathway to another during vocal learning elegantly parallels their anatomical development . hvc does not reach its adult size until the late plastic - song stage ; and establishes synapses in ra later than lman does song maturation and the decrease in vocal variability have thus been attributed to the strengthening of inputs from hvc and the concurrent weakening of inputs from lman . " the authors suggest that rather than a neuronal group selection theory of development ( in which early motor behaviors originate in the same circuits that later produce adult behavior ) , their findings suggest that distinct specialized circuits are dedicated to production of highly variable juvenile behavior . that is , juvenile singing is driven by a circuit , distinct from that which produces adult behavior ( aronov et al . , 2008 ) . ( 2008 ) that distinct cortical / subcortical circuits for the production of infant behavior may be a general feature of developmental learning in the vertebrate brain , is important for the present review . the childhood to adolescent development of cortical / subcortical behavioral circuits involved in infant behaviors such as babbling , free play , and over - activity , with subsequent transition to goal - directed behavior is fundamental to the present concept of motor and language development . like song maturation , the mechanisms by which this development is established may involve a pre - linguistic babbling stage , with subsequent transition to goal - directed language ( doupe and kuhl , 1999 ) . ( 2008 ) describe two distinct populations of projection neurons in the swamp sparrow s telencephalic nucleus ( hvc ) , necessary for singing and normal song perception . these consist of hvcra cells which innervate song premotor neurons in the robust nucleus of the arcopallium ( ra ) , and another hvcx , that innervates a striatal region of the avian basal ganglia ( area x ) , important to song learning and perception . the investigators looked at whether the activity in the hvcx cells during singing was due to auditory feedback or a corollary of the song motor activity . it was noted that a period of playback overlap was locked precisely to features of the syllable being sung , suggesting corollary motor activity . additionally , almost all hvcx cells responded to only naturally occurring sequences , indicating that a sequence of at least two notes was necessary to elicit an auditory response . this selective auditory responsiveness of hvcx cells extended to similar vocal sequences produced by other birds , making auditory vocal hvcx neurons well - suited to a role in communication ( prather et al . , 2008 ) . in many regards , auditory - vocal hvcx cells are similar to visual - motor neurons in the monkey frontal cortex that are hypothesized to play a role in perception of human gestures , including human speech . in that light , the precise temporal alignment of auditory and vocal activity in hvc x cells suggests that auditory - vocal mirror neurons express an additional mode of sensory - motor correspondence not previously reported for visual - motor mirror neurons ( 2009 ) suggest that because hvcx neurons innervate striatal structures important for song learning and perception , the coding strategy employed by hvcx neurons to represent vocal sequences , may have implications for learning and perception of speech in humans . in the human brain , cortical neurons similar to hvc auditory vocal neurons could transmit speech - related auditory and motor information to striatal regions implicated in speech development . vocal mirror neurons with properties similar to the hvcx cells described here could bind sensory and motor features of distinct vocal gestures , providing an efficient substrate for rapid decoding and encoding of speech ( 2009 ) point out that the division of continuously variable acoustic signals into discrete perceptual categories is a fundamental feature of vocal communication , including human speech . despite this , the neural mechanisms involved have been poorly studied ( doupe and kuhl , 1999 ) . the authors point out that swamp sparrows learn their song notes by imitation , a feature of human speech , otherwise rare among animals . behavioral experiments had shown that male swamp sparrows use categorical perception to distinguish fundamental acoustic elements in their species - typical vocal repertoire . these note types ( similar to phones in speech ) are produced with considerable variation by different individuals but are grouped into natural categories . as described above , the investigators had shown that the nucleus hvc contained a certain class of striatum - projecting neurons , hvcx cells that respond to only one song type in the bird s repertoire , and song perception was shown to be impaired by lesions to the striatal portion of an afp into which hvcx cells project their axons . this sensorimotor correspondence was thus suggested by the investigators as being reminiscent of mirror neurons in the monkey cortex , hypothesized to be important in perception ( aronov et al . , 2008 ) . ( 2009 ) recorded from antidromically identified hvc neurons in freely behaving male swamp sparrows , and presented each bird s song types through a speaker located near its perch as above . they presented each hvc cell a set of song stimuli comprising 511 variants of the primary song type , each differing only in the duration of a single replacement note in each trilled syllable , with duration of notes classified as category i to category vi . the procedure revealed that auditory responses of hvcx neurons , but not interneurons were highly sensitive to changes in note duration . robust responses were invariably evoked by stimuli containing replacement notes with durations that fell unambiguously into the same category as the target note in the natural song . in distinction , interneurons responded similarly when the replacement note was the same or a different category as the target note , indicating that categorical responses to changes in note duration are shown by only one subset of auditory responsive hvc neurons , namely those that project to a striatal pathway that is important in song perception " ( prather et al . , 2009 ) . furthermore , the investigators were able to demonstrate geographically distinct populations of swamp sparrows obtained from northwestern pennsylvania vs. upstate new york , in which perceptual categorical note boundaries differed from category i to category vi . this suggested that these differences may have been influenced by learning , and thus vary across populations . according to the investigators , the study provided the first evidence for neurons encoding perceptual information about a phonological feature of learned vocal behavior , specifically information about a categorical perceptual boundary . variation in this perceptual boundary across swamp sparrow populations strongly suggested that both categorical perception and categorical neural responses in sparrows are affected by experience . this observation was linked by the authors to the role of neural experience in shaping human speech perception of categorical boundaries . finally , the establishment that categorical responses are expressed by striatal projecting hvcx neurons , but not by interneurons , was thought to closely parallel the activity of auditory afferents to hvc , where the highly selective auditory responses of hvcx neurons required inhibitory sculpting through interneurons . this sculpting was thought to occur through a process of local inhibition , allowing context sensitivity over hundreds of milliseconds ( prather et al . , 2009 ) . while birdsong may also be built on mirror foundations , the capacity of hvc neurons to innervate striatal structures , important for encoding and decoding of learning and perception , as well as the geographically distinct phonological features of northwestern vs. upstate new york swamp sparrows suggests an indexical function for these songs . werker and tees ( 1999 ) point out newborn infants begin life with a remarkable sensitivity to the acoustic cues that signify different basic elements of speech . by measuring babies sucking response to syllables such as /ba/ vs. /da/ or /ba/ vs. /da/ , infants discriminated consonants most easily that actually occur in most of the world s languages . werker and tees point out that japanese babies were able to hear the distinction between /r/ and /l/ , but japanese adults were unable to hear this distinction . according to werker and tees infants become relatively more sensitive to the phonetic characteristics of the native language , and also to the syllabic context in which that phonetic variation occurs ( werker and lalonde , 1998 ; werker and tees , 1999 ) . the language - general perceptual sensitivities in newborns undergo a change and become more language - specific in the first year of life , thus preparing the infant for the ability to understand and speak his / her native language . during the first 1415 months , infants learn to extract words from the speech stream , and to recognize word forms they have previously heard , and to associate words with objects . coincident with the decline in non - native consonant ( and vowel ) discrimination seen by the end of the first year of life , the ability to co - ordinate two sources of information , such as phonetic detail , and position in a word is developed . the task for the next year of life is to construct a second - order system to effortlessly and efficiently use the medium of speech to map to meaning . with the establishment of a new level of representation , this discontinuity suggests a transformation from iconic mimicry to indexical representation , possibly similar to that which occurs in birdsong . the decline in non - native speech perception at the end of the first year of life , accompanied by the improvement in native speech perception has been found to be predictive of later language development ( kuhl et al . , 2008 ) . critical period have long been of interest to language scientists ( eimas et al . , 1971 ; werker and lalonde , 1998 ; kuhl et al . , 2008 ) . ( 2008 ) suggested that native language perceptual abilities are associated with cognitive control abilities , which play a specific role in the ability to disregard irrelevant phonetic information , while maintaining attention to relevant information . using a conditioned head - turn test of native and non - native speech sound discrimination and non - linguistic object retrieval tasks , sequencing attention , and inhibitory control , the investigators showed that native speech discrimination was positively linked to receptive vocabulary size , but not to cognitive control tasks , whereas non - native speech discrimination was negatively linked to cognitive control scores , but not to vocabulary size . the results suggested specific relationships between the development of native language , speech perception , and vocabulary ( conboy et al . , 2008 ) . ( 2008 ) point out that studies of the maturation of the human auditory cortex show that between the middle of the first year of life and 3 years of age , there is a maturation of axons entering the deeper cortical layers from the subcortical white matter ; and neurofilament - expressing axons appear for the first time in the temporal lobe , with projections to the deep cortical layers of the brain , providing the first highly processed auditory input from the brain stem . the temporal coincidence between this cytoarchitectural change and infants phonetic learning provides a possible maturational factor in the opening of a critical period for phonetic learning ( moore and guan , 2001 ) . the concept of transition between developing brain and native language phonetic ability , as well as the associated concept of a developmental discontinuity to a second - order representational system indicates a possible basis for the understanding of the importance of language in development . the distinction made by prather et al . ( 2008 ) , between visual motor and auditory vocal mirror neurons may be important for human speech development . while the significance of cortico - thalamic - striatal ( ctc ) circuits in visual motor development is well - described , a possibly analogous wernicke circuit , in which auditory vocal mirror neurons play a part is suggested . in both cases , an early process or sculpting of visual representational symbol in the pfc and phonetic category in language circuits , according to fuster , the pfc is phylogenetically one of the latest cortices to develop , having attained maximum relative growth in the human brain ( fuster , 2001 ) . fuster states that by myelogenic and synaptogenic criteria , the pfc is clearly late - maturing , and that the human prefrontal areas do not attain full maturity until adolescence . fuster describes the lateral pfc as the neural substrate for the cognitive functions that support the temporal organization of behavior . to conduct its executive functions , the lateral pfc interacts with subcortical structures and with other parts of the association cortex . a cardinal function of the lateral pfc is the temporal integration of information for the attainment of prospective behavioral goals [ ] there is evidence indicating that activation is maintained through recurrent circuits between pfc cells and posterior cortex [ ] . it is served by two complementary and temporally symmetric functions : working memory and preparatory set . both work together in every sphere of action , including speech thus , fuster suggests an analogous recurrent process is important for both working memory and for speech , which is important in understanding the developmental importance of speech and language development in early childhood . in the mature brain , working memory is thought to depend on an intact dorso - lateral pfc ( dlpfc ; fuster , 2001 ) . according to tau and peterson ( 2010 ) , rudimentary working memory capacities have been observed in infants as young as 6 months of age , but performance on piaget s a not b task ( retrieval of a hidden object after a delay ) is not in place till 9 months , and is not solidly in place for difficult tasks till middle childhood . miller and cohen ( 2001 ) describe the pfc as having the properties required to achieve top - down behavioral control . these include the ability to maintain its activity robustly until a goal is achieved , and second to have interconnections with all sensory systems , cortical and subcortical motor systems , and with limbic and midbrain structures involved in affect , memory , and reward . thus the lateral and mid - dorsal pfc receives visual , somatosensory , and auditory information from the occipital , temporal , and parietal cortices . the dorso - lateral area 46 is connected with premotor areas that send connections to primary motor areas and the spinal cord , as well as cerebellum and superior colliculus . miller and cohen ( 2001 ) also point out that the pfc neurons are both individually selective and others bimodally selective for sensory cues , but in addition pfc neural activity is able to represent rules required to perform a particular task . the miller and cohen model requires feedback signals from the pfc to reach throughout the brain . 1996 ) were able to show that monkeys were able to maintain a working memory of a rewarded stimulus over time , and that target - specific activity appeared simultaneously in the pfc and parietal cortex . while other brain areas can sustain activity up to several seconds , the pfc is distinguished by the ability to sustain such activity in the face of intervening distractions ( hopfield , 1982 ) . thus the pfc exhibits sustained activity that is robust to interference : multimodal convergence and integration of behaviorally relevant information ; feedback pathways that can exert biasing influences on other structures throughout the brain ; and ongoing plasticity that is adaptive to the demands of new tasks . this specialization is optimal for a role in the brain - wide control and coordination of processing . the mechanisms responsible for updating representations in the pfc must be responsive to changes in the environment , as well as resistant to updating irrelevant changes . miller and cohen hypothesized that dopamine ( da ) might play an important role in this gating function . they suggested that dual concurrent influences on midbrain da allow the system to learn while it gates , and where a da - mediated gating signal leads to a successful behavior , its concurrent reinforcing effects will strengthen the association of the signal with cues representing the pattern of activity that produced the behavior . thus this self - organizing boot - strapping mechanism averted the invocation of a homunculus to control behavioral selection ( miller and cohen , 2001 ) . ( 1990 ) have described the anatomical overlap of different mono - aminergic receptors in the same cortical strata , suggesting that there may be families of receptors linked by localization on common targets . arnsten points out that although goldman - rakic ( 1994 ) used spatial working memory as a model system for examining functional circuitry , she proposed that these principles applied to other sensory and affective domains , and described the process as representational knowledge within parallel processing streams . according to arnsten , goldman - rakic spoke of pfc network activity as a fundamental contribution to mind , and the disruption of this process as a primary contribution to thought disorder in mental illness . she used the term working memory to describe a building block of cognition : the ability to represent information no longer in the environment through recurrent excitation of pyramidal cells with shared stimulus properties arnsten described the role of the pfc in working memory , as applying representational knowledge to inhibit inappropriate action , thought , and feelings , as well as inhibiting responses to distracting stimuli . however , arnsten and goldman - rakic ( 1985 ) were able to show that many effects formerly attributed solely to da , involved both ne and da actions . according to arnsten ( 2007 ) , both da and ne exhibit an inverted-u dose / response , where either too little or too much arousal impairs working memory . an important distinction outlined by arnsten relates to the location of d1 and alpha-2a signaling mechanisms on dendritic spines . she points out that under optimal neurochemical conditions , moderate levels of ne engage alpha-2a receptors , and increase signals , whereas moderate levels of dad1 receptor stimulation decrease noise . these beneficial effects of alpha-2a vs. dad1 arise from opposing effects on camp signaling , where alpha-2a stimulation inhibits , while dad1 activates camp production . thus d1/alpha-2a signaling appears to have an important representational role in visuo - spatial working memory ( arnsten and goldman - rakic , 1985 ) . the demonstration by the work of goldman - rakic and arnsten ( arnsten and goldman - rakic , 1985 ; goldman - rakic , 1994 ; arnsten , 2007 ) on the importance of symbolic representational capacity in the human prefrontal cortex for working memory has implications for language development . it is likely that human language is distinguished from primate indexical reference by its capacity to recursively encode , incorporate , and combine visual and auditory symbols in working memory , basic for human language functions . the classification of referential capacity in terms of hierarchical iconic , indexical , and symbolic levels allows an understanding of the hierarchical nature of mirror , birdsong , and human language capacity . birdsong studies have revealed that the hvc was not necessary for the generation of subsong or early babbling , whereas the generation of adult syllabic complex stereotyped motifs required an inhibitory output from the hvc to brainstem motor nuclei , but nonetheless remain stereotypic and indexical . from the birdsong analogy , stage represents a sculpting of categorical native phoneme recognition , while mature song requires stabilization of hvcx neurons may play a part in the development of capacity for symbolic representation in the dlpfc . the present review suggests that similar processes occur in human language development , where categorical native phoneme recognition is sculpted in a wernicke / broca circuit en route to sequential language capacity . studies of human language development reveal an analogous early babbling stage , during which there is a transition from generalized to native phonemic usage , with a subsequent childhood transition to second - order representational capacity . the capacity for association visual and auditory symbols in working memory providing a basis for human language development . adequate pfc functioning appears critical for not only mature reasoning , but also involves behavioral functions , including inhibition of task - irrelevant behaviors , processing of affect , motivation , and reward attainment by virtue of connections with wide - ranging cortical centers . a consequence of such deficits in pfc development is an incapacity for sequential reasoning , lack of affect regulation , a lack of capacity for working on sustained goal achievement , and a tendency for impulsive and repetitive behaviors , under either environmental , or subcortical control . it can thus be argued that the process of development is closely dependant on adequate pfc development , and many if not most behavioral syndromes of childhood reflect deficits in cortical development . importantly this includes language development , where auditory vocal mirror neurons may have an important role in the transition from babbling to goal - directed language ( levy et al . three fundamental forms of cognitive reference : iconic , indexical , and symbolic are described in relation to mirror systems , birdsong , and human language . the process of human development is closely dependant on adequate pfc development , ( and many if not most behavioral syndromes of childhood reflect deficits in cortical development ) . importantly this includes language development , where auditory vocal mirror neurons may have an important role in the transition from babbling to goal - directed language . while the significance of ctc circuits in visual motor development is well - described , a possibly analogous broca wernicke circuit , in which auditory vocal mirror neurons play an initial part is suggested . in both cases , an early process or sculpting of visual and auditory representational symbols in 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 .

the mirror system hypothesis and investigations of birdsong are reviewed in relation to the significance for the development of human symbolic and language capacity , in terms of three fundamental forms of cognitive reference : iconic , indexical , and symbolic . mirror systems are initially iconic but can progress to indexical reference when produced without the need for concurrent stimuli . developmental stages in birdsong are also explored with reference to juvenile subsong vs complex stereotyped adult syllables , as an analogy with human language development . while birdsong remains at an indexical reference stage , human language benefits from the capacity for symbolic reference . during a pre - linguistic babbling stage , recognition of native phonemic categories is established , allowing further development of subsequent prefrontal and linguistic circuits for sequential language capacity .

Mirror System Hypothesis Birdsong Analogy Human Language Maturation PFC Development Discussion Summary Conflict of Interest Statement

while the circuitry for motor imitation is described as encompassing the posterior inferior gyrus ( including area f5 ) , and adjacent ventral premotor cortex , as well as a posterior area located in the rostral part of the inferior parietal lobule , mirror neurons are also thought to have importance in language development . iacoboni and dapretto ( 2006 ) also point out while trans - cranial magnetic stimulation ( tms ) has demonstrated bilaterality for the human mirror system ( mns ) , the question is raised how a relatively bilateral system for action observation and imitation contributes to a left - lateralized system for language . the authors point out that as mirror neurons also respond to the sound of an action , the motor system of listeners should be facilitated when listening to the sounds produced by actions , but such facilitation only occurred in the left hemisphere . in humans , the shift from a purely visual to a multimodal mns , could have determined functional changes that could have facilitated language and a left - lateralization of language functions arbib ( 2010 ) has proposed a mirror system hypothesis ( msh ) of language evolution . he suggests that the mechanisms which support language in the human evolved atop a basic mechanism not originally related to communication , namely the mirror system for grasping . similarly , a mirror system for grasping has been shown in the human brain . arbib hypothesized seven stages in the evolution of languages , with the first three stages s1 ( grasping ) , s2 ( grasping shared with the common ancestor of human and monkey ) , s3 ( a simple imitation system for grasping shared with the common ancestor ) thought to be pre - hominid , and the next three s4 ( a complex imitation system for grasping ) , s5 ( protosign , a manual - based communication system , breaking through the fixed repertoire of primate vocalizations to yield an open repertoire ) , s6 ( protospeech , the ability of control mechanisms evolved for protosign coming to control the vocal apparatus with increasing flexibility , thought to distinguish the hominid line from that of the great apes ) , while the final stage s7 is that of language ( arbib and rizzolatti , 1997 ) . according to arbib , msh is simply the assertion that the mechanisms which get us to the role of broca s area in language depend in a crucial way on the mechanisms established in stage 2 , namely mirror mechanisms . while the mirror system for grasping is described as evolving to support protosign and protospeech in humans ( arbib and rizzolatti , 1997 ; rizzolatti and arbib , 1998 ; iacoboni and dapretto , 2006 ; arbib , 2010 ) . broca s area must be linked into prefrontal cortical ( pfc ) planning ( and its administration by the basal ganglia ) to assemble verb - argument and more complex hierarchical structures , finding the words , and binding them correctly . " by implication , the mirror aspect of language suggests an early feed - forward pantomime stage , while the development of grammar implies a sequential recursive capacity based on pfc developments ( arbib and bota , 2010 ) . fitch ( 2005 ) has pointed out that the weakest link in the arbib mns model is the crucial link from protosign to protospeech , " specifically his elison between two distinct forms of imitation : vocal and manual . by this hypothesis , the crucial first step in human evolution from our last common ancestor with chimpanzees was the development of vocal imitation , similar in form and function to that independently evolved in many other vertebrate lineages ( including cetaceans , pinnipeds , and multiple avian lineages ) this hypothetical musical protolanguage preceded any truly linguistic system , capable of communicating particulate propositional meanings while dogs , birds , and apes can learn to map between meanings and words presented in isolation , the ability to extract words from arbitrary complex contexts and to recombine them in equally complex novel contexts is unattested in any non - human animal ( fitch , 2005 ) . leap by the age of three , without tutelage , feedback , or specific scaffolding , in contrast with skills such as alphabetic writing . in terms of deacon s ( 1997 ) classification above , mirror mechanisms should originally be classified as iconic , but according to arbib and bota ( 2010 ) , they provide a platform on which symbolic language representation may be built . where mirror systems reproduce a motor or vocal action in the absence of a concurrent stimulus , a further analogy with early language development is provided by the investigation of birdsong . however , there are large nuclear masses , which resemble the mammalian basal ganglia ( striatum ) , and a laminated isocortex ( gray matter ) , which is separated from the underlying basal ganglia by a band of myelinated axons ( white matter ) . like humans , birdsong and language both consist of ordered strings of sounds , separated by brief silent intervals . according to the authors , plastic - song is by 90 dph gradually transformed into highly complex , stereotyped , motifs , or sequences of syllables that constitute adult song . the premotor circuit for adult song production is believed to consist of the high vocal center ( hvc ) , robust nucleus of the archipallium ( ra ) , and brainstem motor nuclei . this motor pathway is crucial for generating stereotyped , learned vocalizations , and exhibits firing , that is precisely time - locked to the song output ( aronov et al . ( 2008 ) another circuit , the anterior forebrain pathway ( afp ) is homologous to the basal ganglia thalamo - cortical loops in mammals , and projects to ra through a forebrain nucleus , lateral magnocellular nucleus of the nidopallium ( lman ) . although lman is not required for singing in adult birds , it is necessary for normal song learning in juveniles , and plays a role in producing song variability in adult and juvenile birds aronov and colleagues eliminated the hvc bilaterally ( important in adult singing ) in nine subsong ( 3344 dph ) , and in three additional birds in which they left the hvc intact , but specifically eliminated its projection to ra . the investigators posited three possibilities in relation to subsong : it is entirely produced by the midbrain or brainstem ; it is driven by circuitry intrinsic to ra , even in the absence of hvc and lman ; and third it is driven by or requires inputs from lman or ra . the above authors concluded that lman and possibly other components of the afp constitute an essential premotor circuit for the production of early babbling . at the same time , the classical premotor nucleus hvc was not necessary for the generation of subsong . hvc does not reach its adult size until the late plastic - song stage ; and establishes synapses in ra later than lman does song maturation and the decrease in vocal variability have thus been attributed to the strengthening of inputs from hvc and the concurrent weakening of inputs from lman . " ( 2008 ) that distinct cortical / subcortical circuits for the production of infant behavior may be a general feature of developmental learning in the vertebrate brain , is important for the present review . the childhood to adolescent development of cortical / subcortical behavioral circuits involved in infant behaviors such as babbling , free play , and over - activity , with subsequent transition to goal - directed behavior is fundamental to the present concept of motor and language development . like song maturation , the mechanisms by which this development is established may involve a pre - linguistic babbling stage , with subsequent transition to goal - directed language ( doupe and kuhl , 1999 ) . in many regards , auditory - vocal hvcx cells are similar to visual - motor neurons in the monkey frontal cortex that are hypothesized to play a role in perception of human gestures , including human speech . vocal mirror neurons with properties similar to the hvcx cells described here could bind sensory and motor features of distinct vocal gestures , providing an efficient substrate for rapid decoding and encoding of speech ( 2009 ) point out that the division of continuously variable acoustic signals into discrete perceptual categories is a fundamental feature of vocal communication , including human speech . the authors point out that swamp sparrows learn their song notes by imitation , a feature of human speech , otherwise rare among animals . as described above , the investigators had shown that the nucleus hvc contained a certain class of striatum - projecting neurons , hvcx cells that respond to only one song type in the bird s repertoire , and song perception was shown to be impaired by lesions to the striatal portion of an afp into which hvcx cells project their axons . furthermore , the investigators were able to demonstrate geographically distinct populations of swamp sparrows obtained from northwestern pennsylvania vs. upstate new york , in which perceptual categorical note boundaries differed from category i to category vi . this suggested that these differences may have been influenced by learning , and thus vary across populations . according to the investigators , the study provided the first evidence for neurons encoding perceptual information about a phonological feature of learned vocal behavior , specifically information about a categorical perceptual boundary . this observation was linked by the authors to the role of neural experience in shaping human speech perception of categorical boundaries . this sculpting was thought to occur through a process of local inhibition , allowing context sensitivity over hundreds of milliseconds ( prather et al . while birdsong may also be built on mirror foundations , the capacity of hvc neurons to innervate striatal structures , important for encoding and decoding of learning and perception , as well as the geographically distinct phonological features of northwestern vs. upstate new york swamp sparrows suggests an indexical function for these songs . werker and tees ( 1999 ) point out newborn infants begin life with a remarkable sensitivity to the acoustic cues that signify different basic elements of speech . according to werker and tees infants become relatively more sensitive to the phonetic characteristics of the native language , and also to the syllabic context in which that phonetic variation occurs ( werker and lalonde , 1998 ; werker and tees , 1999 ) . the language - general perceptual sensitivities in newborns undergo a change and become more language - specific in the first year of life , thus preparing the infant for the ability to understand and speak his / her native language . during the first 1415 months , infants learn to extract words from the speech stream , and to recognize word forms they have previously heard , and to associate words with objects . coincident with the decline in non - native consonant ( and vowel ) discrimination seen by the end of the first year of life , the ability to co - ordinate two sources of information , such as phonetic detail , and position in a word is developed . the task for the next year of life is to construct a second - order system to effortlessly and efficiently use the medium of speech to map to meaning . with the establishment of a new level of representation , this discontinuity suggests a transformation from iconic mimicry to indexical representation , possibly similar to that which occurs in birdsong . the decline in non - native speech perception at the end of the first year of life , accompanied by the improvement in native speech perception has been found to be predictive of later language development ( kuhl et al . using a conditioned head - turn test of native and non - native speech sound discrimination and non - linguistic object retrieval tasks , sequencing attention , and inhibitory control , the investigators showed that native speech discrimination was positively linked to receptive vocabulary size , but not to cognitive control tasks , whereas non - native speech discrimination was negatively linked to cognitive control scores , but not to vocabulary size . the results suggested specific relationships between the development of native language , speech perception , and vocabulary ( conboy et al . ( 2008 ) point out that studies of the maturation of the human auditory cortex show that between the middle of the first year of life and 3 years of age , there is a maturation of axons entering the deeper cortical layers from the subcortical white matter ; and neurofilament - expressing axons appear for the first time in the temporal lobe , with projections to the deep cortical layers of the brain , providing the first highly processed auditory input from the brain stem . the concept of transition between developing brain and native language phonetic ability , as well as the associated concept of a developmental discontinuity to a second - order representational system indicates a possible basis for the understanding of the importance of language in development . while the significance of cortico - thalamic - striatal ( ctc ) circuits in visual motor development is well - described , a possibly analogous wernicke circuit , in which auditory vocal mirror neurons play a part is suggested . fuster states that by myelogenic and synaptogenic criteria , the pfc is clearly late - maturing , and that the human prefrontal areas do not attain full maturity until adolescence . fuster describes the lateral pfc as the neural substrate for the cognitive functions that support the temporal organization of behavior . a cardinal function of the lateral pfc is the temporal integration of information for the attainment of prospective behavioral goals [ ] there is evidence indicating that activation is maintained through recurrent circuits between pfc cells and posterior cortex [ ] . both work together in every sphere of action , including speech thus , fuster suggests an analogous recurrent process is important for both working memory and for speech , which is important in understanding the developmental importance of speech and language development in early childhood . according to tau and peterson ( 2010 ) , rudimentary working memory capacities have been observed in infants as young as 6 months of age , but performance on piaget s a not b task ( retrieval of a hidden object after a delay ) is not in place till 9 months , and is not solidly in place for difficult tasks till middle childhood . these include the ability to maintain its activity robustly until a goal is achieved , and second to have interconnections with all sensory systems , cortical and subcortical motor systems , and with limbic and midbrain structures involved in affect , memory , and reward . thus the lateral and mid - dorsal pfc receives visual , somatosensory , and auditory information from the occipital , temporal , and parietal cortices . the dorso - lateral area 46 is connected with premotor areas that send connections to primary motor areas and the spinal cord , as well as cerebellum and superior colliculus . the miller and cohen model requires feedback signals from the pfc to reach throughout the brain . 1996 ) were able to show that monkeys were able to maintain a working memory of a rewarded stimulus over time , and that target - specific activity appeared simultaneously in the pfc and parietal cortex . thus the pfc exhibits sustained activity that is robust to interference : multimodal convergence and integration of behaviorally relevant information ; feedback pathways that can exert biasing influences on other structures throughout the brain ; and ongoing plasticity that is adaptive to the demands of new tasks . the mechanisms responsible for updating representations in the pfc must be responsive to changes in the environment , as well as resistant to updating irrelevant changes . they suggested that dual concurrent influences on midbrain da allow the system to learn while it gates , and where a da - mediated gating signal leads to a successful behavior , its concurrent reinforcing effects will strengthen the association of the signal with cues representing the pattern of activity that produced the behavior . arnsten points out that although goldman - rakic ( 1994 ) used spatial working memory as a model system for examining functional circuitry , she proposed that these principles applied to other sensory and affective domains , and described the process as representational knowledge within parallel processing streams . according to arnsten , goldman - rakic spoke of pfc network activity as a fundamental contribution to mind , and the disruption of this process as a primary contribution to thought disorder in mental illness . she used the term working memory to describe a building block of cognition : the ability to represent information no longer in the environment through recurrent excitation of pyramidal cells with shared stimulus properties arnsten described the role of the pfc in working memory , as applying representational knowledge to inhibit inappropriate action , thought , and feelings , as well as inhibiting responses to distracting stimuli . an important distinction outlined by arnsten relates to the location of d1 and alpha-2a signaling mechanisms on dendritic spines . she points out that under optimal neurochemical conditions , moderate levels of ne engage alpha-2a receptors , and increase signals , whereas moderate levels of dad1 receptor stimulation decrease noise . the demonstration by the work of goldman - rakic and arnsten ( arnsten and goldman - rakic , 1985 ; goldman - rakic , 1994 ; arnsten , 2007 ) on the importance of symbolic representational capacity in the human prefrontal cortex for working memory has implications for language development . it is likely that human language is distinguished from primate indexical reference by its capacity to recursively encode , incorporate , and combine visual and auditory symbols in working memory , basic for human language functions . the classification of referential capacity in terms of hierarchical iconic , indexical , and symbolic levels allows an understanding of the hierarchical nature of mirror , birdsong , and human language capacity . birdsong studies have revealed that the hvc was not necessary for the generation of subsong or early babbling , whereas the generation of adult syllabic complex stereotyped motifs required an inhibitory output from the hvc to brainstem motor nuclei , but nonetheless remain stereotypic and indexical . from the birdsong analogy , stage represents a sculpting of categorical native phoneme recognition , while mature song requires stabilization of hvcx neurons may play a part in the development of capacity for symbolic representation in the dlpfc . the present review suggests that similar processes occur in human language development , where categorical native phoneme recognition is sculpted in a wernicke / broca circuit en route to sequential language capacity . studies of human language development reveal an analogous early babbling stage , during which there is a transition from generalized to native phonemic usage , with a subsequent childhood transition to second - order representational capacity . the capacity for association visual and auditory symbols in working memory providing a basis for human language development . adequate pfc functioning appears critical for not only mature reasoning , but also involves behavioral functions , including inhibition of task - irrelevant behaviors , processing of affect , motivation , and reward attainment by virtue of connections with wide - ranging cortical centers . a consequence of such deficits in pfc development is an incapacity for sequential reasoning , lack of affect regulation , a lack of capacity for working on sustained goal achievement , and a tendency for impulsive and repetitive behaviors , under either environmental , or subcortical control . it can thus be argued that the process of development is closely dependant on adequate pfc development , and many if not most behavioral syndromes of childhood reflect deficits in cortical development . importantly this includes language development , where auditory vocal mirror neurons may have an important role in the transition from babbling to goal - directed language ( levy et al . three fundamental forms of cognitive reference : iconic , indexical , and symbolic are described in relation to mirror systems , birdsong , and human language . the process of human development is closely dependant on adequate pfc development , ( and many if not most behavioral syndromes of childhood reflect deficits in cortical development ) . importantly this includes language development , where auditory vocal mirror neurons may have an important role in the transition from babbling to goal - directed language . while the significance of ctc circuits in visual motor development is well - described , a possibly analogous broca wernicke circuit , in which auditory vocal mirror neurons play an initial part is suggested .

in most protein - coding genes of eukaryotes the coding exons alternate with noncoding introns . the nuclear pre - mrna is a transcript of the whole gene , including the introns . however , before it is exported to the cytoplasm , the introns are removed through a process called pre - mrna splicing and the exons orderly joined to form the mature coding mrna . cutting at splicing sites is usually accomplished with a high degree of precision , as needed for the synthesis of the correct protein products in the process of translation . precision splicing requires the existence of specific sequence arrangements at appropriate pre - mrna sites ( signals ) and is affected by a massive ribonucleoprotein complex , the spliceosome , which has evolved to interact with these sequences . most often the splicing signal is univocal and robust enough to allow one single splicing pattern only at each site . but when the splicing signals are less robust or possibly not univocal , physiological alternative splicing patterns may occur , with total or partial deletion of some exons or retention of in - frame introns resulting in alterations of the encoded protein product . in most of these cases the generated protein either retains a similar function to that of the default protein or may acquire a different biological function [ 13 ] . in other instances unsuited mrnas are prevented from crossing the nuclear membrane , a selection structure which emerged in eukaryotes to separate intron - containing rnas from the translation apparatus . in addition , other cellular systems may degrade irregularly spliced or mutated mrnas ( nonsense - mediated decay , nmd ) [ 46 ] or , eventually , altered proteins may be ubiquitinated and proteasome - degraded . however , in some cases , despite all these control mechanisms , an irregular splicing or a disruption of the physiological alternative splicing may impair cell functions and bring about severe illnesses [ 810 ] . special strategies to allow some unspliced virus - derived mrnas ( required for the synthesis of some envelope and capsid proteins ) to be exported out of the nucleus are adopted by human immunodeficiency virus type 1 . the main intronic splicing signals are the 5 splice site ( 5ss ) or splice donor site ; the branch site ; the polypyrimidine tract ; and the 3 splice site ( 3ss ) or splice acceptor site . the 5ss marks the 5 end ( beginning ) of the intron and in almost 99% of cases begins with the canonical the polypyrimidine tract is a ~15-nucleotide sequence with a rich content of cs and ts . the 3ss marks the 3 end of the intron and in almost 99% of cases it ends with the canonical dinucleotide ag , which is preceded by a few varying nucleotides . the polypyrimidine tract is located immediately upstream of the 3ss and the branch site lies further upstream at a short distance . in 1% of the introns the canonical 5ss-3ss combination gt ag is replaced by noncanonical combinations , such as gc ag or at ac . besides the above - mentioned main splicing signals , it is believed that other sections of the intron may harbor additional splicing motifs which may contain consensus sequences for specific nuclear proteins . in addition , both introns ( i ) and exons ( e ) possibly harbor splicing enhancer ( ise and ese ) and splicing silencer ( iss and ess ) motifs . however , these splicing motifs are neither specific nor constantly present and the splicing code may be context - dependent and result from an integration of several different inputs [ 1223 ] . the spliceosome is a massive ribonucleoprotein complex comprising some small us nuclear rnas ( snrnas ) plus different specific proteins , these complexes being referred to as small nuclear ribonucleoproteins ( snrnps or snurps ) . in addition , more than a hundred other protein factors cooperate in splicing . two types of spliceosomal introns have been described : u2 snrnp - dependent introns ( the main group ) which use u1 , u2 , u4 , u5 and u6 snrnps ( with three subtypes , according to the terminal dinucleotides : gt ag , gc ag and at ac ) and u12 snrnp - dependent introns which use u11 , u12 , u4atac , u6atac and u5 , snrnps ( with two subtypes , according to the terminal dinucleotides : at ac and gt ag ) [ 1215 , 2427 ] . large - scale statistical analyses of the splice sites have been made in model species of vertebrates , invertebrates , fungi , protozoa , and plants [ 2530 ] . comprehensive databases have also been generated , for example , http://www.softberry.com/spldb/splicedb.html [ 2931 ] , and . cumulatively , the above - quoted papers report data on the global nucleotide patterns at the splice sites in each species , that is , the frequency of each base at a given position with reference to the intron boundaries ( including the flanking sections of the neighboring exons ) , the information content of the intronic sequences which are bound by the splicing factors ( as a measure of their conservation ) , and the evolution of the splicing factors in parallel with the evolution of the intronic splicing signals . we deemed that a comparative analysis of the intronic splice signals in orthologous genes ( i.e. , genes which derived from a common ancestor and diverged following speciation events ) at the individual orthologous splice sites could throw further light on the evolution of the splice sites during the process of speciation . in some topographically corresponding splice sites of orthologous genes of mouse and human , the signaling sequences are identical , whereas in others they are different . it is likely that , at least in the great majority of cases , the unaltered sequences are derived from the common ancestor and were conserved during the process of divergent speciation . thus , comparative analysis of orthologous splice sites could indicate which specific signaling sequences tend to be more conserved and thence the direction of the selective pressure in the time interval from the beginning of the speciation process to the present day . to this end we considered a group of cytokine receptor genes which are orthologous in mouse and human , two species which diverged quite recently , some 6585 mya ( million years ago ) [ 32 , 33 ] . we also examined the mouse and human transcript variants in this group of receptors with the aim of spotting those splicing signals which are more frequently silenced or replaced by other potential splicing signals at a different position in the gene . this study was made on a group of orthologous genes of mouse and human coding for cytokine receptors [ 34 , 35 ] . ( a database of homologous genes is available at http://www.ncbi.nlm.nih.gov/homologene/ ; a database of orthologous genes is available at http://inparanoid.sbc.su.se / cgi - bin / index.cgi/. ) a structural classification of cytokine receptors ( mainly according to coico and sunshine 2009 ) divides the receptors into the following groups : ( 1 ) immunoglobulin superfamily receptors ; ( 2 ) class i cytokine receptor family ; ( 3 ) class ii cytokine receptor family ( interferon receptor family ) ; ( 4 ) tumor necrosis factor receptor superfamily ; ( 5 ) chemokine receptor family ; ( 6 ) transforming growth factor beta receptor family . in this study we analyzed 26 receptors , belonging to all these groups , as indicated in table 1 . we selected the splice variants of these genes which showed superimposable exon - intron arrangements in mouse and human and exons aligning with nucleotide identities of 70% or higher . in addition , we compared these canonical transcripts with the mouse and human transcript variants of the same genes . nucleotide sequence alignments without gaps of each couple of orthologous introns were made using a program available at http://multalin.toulouse.inra.fr/multalin/ . all probabilistic comparisons between percentages were made using the binomial distribution ( one - tailed tests ) with the original actual figures . nucleotide identities were recorded in orderly manner in the first 50 nucleotides of each mouse / human couple of introns ( figure 1 ) . all the 216 couples of introns considered started with the canonical dinucleotide gt and identity was , of course , 100% at positions 1 and 2 . then identities averaged 84.3% , 90.3% , 93.5% , and 73.6% at positions 3 , 4 , 5 , and 6 , respectively . in positions 7 to 50 , the average identities were lower and remained relatively constant with a mean value of 55.4% ( the horizontal line in figure 1 ) . nucleotide identities were also recorded in the last 50 nucleotides ( numbered from 50 to 1 ) of each mouse / human couple of introns ( figure 2 ) . all couples of introns ended with the canonical dinucleotide ag and thus identity was 100% at positions 1 and 2 . then identities averaged 77.3% , 59.7% , 72.7% , and 72.2% at positions 3 , 4 , 5 , and 6 , respectively . in positions 7 to 21 the average identities remained relatively constant with a mean value of 63.1% ( the right horizontal line in figure 2 ) . in the last positions ( 22 to 50 ) the average identities remained relatively constant with a mean value of 56.0% ( the left horizontal line in figure 2 ) . from these preliminary data as well as from other literature data the initial and terminal hexanucleotides of the introns appeared to be the best characterized components of the 5ss and the 3ss , respectively . the possible number of hexanucleotides beginning with gt is 4 = 256 . of these , 51 ( cumulatively in mouse and human ) were found in our sample , which does not exclude the possibility that other hexanucleotides are used in other genes . the per cent incidences of these hexanucleotides in mouse and human do not differ significantly ( p > 0.05 ) . the nucleotide composition according to the position in the initial hexanucleotides in mouse and human is shown in table 3 , columns 14 . the per cent incidences of the different nucleotides in mouse and human at each position did not differ significantly ( p > 0.05 ) except in the case of the sixth nucleotide , where c was significantly more represented in mouse as compared to human . of the 256 possible hexanucleotides ending with ag , 94 ( cumulatively in mouse and human ) were found in our sample , which does not exclude the possibility that other hexanucleotides are used in other genes . the per cent incidences of these hexanucleotides in mouse and human did not differ significantly ( p > 0.05 ) . the nucleotide composition according to the position in the terminal hexanucleotides in mouse and human is shown in table 5 , columns 14 . the per cent incidences of the different nucleotides in mouse and human at each position did not differ significantly ( p > 0.05 ) except in the case of the first nucleotide , where g was significantly more represented in mouse as compared to human . it is noteworthy that in our sample the nucleotide g was never present in the fourth position so that no hexanucleotide ended by gag . the limits of the polypyrimidine tract are not well defined and the two leftmost nucleotides ( c- and t - rich positions 1 and 2 ; table 4 ) of the terminal hexanucleotide might , in fact , be the rightmost part of the polypyrimidine tract . all the 16 ( 4 ) hexanucleotides beginning by gt and ending by ag may in theory act as both 5ss and 3ss ( bifunctional hexanucleotides ) . of these , 8 never appeared as initial or terminal hexanucleotides in our sample and 3 ( gtaaag , gtacag , and gttaag ) appeared at the beginning ( but never at the end ) of some introns , while 5 ( gtccag , gtgcag , gtgtag , gttcag , and gtttag ) appeared at the end ( but never at the beginning ) of other introns , in both mouse and human . the structural conservation / mutation in the couples of mouse / human orthologous initial hexanucleotides was studied . in more than half of the couples ( 57.4% ) a decreasing percentage of couples exhibited one , two , or three nucleotide differences ( regardless of position ) ; in no instance all four variable nucleotides changed ( figure 3 ) . we calculated the probability of random occurrence of the same nucleotide at a given position in orthologous mouse / human hexanucleotides ( table 3 , column 5 ) . for instance , a in the third position is present with probability 0.597 in the mouse and 0.570 in the human ( table 3 , gtaxxx , columns 2 and 3 ) ; then the probability of random occurrence of a at that position in both mouse and human is 0.597 0.570 = 0.3403 ( or 34.03% ) ( binomial distribution ; n = 216 ) . the actual occurrence ( % ) of nucleotide identities at each position is reported in column 6 of table 3 . the percentages of actual identities were always significantly ( p < 0.01 ) higher than the percentages of random identities and the difference expresses the level of nucleotide biological conservation . in the nucleotides of the initial hexanucleotides the actual total conservation was about 53% higher than the calculated purely random conservation . the contribution of groups of bases in the initial hexanucleotides to enhancing or reducing the level of mouse / human conservation was then studied . the random probability of occurrence of complete hexanucleotides or groups of three or two nucleotides at given positions was calculated on the basis of the percentages of the actual conservation of the individual component nucleotides at the corresponding positions , as reported in table 3 ( column 6 ) . for example , the random probability of the hexanucleotide gtaagt is ( 1)1.00001.00000.50930.7778 0.82410.4537=0.1481 ( or 14.81% ) ( binomial distribution ; n = 216 ) and the random probability of the sequence gtaagx is ( 2)1.00001.00000.50930.77780.8241 = 0.3265 ( or 32.65% ) . analysis of the initial hexanucleotides showed that the sequences gtgagx , gtgaxt , and gtgxgt are significantly more expressed than could be expected ( table 6 ) . analysis of all the hexanucleotides which included these sequences demonstrated that in gtgagt ( which included all three overexpressed sequences ) the overexpression was highly significant ( expected 8.75% ; actual : 13.89% ; p < 0.01 ) ; overexpression of gtgagg ( which included the gag motif only ) was less significant , possibly due to the lower frequency ( expected 1.61% ; actual 3.24% ; p = 0.06 ) . none of the other hexanucleotides which included some of the overexpressed sequences exhibited significantly different expression levels from those expected , due to the fact that they contained other not overexpressed or underexpressed motifs . sequences gtaagx and gtaxgg were found to be significantly less expressed than could be expected ( table 6 ) . the hexanucleotide gtaagg , which included both underexpressed sequences , was clearly underexpressed , although at a low significance level due to the low frequency ( expected 2.72% ; actual 0.93% ; p = 0.07 ) . all other hexanucleotides which included some of the underexpressed sequences did not exhibit significantly different expression levels from those expected . another observation supports the role of the trinucleotides gag and aag in the association to overexpression and underexpression , respectively . as at the third nucleotide the actual conservation of a is 1.69 times the actual conservation of g ( 50.93/30.09 ; table 3 ) , gtaagt could be expected to be more expressed than gtgagt by about 70% . on the contrary , gtgagt was actually more expressed than gtaagt by about 2% ( 18.5/18.1 ; table 2 ) and indeed it was the most frequently expressed initial hexanucleotide . similarly , gtaagg should be more expressed than gtgagg by 70% , but actually gtgagg was more expressed than gtaagg by about 27% ( 5.6/4.4 ; table 2 ) . conservation / mutation in the couples of mouse / human orthologous terminal hexanucleotides was also studied . in 28.2% only of the couples was there a complete identity between the mouse and human orthologous hexanucleotides . in a higher percentage of cases ( 37.9% ) one nucleotide ( regardless of position ) was changed . a decreasing percentage of couples exhibited two or three nucleotide differences and in only 0.9% of cases were all four variable nucleotides changed ( figure 4 ) . the probability of random occurrence of the same nucleotide at a given position in orthologous mouse / human hexanucleotides was calculated as previously reported ( table 5 , column 5 ) . the actual occurrence ( % ) of nucleotide identities at each position the percentages of actual identities were always significantly ( p < 0.01 ) higher than the percentages of random identities . in the nucleotides of the terminal hexanucleotides the actual total conservation was about 80% higher than the calculated purely random conservation . the contribution of groups of bases in the terminal hexanucleotides in favoring the mouse / human conservation was studied by comparing their expected random probabilities with the observed actual frequencies ( see previous paragraph for the computing of random probabilities and table 5 , column 6 , for the actual conservation of the individual nucleotides at the different positions ) . analysis of the terminal hexanucleotides showed that the nucleotide sequences tttxag , ttxtag , xtttag , txttag , and xtgcag are significantly more expressed than could be expected ( table 7 ) . furthermore , some dinucleotides derived from the above sequences are themselves overexpressed : ttxxag ( derived from tttxag and ttxtag ) , xxttag ( derived from xtttag and txttag ) , and xtgxag ( derived from xtgcag ) ( table 7 ) . three hexanucleotides were found to be significantly overexpressed as compared to the random probability : tttcag ( expected 1.74% ; actual 4.63% ; p < 0.01 ) ; ttttag ( expected 0.72% ; actual 3.70% ; p < 0.01 ) ; and ctgcag ( expected 0.54% ; actual 1.85% ; p = 0.03 ) . other hexanucleotides , although containing some of the overexpressed trinucleotides , did not exhibit significantly different conservation levels from those of the random probability , and in no case did the occurrence of one of the conserved dinucleotides determine a higher conservation of the corresponding hexanucleotides . no motif associated with a significant underexpression splicing variants of the individual genes are usually of a different type in mouse and human so the positions of the splicing sites no longer correspond in the two species ( i.e. , the orthology of the splicing sites is lost ) , even though the main traits of the protein products are preserved . in only one instance , in receptor kit , mouse and human exhibited the same type of variant , that is , the use of another 5ss 12 nucleotides upstream of the canonical site . in several cases one entire exon present in the canonical form is lacking in a variant although the other downstream exons are regularly transcribed , indicating that the constitutive 3ss at the end of the preceding intron did not operate . as an example , in the transcript variant x1 ( xm_005252446 ) of the human il2ra the canonical 3ss ttccag immediately upstream of exon 4 is silenced and the fourth exon ( 216 nt ; 72 aa ) of the canonical sequence ( nm_000417 ) is lacking in this variant . in other cases up to three consecutive 3sss are silenced : in the human il2rg transcript variant x2 ( xm_005262262 ) 3sss atctag , ctctag , and ctccag are all silenced , with loss of exons 2 , 3 , and 4 , while exons 5 , 6 , 7 , and 8 are transcribed as in the canonical form ( nm_000206 ) without alteration of the reading frame . in other cases the active alternative ss may be located upstream or downstream of the canonical ss , resulting in an alteration of the length of individual exons , usually without frameshifts . for example , in the human variant x1 of csf2rb ( xm_005261340 ) the intron upstream of exon 7 ends by an early cctcag while the corresponding intron in the canonical sequence ( nm_007780 ) ends at another cctcag 18 nt downstream , resulting in an 18-nt longer exon in the variant . although variations of the 3sss are more frequent , the 5sss may also be variable . as an example , in human transcript variant x1 of cd40 ( xm_005260617 ) the 5ss after exon 7 is gtgggg , replacing the gtgagt of the canonical variant ( nm_001250 ) which occurs 12 nt upstream . we studied the ungapped base - pairing between the u1 snrna sequence acttac ( ncbi genbank accession code nr_004430 ) , conserved in mouse and human , and the last three nucleotides of the exons plus the corresponding 5ss hexanucleotide . the 5ss gtaagt , which exactly base - pairs with the u1 , is expressed in 19.0% of cases in mouse and 17.1% of cases in human ( table 2 ) . in all other cases a complementary match between the u1 nucleotides and the four variable nucleotides of the 5ss is achieved in 67% of nucleotides . the highest match is with the second variable nucleotide ( the fourth of the 5ss ) with 78% of occurrences , while the matching of all the other variable nucleotides averages 50% . indeed , all seven more expressed 5ss of mouse and human have an a in the fourth position ( table 2 ) . however , the number of matches in the individual 5sss is very variable , and in a few cases ( e.g. , gtttcg ) only the initial gt matches with u1 . in very few cases we observed that the optimal ungapped base - pairing of u1 could be achieved by partly binding the last three nucleotides of the exon . this study was aimed at describing the conservation / mutation dynamics of the intron ends of the cytokine receptor genes during the speciation processes to mouse and human which began , starting from a common ancestor , 6585 mya [ 32 , 33 ] . we selected 26 orthologous genes of the two species in which the mouse / human topographic correspondence of exons had been consistently preserved . the selected genes coded for receptors belonging to different cytokine receptor groups ( table 1 ) . we firstly identified the nucleotide identities at the corresponding positions in mouse and human in the first 50 and last 50 nucleotides of each intron . all introns studied ( 216 couples ) started and ended with the canonical dinucleotides gt and ag , respectively , conforming to the usual situation . this preliminary analysis demonstrated that , in general , the four nucleotides following gt at the 5ss and the four nucleotides preceding ag at the 3ss were more highly conserved , as compared to the other nucleotides at both ends of the introns ( figures 1 - 2 ) . all the 16 hexanucleotides beginning by gt and ending by ag might act both as 5ss and 3ss . in our sample 8 of these sequences appeared either at 5 or at 3 , but never in both . it may thus be hypothesized that some mechanism of disambiguation may operate possibly related to other exonic or intronic signals involved in the splicing processes . the structure of the hexanucleotides actually expressed at the beginning and end of the introns is quite variable even if some configurations are more frequent ( tables 2 and 4 ) . in our sample the initial hexanucleotide appeared in 51 different configurations while the terminal hexanucleotide appeared in 94 different configurations , indicating a lower selective pressure on the terminal hexanucleotide . this is confirmed by the mouse / human conservation / mutation data : the average mutation per hexanucleotide was 0.58 in the initial hexanucleotides and 1.17 in the terminal hexanucleotides , and the total hexanucleotide conservation was 57.4% for initial hexanucleotides and only 28.2% for terminal hexanucleotides . the percentages of occurrence of the more expressed initial and terminal hexanucleotides did not differ significantly in mouse and human ( tables 2 and 4 ) . the percentage of the individual nucleotides at each position in the initial and terminal hexanucleotides was also similar in mouse and human , and our results are in general comparable to those reported in the literature [ 25 , 27 , 28 ] . in our sample , in the sixth nucleotide of the initial hexanucleotides c was significantly more expressed in the mouse than in human , and in the first nucleotide of the terminal hexanucleotides g was significantly more expressed in the mouse than in human ( tables 3 and 5 ) . in our sample the nucleotide g was never present in the fourth position of terminal hexanucleotides . from the data on the actual frequencies of the individual nucleotides at each position in the initial and terminal hexanucleotides in mouse and human separately we calculated the probability of random conservation of any given nucleotide at a given position in both species . the results are shown in table 3 ( initial hexanucleotides ) and table 5 ( terminal hexanucleotides ) , together with the results obtained on the actual conservation of each nucleotide at a given position in both mouse and human . as shown in the tables the actual conservation was always significantly higher than the random conservation . on average , the actual conservation exceeded by 53% the random conservation in the initial hexanucleotides and by 80% in the terminal hexanucleotides . these gains express the conservation due to a selective evolutionary pressure to keep the configuration of the common ancestor at the level of the individual nucleotides . we also analyzed the conservation of groups of the variable nucleotides in initial and terminal hexanucleotides . the random conservation of groups of three or two nucleotides was calculated and compared with the actual frequencies . all the motifs of the initial hexanucleotides found to be associated with a higher or a lower conservation are reported in the results section . since in the third position a is more conserved than g by about 70% , gtgagt and gtgagg should be less expressed than gtaagt and gtaagg , respectively , by a similar percentage . actually gtgagt and gtgagg were more expressed than the similar sequences with a in the third position so that in our sample gtgagt was the most frequently expressed initial hexanucleotide and gtaagt was only the second most frequently occurring sequence ( table 2 ) . in the terminal hexanucleotides the sequences tttxag and xtgcag were overexpressed , and accordingly tttcag , ttttag , and ctgcag were more expressed than the random expectance . it should be remarked that the presence of overexpressed or underexpressed trinucleotides seems to be a condition which , although necessary , is not sufficient per se to entail over- or underexpression of the corresponding hexanucleotides since the outcome also depends on the other variable nucleotide . the present results suggest that the conservation of each nucleotide of the initial and terminal hexanucleotides is dependent upon the conservation / mutation of the other nucleotides , and an evolutionary selective pressure operates to keep certain global configurations derived from the mouse / human common ancestor while other configurations tend to be less conserved . in particular , the initial configuration gtaagt , which corresponded to the maximal conservation of the individual nucleotides at positions 3 to 6 ( table 3 , column 6 ) and is the one which exactly base - pairs with u1 snrna , appeared to be negatively biased . in general , the requirement for base - pairing of the initial hexanucleotide with u1 seems not to be stringent since in our sample complete matching was observed in 18% only of the 5sss and 5sss with as few as three matching nucleotides being effective ( 13% of cases ) . in very few cases , optimal base - pairing of u1 could be achieved by partly binding the terminal trinucleotide of the exon . analysis of the transcript variants of the genes considered in the present study revealed that no type of variant is common in mouse and human , except in one single case . this suggests that these variants were not inherited from the common ancestor but emerged only later during the speciation process . one frequent variant is the silencing of a constitutive 3ss which leads to the skipping of the immediately downstream exon , with transcription being then resumed for the other downstream exons . another type of variant is the activation of an alternative 5ss or 3ss , usually located a few nucleotides upstream or downstream of the canonical splicing site , the latter being silenced . this leads to alterations of the nucleotide number of the neighboring exons , but usually without frameshifts . most constitutive sss , especially at 5 , are robust enough to be maintained steadily active , but 10.4% of the 3sss and 3.2% of the 5sss were found to be variable . our analysis could not reveal any specific trend towards the silencing of constitutive sss or the activation of alternative sss , as certain hexanucleotides which are silenced at given sites are , on the contrary , activated at other sites to replace constitutive sss , as , for example , the 5sss gtgaga and gtggga and the 3ss ctccag . possibly , silencing or activation may depend in a complex way on the general context . to conclude , analysis of the nucleotide conservation in the 5ss and 3ss hexanucleotides of mouse and human introns reveals definite evolutionary positive biases towards the conservation of some specific configurations and negative biases against other configurations . however , the 5ss hexanucleotides and especially the 3ss hexanucleotides still exhibit a wide structural variability , confirming the contention that splicing depends on a complex code which , besides the 5ss and 3ss , possibly involves additional signals from the neighboring exons or from sections of the intron other than the splice sites and might also be regulated by the secondary and tertiary structures forming in the pre - mrna molecule [ 1214 , 1623 ] .

conservation / mutation in the intronic initial and terminal hexanucleotides was studied in 26 orthologous cytokine receptor genes of mouse and human . introns began and ended with the canonical dinucleotides gt and ag , respectively . identical configurations were found in 57% of the 5 hexanucleotides and 28% of the 3 hexanucleotides . the actual conservation percentages of the individual variable nucleotides at each position in the hexanucleotides were determined , and the theoretical rates of conservation of groups of three nucleotides were calculated under the hypothesis of a mutual evolutionary independence of the neighboring nucleotides ( random association ) . analysis of the actual conservation of groups of variable nucleotides showed that , at 5 , gtgagx was significantly more expressed and gtaagx was significantly less expressed , as compared to the random association . at 3 , tttxag and xtgcag were overexpressed as compared to a random association . study of mouse and human transcript variants involving the splice sites showed that most variants were not inherited from the common ancestor but emerged during the process of speciation . in some variants the silencing of a terminal hexanucleotide determined skipping of the downstream exon ; in other variants the constitutive splicing hexanucleotide was replaced by another potential , in - frame , splicing hexanucleotide , leading to alterations of exon lengths .

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

cutting at splicing sites is usually accomplished with a high degree of precision , as needed for the synthesis of the correct protein products in the process of translation . but when the splicing signals are less robust or possibly not univocal , physiological alternative splicing patterns may occur , with total or partial deletion of some exons or retention of in - frame introns resulting in alterations of the encoded protein product . however , in some cases , despite all these control mechanisms , an irregular splicing or a disruption of the physiological alternative splicing may impair cell functions and bring about severe illnesses [ 810 ] . the main intronic splicing signals are the 5 splice site ( 5ss ) or splice donor site ; the branch site ; the polypyrimidine tract ; and the 3 splice site ( 3ss ) or splice acceptor site . the 5ss marks the 5 end ( beginning ) of the intron and in almost 99% of cases begins with the canonical the polypyrimidine tract is a ~15-nucleotide sequence with a rich content of cs and ts . the 3ss marks the 3 end of the intron and in almost 99% of cases it ends with the canonical dinucleotide ag , which is preceded by a few varying nucleotides . in 1% of the introns the canonical 5ss-3ss combination gt ag is replaced by noncanonical combinations , such as gc ag or at ac . large - scale statistical analyses of the splice sites have been made in model species of vertebrates , invertebrates , fungi , protozoa , and plants [ 2530 ] . cumulatively , the above - quoted papers report data on the global nucleotide patterns at the splice sites in each species , that is , the frequency of each base at a given position with reference to the intron boundaries ( including the flanking sections of the neighboring exons ) , the information content of the intronic sequences which are bound by the splicing factors ( as a measure of their conservation ) , and the evolution of the splicing factors in parallel with the evolution of the intronic splicing signals . , genes which derived from a common ancestor and diverged following speciation events ) at the individual orthologous splice sites could throw further light on the evolution of the splice sites during the process of speciation . in some topographically corresponding splice sites of orthologous genes of mouse and human , the signaling sequences are identical , whereas in others they are different . it is likely that , at least in the great majority of cases , the unaltered sequences are derived from the common ancestor and were conserved during the process of divergent speciation . thus , comparative analysis of orthologous splice sites could indicate which specific signaling sequences tend to be more conserved and thence the direction of the selective pressure in the time interval from the beginning of the speciation process to the present day . to this end we considered a group of cytokine receptor genes which are orthologous in mouse and human , two species which diverged quite recently , some 6585 mya ( million years ago ) [ 32 , 33 ] . we also examined the mouse and human transcript variants in this group of receptors with the aim of spotting those splicing signals which are more frequently silenced or replaced by other potential splicing signals at a different position in the gene . this study was made on a group of orthologous genes of mouse and human coding for cytokine receptors [ 34 , 35 ] . in addition , we compared these canonical transcripts with the mouse and human transcript variants of the same genes . then identities averaged 84.3% , 90.3% , 93.5% , and 73.6% at positions 3 , 4 , 5 , and 6 , respectively . then identities averaged 77.3% , 59.7% , 72.7% , and 72.2% at positions 3 , 4 , 5 , and 6 , respectively . from these preliminary data as well as from other literature data the initial and terminal hexanucleotides of the introns appeared to be the best characterized components of the 5ss and the 3ss , respectively . of these , 51 ( cumulatively in mouse and human ) were found in our sample , which does not exclude the possibility that other hexanucleotides are used in other genes . the nucleotide composition according to the position in the initial hexanucleotides in mouse and human is shown in table 3 , columns 14 . the per cent incidences of the different nucleotides in mouse and human at each position did not differ significantly ( p > 0.05 ) except in the case of the sixth nucleotide , where c was significantly more represented in mouse as compared to human . of the 256 possible hexanucleotides ending with ag , 94 ( cumulatively in mouse and human ) were found in our sample , which does not exclude the possibility that other hexanucleotides are used in other genes . the nucleotide composition according to the position in the terminal hexanucleotides in mouse and human is shown in table 5 , columns 14 . the per cent incidences of the different nucleotides in mouse and human at each position did not differ significantly ( p > 0.05 ) except in the case of the first nucleotide , where g was significantly more represented in mouse as compared to human . the limits of the polypyrimidine tract are not well defined and the two leftmost nucleotides ( c- and t - rich positions 1 and 2 ; table 4 ) of the terminal hexanucleotide might , in fact , be the rightmost part of the polypyrimidine tract . of these , 8 never appeared as initial or terminal hexanucleotides in our sample and 3 ( gtaaag , gtacag , and gttaag ) appeared at the beginning ( but never at the end ) of some introns , while 5 ( gtccag , gtgcag , gtgtag , gttcag , and gtttag ) appeared at the end ( but never at the beginning ) of other introns , in both mouse and human . the structural conservation / mutation in the couples of mouse / human orthologous initial hexanucleotides was studied . in more than half of the couples ( 57.4% ) a decreasing percentage of couples exhibited one , two , or three nucleotide differences ( regardless of position ) ; in no instance all four variable nucleotides changed ( figure 3 ) . we calculated the probability of random occurrence of the same nucleotide at a given position in orthologous mouse / human hexanucleotides ( table 3 , column 5 ) . for instance , a in the third position is present with probability 0.597 in the mouse and 0.570 in the human ( table 3 , gtaxxx , columns 2 and 3 ) ; then the probability of random occurrence of a at that position in both mouse and human is 0.597 0.570 = 0.3403 ( or 34.03% ) ( binomial distribution ; n = 216 ) . the actual occurrence ( % ) of nucleotide identities at each position is reported in column 6 of table 3 . in the nucleotides of the initial hexanucleotides the actual total conservation was about 53% higher than the calculated purely random conservation . the contribution of groups of bases in the initial hexanucleotides to enhancing or reducing the level of mouse / human conservation was then studied . the random probability of occurrence of complete hexanucleotides or groups of three or two nucleotides at given positions was calculated on the basis of the percentages of the actual conservation of the individual component nucleotides at the corresponding positions , as reported in table 3 ( column 6 ) . analysis of the initial hexanucleotides showed that the sequences gtgagx , gtgaxt , and gtgxgt are significantly more expressed than could be expected ( table 6 ) . analysis of all the hexanucleotides which included these sequences demonstrated that in gtgagt ( which included all three overexpressed sequences ) the overexpression was highly significant ( expected 8.75% ; actual : 13.89% ; p < 0.01 ) ; overexpression of gtgagg ( which included the gag motif only ) was less significant , possibly due to the lower frequency ( expected 1.61% ; actual 3.24% ; p = 0.06 ) . sequences gtaagx and gtaxgg were found to be significantly less expressed than could be expected ( table 6 ) . another observation supports the role of the trinucleotides gag and aag in the association to overexpression and underexpression , respectively . as at the third nucleotide the actual conservation of a is 1.69 times the actual conservation of g ( 50.93/30.09 ; table 3 ) , gtaagt could be expected to be more expressed than gtgagt by about 70% . conservation / mutation in the couples of mouse / human orthologous terminal hexanucleotides was also studied . the probability of random occurrence of the same nucleotide at a given position in orthologous mouse / human hexanucleotides was calculated as previously reported ( table 5 , column 5 ) . the actual occurrence ( % ) of nucleotide identities at each position the percentages of actual identities were always significantly ( p < 0.01 ) higher than the percentages of random identities . in the nucleotides of the terminal hexanucleotides the actual total conservation was about 80% higher than the calculated purely random conservation . the contribution of groups of bases in the terminal hexanucleotides in favoring the mouse / human conservation was studied by comparing their expected random probabilities with the observed actual frequencies ( see previous paragraph for the computing of random probabilities and table 5 , column 6 , for the actual conservation of the individual nucleotides at the different positions ) . analysis of the terminal hexanucleotides showed that the nucleotide sequences tttxag , ttxtag , xtttag , txttag , and xtgcag are significantly more expressed than could be expected ( table 7 ) . three hexanucleotides were found to be significantly overexpressed as compared to the random probability : tttcag ( expected 1.74% ; actual 4.63% ; p < 0.01 ) ; ttttag ( expected 0.72% ; actual 3.70% ; p < 0.01 ) ; and ctgcag ( expected 0.54% ; actual 1.85% ; p = 0.03 ) . other hexanucleotides , although containing some of the overexpressed trinucleotides , did not exhibit significantly different conservation levels from those of the random probability , and in no case did the occurrence of one of the conserved dinucleotides determine a higher conservation of the corresponding hexanucleotides . no motif associated with a significant underexpression splicing variants of the individual genes are usually of a different type in mouse and human so the positions of the splicing sites no longer correspond in the two species ( i.e. in only one instance , in receptor kit , mouse and human exhibited the same type of variant , that is , the use of another 5ss 12 nucleotides upstream of the canonical site . in several cases one entire exon present in the canonical form is lacking in a variant although the other downstream exons are regularly transcribed , indicating that the constitutive 3ss at the end of the preceding intron did not operate . as an example , in the transcript variant x1 ( xm_005252446 ) of the human il2ra the canonical 3ss ttccag immediately upstream of exon 4 is silenced and the fourth exon ( 216 nt ; 72 aa ) of the canonical sequence ( nm_000417 ) is lacking in this variant . in other cases up to three consecutive 3sss are silenced : in the human il2rg transcript variant x2 ( xm_005262262 ) 3sss atctag , ctctag , and ctccag are all silenced , with loss of exons 2 , 3 , and 4 , while exons 5 , 6 , 7 , and 8 are transcribed as in the canonical form ( nm_000206 ) without alteration of the reading frame . for example , in the human variant x1 of csf2rb ( xm_005261340 ) the intron upstream of exon 7 ends by an early cctcag while the corresponding intron in the canonical sequence ( nm_007780 ) ends at another cctcag 18 nt downstream , resulting in an 18-nt longer exon in the variant . as an example , in human transcript variant x1 of cd40 ( xm_005260617 ) the 5ss after exon 7 is gtgggg , replacing the gtgagt of the canonical variant ( nm_001250 ) which occurs 12 nt upstream . we studied the ungapped base - pairing between the u1 snrna sequence acttac ( ncbi genbank accession code nr_004430 ) , conserved in mouse and human , and the last three nucleotides of the exons plus the corresponding 5ss hexanucleotide . the highest match is with the second variable nucleotide ( the fourth of the 5ss ) with 78% of occurrences , while the matching of all the other variable nucleotides averages 50% . indeed , all seven more expressed 5ss of mouse and human have an a in the fourth position ( table 2 ) . this study was aimed at describing the conservation / mutation dynamics of the intron ends of the cytokine receptor genes during the speciation processes to mouse and human which began , starting from a common ancestor , 6585 mya [ 32 , 33 ] . we selected 26 orthologous genes of the two species in which the mouse / human topographic correspondence of exons had been consistently preserved . we firstly identified the nucleotide identities at the corresponding positions in mouse and human in the first 50 and last 50 nucleotides of each intron . all introns studied ( 216 couples ) started and ended with the canonical dinucleotides gt and ag , respectively , conforming to the usual situation . this preliminary analysis demonstrated that , in general , the four nucleotides following gt at the 5ss and the four nucleotides preceding ag at the 3ss were more highly conserved , as compared to the other nucleotides at both ends of the introns ( figures 1 - 2 ) . in our sample 8 of these sequences appeared either at 5 or at 3 , but never in both . this is confirmed by the mouse / human conservation / mutation data : the average mutation per hexanucleotide was 0.58 in the initial hexanucleotides and 1.17 in the terminal hexanucleotides , and the total hexanucleotide conservation was 57.4% for initial hexanucleotides and only 28.2% for terminal hexanucleotides . the percentages of occurrence of the more expressed initial and terminal hexanucleotides did not differ significantly in mouse and human ( tables 2 and 4 ) . the percentage of the individual nucleotides at each position in the initial and terminal hexanucleotides was also similar in mouse and human , and our results are in general comparable to those reported in the literature [ 25 , 27 , 28 ] . in our sample , in the sixth nucleotide of the initial hexanucleotides c was significantly more expressed in the mouse than in human , and in the first nucleotide of the terminal hexanucleotides g was significantly more expressed in the mouse than in human ( tables 3 and 5 ) . from the data on the actual frequencies of the individual nucleotides at each position in the initial and terminal hexanucleotides in mouse and human separately we calculated the probability of random conservation of any given nucleotide at a given position in both species . the results are shown in table 3 ( initial hexanucleotides ) and table 5 ( terminal hexanucleotides ) , together with the results obtained on the actual conservation of each nucleotide at a given position in both mouse and human . as shown in the tables the actual conservation was always significantly higher than the random conservation . on average , the actual conservation exceeded by 53% the random conservation in the initial hexanucleotides and by 80% in the terminal hexanucleotides . these gains express the conservation due to a selective evolutionary pressure to keep the configuration of the common ancestor at the level of the individual nucleotides . we also analyzed the conservation of groups of the variable nucleotides in initial and terminal hexanucleotides . the random conservation of groups of three or two nucleotides was calculated and compared with the actual frequencies . since in the third position a is more conserved than g by about 70% , gtgagt and gtgagg should be less expressed than gtaagt and gtaagg , respectively , by a similar percentage . in the terminal hexanucleotides the sequences tttxag and xtgcag were overexpressed , and accordingly tttcag , ttttag , and ctgcag were more expressed than the random expectance . the present results suggest that the conservation of each nucleotide of the initial and terminal hexanucleotides is dependent upon the conservation / mutation of the other nucleotides , and an evolutionary selective pressure operates to keep certain global configurations derived from the mouse / human common ancestor while other configurations tend to be less conserved . in particular , the initial configuration gtaagt , which corresponded to the maximal conservation of the individual nucleotides at positions 3 to 6 ( table 3 , column 6 ) and is the one which exactly base - pairs with u1 snrna , appeared to be negatively biased . analysis of the transcript variants of the genes considered in the present study revealed that no type of variant is common in mouse and human , except in one single case . this suggests that these variants were not inherited from the common ancestor but emerged only later during the speciation process . one frequent variant is the silencing of a constitutive 3ss which leads to the skipping of the immediately downstream exon , with transcription being then resumed for the other downstream exons . this leads to alterations of the nucleotide number of the neighboring exons , but usually without frameshifts . most constitutive sss , especially at 5 , are robust enough to be maintained steadily active , but 10.4% of the 3sss and 3.2% of the 5sss were found to be variable . our analysis could not reveal any specific trend towards the silencing of constitutive sss or the activation of alternative sss , as certain hexanucleotides which are silenced at given sites are , on the contrary , activated at other sites to replace constitutive sss , as , for example , the 5sss gtgaga and gtggga and the 3ss ctccag . to conclude , analysis of the nucleotide conservation in the 5ss and 3ss hexanucleotides of mouse and human introns reveals definite evolutionary positive biases towards the conservation of some specific configurations and negative biases against other configurations . however , the 5ss hexanucleotides and especially the 3ss hexanucleotides still exhibit a wide structural variability , confirming the contention that splicing depends on a complex code which , besides the 5ss and 3ss , possibly involves additional signals from the neighboring exons or from sections of the intron other than the splice sites and might also be regulated by the secondary and tertiary structures forming in the pre - mrna molecule [ 1214 , 1623 ] .

medical doctors move across borders being motivated by higher salaries , better working conditions , new professional experience , and training and career opportunities . in europe , migration of medical doctors has been observed since the 1940s and has shown various dynamics over the years . european integration has offered new possibilities for medical doctors to improve their skills , to study or to work in other countries . however , outflows from eastern europe started before accession , due to the political transitions of the late 1980s and the last 1990s . the eu enlargement , first in 2004 and then in 2007 , has generated increased mobility , especially from east to west , namely from eu-12 to eu-15 . studies have shown that migration of physicians from new member states has been lower than the leaving intentions . given the increasing trends in health professionals mobility , the european union has established a legal framework to regulate both the recognition of professional qualification and the free mobility of doctors and patients within europe . however , a legal framework for the recognition of professional qualification for physicians willing to work outside europe and for those coming from non - european countries is still lacking . migration of health professionals in generally and of medical doctors in particular has its roots in current problems of healthcare systems . medical doctors decide to move from one country to another not only because of higher incomes , but in search of better working environments , career opportunities and social recognition . thus , mobility of medical doctors is seen as a symptom of more fundamental health systems problems . these need to be addressed by policy makers in an integrated manner because health professional mobility can not be considered in isolation . while some european countries have to deal with major shortages of medical doctors , other are confronted with increasing pressures to manage maldistribution , both geographically and in terms of specialities needed . usually the positive effects occur when mobility is temporary and with the purpose of achieving new experiences , new specialities and training , followed by return in home country . in addition , mobility of doctors impacts positively the patients access to medical treatment and medical service , because patients can benefit of the knowledge and training achieved by medical doctors in other countries . if mobility is for long - term and outflows occur in countries struggling with shortages of medical doctors , then the negative impacts on the healthcare systems are felt both at macro level - financial loss for the country that has paid for the education of the physicians , national health system has to be adapted to the new situation , and at micro level lack of sufficient medical doctors or maldistribution will impact patients safety and access to care , and finally patients health . among the most - cited factors for physicians mobility is the financial motivation . as regards the salaries , major differences between european countries can be observed . especially since the eu became more diverse in socio - economic terms , with larger salary differentials , incentives to seek employment in another member state have increased . for example , an estonian medical doctor can earn six times more in finland while a romanian general practitioner can earn ten times more in france . in 2009 , a 25% cut in the salaries of health professionals in romania has led to an increase in the number of doctors seeking work abroad . a reverse effect has been observed in poland , lithuania and slovenia , where annual salary increases have helped to diminish the outflow of medical doctors . high salary differentials exist not only between eu-12 and eu-15 , but also between eu-15 and non - eu countries , such as us , new zeeland and australia . therefore , many doctors from western european countries ( eu-15 ) may be motivated to seek work overseas . the economic situation of a country has a major impact on the quality and standards of healthcare facilities and on the social benefits offered for health professionals . while some european countries , mostly from eu-15 , have developed high standards for healthcare such as better equipped hospitals , introduction of high technologies for diagnosis and surgery , and new tools for testing , other countries struggle with major shortages as regards the facilities named above . this is usually the case of new member states , which had to pass through a difficult transition period and are affected by economic problems that do not leave room for further development of the existent healthcare facilities . therefore , when medical doctors are forced to deal with a lower number of hospital beds than the number of patients , with old diagnostic tools , with shortage of medicines and instruments available in hospital pharmacies , or with inappropriate conditions for carrying out complex surgeries , they often decide to work in an environment that can offer them better conditions for doing their job . however , better working conditions do not only refer to access to better healthcare facilities but also to social and economic incentives , working schedule and promotion opportunities . training and career opportunities are also among the relevant decision - making factors for physicians who consider leaving their country of origin , either temporary or for a long period of time . again , the available training opportunities vary considerably across europe and there are also differences between europe and countries like us or japan . therefore , doctors migrate because they may wish to specialise in a certain medical filed which is not available in their country or , if available , it is not yet sufficiently developed . but doctors may seek training and career opportunities in other countries not only because such opportunities lack in their home country , but because the experience and the know - how achieved abroad is often very enriching and acts as a boost for their career . in addition , new professional and personal experiences achieved across the borders may widen their horizon in the medical field . finally , some medical doctors also wish to migrate because they simply want to make a change in their lifestyle . for instance , a physician from denmark or sweden could seek working in spain or in italy , because the southern european countries are well known for their warm climate , relaxed daily life , rich culture and tasty foods . similarly , a physician from bulgaria could wish to work in austria not necessarily for a higher salary but for the well organised system and infrastructure , for the austrian high living standards or for the great mountains . according to a who report on healthcare workforce migration in europe , there are also other factors associated with migration flows that can stimulate migration and affect the choice of a destination country : organizational factors , such as heavy workload , occupational risks , poor management , favouritism or lack of due process , lack of recognition;healthcare system factors , such as the absence or inadequacy of human resource policies , insufficient funding of health services , and centralised decision - making;general environmental factors , such as poor economic conditions and lack of security . organizational factors , such as heavy workload , occupational risks , poor management , favouritism or lack of due process , lack of recognition ; healthcare system factors , such as the absence or inadequacy of human resource policies , insufficient funding of health services , and centralised decision - making ; general environmental factors , such as poor economic conditions and lack of security . the mobility of doctors within the european region was to a large extent influenced by the eu enlargement in 2004 and 2007 . the eu enlargement triggered east west asymmetries in terms of inflows and outflows of health professionals , with migrants from new member states moving to countries from eu-15 . however , many of the eu-15 countries have outflows of the same magnitude as the eu-12 , but unlike the eu-15 , the eu-12 countries have only negligible inflows . in europe , among the major destination countries are germany , france , italy , uk and spain . they receive doctors from countries like poland , greece , romania , switzerland and the czech republic . germany is at the same time a big source and destination country , german doctors choosing to migrate mainly to the united kingdom and italy , followed by switzerland and us . outflows have also been observed in the uk , mainly toward neighbouring countries like ireland and france , but also to spain and the us . according to a who study on health personnel migration , the flow of migrant doctors is very dynamic in the european region . this situation is illustrated in the fig . 1 , which shows the inflows and outflow of physicians from selected european countries .fig . 1migration of physicians in the who european region ( red arrows indicate two - way flows ) , taken from migration of physicians in the who european region ( red arrows indicate two - way flows ) , taken from in fig . 1 one can observe not only a flow from east to west , but also a dynamic exchange between western countries . thus , the flow of migrant doctors among belgium , france and the netherlands is also multidirectional . although there have been several recent studies examining the migration of healthcare workforce in europe , data are still limited . policy makers , workforce planners and healthcare managers need to better understand the mobility of trends in order to react with the right measures . due to the european integration and eu enlargement , which brought more flexibility for travelling and working in europe , it became obvious that an eu framework for professional mobility is needed . in response to this situation , the european parliament and the european council adopted the directive 2005/36/ec on recognition of professional qualifications , which affects more than 800 different professions regulated by member states across the eu , including medical doctors , nurses , midwives , and dentists . the directive sets the european legal framework for the recognition of professional qualifications obtained in a member state other than the one where the person wishes to work . with directive 2005/36/ec the eu has reformed the system for recognition of professional qualifications , in order to help make labour markets more flexible , further liberalise the provision of services , encourage more automatic recognition of qualifications , and simplify administrative procedures . the most recent consolidated version of the directive was made available on 24 march 2011 . until 20 october 2007 when the transposition period ended , this directive has replaced 15 existing directives in the field of recognition of professional qualifications , providing the first comprehensive modernisation of the eu system since its introduction over 40 years ago . according to the european commission , only 22 out of 27 member states fully transposed the directive within the given timeframe . considering the complexity of directive and the degree it impacts national legislation , some member states had to adapt a significant number of measures in order to complete the transposition . directive 2005/36/ec facilitates temporary provision of services ( tps ) by replacing the previous system of prior check of qualifications by the simpler , optional , system of prior declaration . however , due to the need to protect consumers , a prior check of qualifications may be maintained for professions having public health or safety implications ( article 7(4 ) of the directive ) . according to the european commission , 26 member states have implemented article 7(4 ) of the directive by april 2010 , apart from greece where the situation was still unclear . , professionals can in principle work on the basis of a declaration made in advance . the directive also applies to professionals wishing to establish themselves in an eu country other than that in which they obtained their professional qualifications , either as an employed or self - employed person , either on a permanent basis . directive 2005/36/ec sets out three systems for the recognition of qualifications : automatic recognition for professions for which the minimum training conditions have been harmonised ( health professionals , architects , veterinary surgeons).basic medical training and general practitioner qualifications ( annex v.5.1.1 and v.5.1.4)specialist doctors qualifications are automatically recognised in certain eu countries ( annex v.5.1)the general system for other regulated professions ( including non - automatic recognition - art . . automatic recognition for professions for which the minimum training conditions have been harmonised ( health professionals , architects , veterinary surgeons).basic medical training and general practitioner qualifications ( annex v.5.1.1 and v.5.1.4)specialist doctors qualifications are automatically recognised in certain eu countries ( annex v.5.1 ) basic medical training and general practitioner qualifications ( annex v.5.1.1 and v.5.1.4 ) specialist doctors qualifications are automatically recognised in certain eu countries ( annex v.5.1 ) the general system for other regulated professions ( including non - automatic recognition - art . 10 to 15 ) . recognition on the basis of professional experience for certain professional activities . title ii of directive 2005/36/ec governs the recognition of professional qualifications in the context of a temporary move to the territory of another eu country . the temporary and occasional nature of the activities of a self - employed or employed person is assessed on a case - by - case basis , in light of the duration of the activity , its frequency , regularity and continuity . but the current system must be evaluated in order to verify whether full use has been made of all the opportunities offered by directive 2005/36/ec . the system must also take account of the considerable changes that have occurred in the member states educational and training systems . for this reason , the commission has begun working on evaluating the 2005 directive which culminated in a green paper modernising the professional qualifications directive in june 2011 . the public consultation on the green paper was launched by the commission in january 2011 and a summary report with responses is already available . specifically the mobility of health professionals , the european commission issued a green paper on european workforce for health in december 2008 and invited all interested organisation to a public consultation , which closed on 31 march 2009 . in addition to directive 2005/36/ec addressing cross - border workforce mobility across europe , on 9 march 2011 the european parliament and council adopted directive 2011/24/eu on the application of patients rights in cross - border healthcare . the directive addresses in particular the mobility of patients seeking treatment in another member state and introduces rules regarding the reimbursement of treatments and medical care or investigations received abroad . as regards the impact of directive 2011/24/eu on the mobility of health professionals , the directive stipulates that member states should facilitate cooperation between healthcare providers , purchasers and regulators of different member states at national , regional or local level in order to ensure safe , high - quality and efficient cross - border healthcare . this could be of particular importance in border regions , where cross - border provision of services may be the most efficient way of organising health services for the local population , but where achieving such cross - border provision on a sustained basis requires cooperation between the health systems of different member states . according to directive 2011/24/eu , cooperation between member states may concern practical mechanisms to ensure continuity of care or practical facilitating of cross - border provision of healthcare by health professionals on a temporary or occasional basis . however , in directive 2011/24/eu it is mentioned that this directive should be without prejudice to directive 2005/36/ec on the recognition of professional qualifications . this means that free provision of services of a temporary or occasional nature , including services provided by health professionals in another member state is not subject to specific provisions of union law and is to be restricted for any reason relating to professional qualifications . although the mobility of doctors within the european union is to an extent regulated by eu law , it is also necessary to take into consideration the national legislation from european member states . as mentioned in previous paragraphs , the existing legal framework addresses only the mobility of health professionals ( and other ) when they cross borders within europe . but thus , if a german physician would like to work in china he / she may encounter difficulties in acquiring the recognition of his / her qualifications allowing working in this country . or , if a physician from georgia is seeking work in france , again , there is to date no european legal act which can help in such cases . therefore , the european union should consider introducing further regulations or developing legal instruments aiming at solving this challenge healthcare workforce migration beyond europe s borders . besides the legislative framework aiming at regulating the mobility of doctors in europe , the world health organisation developed a non - regulatory instrument on the international recruitment of health personnel - who global code of practice on the international recruitment of health personnel . the who code of practice was adopted at the sixty - third world health assembly in geneva on 21 may 2010 and its key components are : in destination countries : ethical recruitment practices;protection of the rights of foreign healthcare workers;increased education and training for health sector students;pairing of needs and supply.in source countries : improved conditions for healthcare professionals;continued medical training and increased opportunities;incentives to retain physicians and nurses in countries and regions with human resource shortages . in destination countries : ethical recruitment practices;protection of the rights of foreign healthcare workers;increased education and training for health sector students;pairing of needs and supply . ethical recruitment practices ; protection of the rights of foreign healthcare workers ; increased education and training for health sector students ; pairing of needs and supply . in source countries : improved conditions for healthcare professionals;continued medical training and increased opportunities;incentives to retain physicians and nurses in countries and regions with human resource shortages . improved conditions for healthcare professionals ; continued medical training and increased opportunities ; incentives to retain physicians and nurses in countries and regions with human resource shortages . this initiative of who comes also in response to the consequences of the financial crisis on labour markets and addresses the need to mitigate the negative effects of migration on health systems in developing countries and to ensure equitable access to health care services while minimizing the need to rely on the immigration of health personnel from other countries . the resolution ( eur / rc59/r4 ) adopted by the regional committee urges member states to increase their efforts to develop and implement sustainable health workforce policies , strategies and plans as a critical component of health systems strengthening and to advocate the adoption of a global code of practice on the international recruitment of health personnel in line with the european values of solidarity , equity and participation , both within the who european region and globally . in order to assist the medical doctors ( and other professionals ) who intend to work across borders , national contact points have been established at the initiative of the european commission s service free movement of professionals , directorate general internal market . there are contact points in every eu country that can give information on the recognition of professional qualifications according to the national law and procedures to be followed . contact points may be ministries of education , research or science or other national institutions . the contact points also serve as a guide for the applicants , helping them to complete the required administrative formalities . but contact points can only assist the applicants with their requests and are not in charge for deciding whether the recognition of a certain profession should be granted . the decision - makers are the national competent authorities , who decide whether or not to recognise professional qualifications obtained in other eu countries , in accordance with european and national legislation . the competent authorities in member states are expected to use a set of common rules laid down by a code of conduct . as regards the procedure to be followed for the recognition of professional qualifications , the applicant must apply to the authority that oversees the doctor profession in that country and provide the authority with the proof of qualifications . the competent authority must acknowledge the application within 1 month of receiving it , and ask for missing but necessary documents to process the application . then the authority assesses the qualifications and decides whether to grant the application within 3 months . for complicated cases in the area of non - automatic recognition this procedure may last 4 months . if the applicant does not accept the decision of the competent authority , he / she can appeal to the relevant court in that country . with the aim of ensuring better coordination both among member states and between member states and ec , the tasks of this group include helping national authorities and the commission work together better , monitoring policies with a bearing on qualifications for regulated professions , and exchanging experiences and good practices in the recognition of qualifications . group s members and alternate members are appointed by national governments and they meet several times a year . experts and observers are invited to take part in the group s meetings , which are chaired by the european commission . further support for workforce mobility within europe was made available by the european commission directorate general on employment , social affairs and equal opportunities , who has established eures the european job mobility portal . the purpose of eures is to provide information , advice and recruitment / placement ( job - matching ) services for the benefit of workers and employers as well as any citizen wishing to benefit from the principle of the free movement of persons . there are currently over 20 eures cross - border partnerships , spread geographically throughout europe and involving more than 13 countries . eures plays an important especially in cross - border regions , areas in which there are significant levels of cross - border mobility . more than 600 000 people live in one eu country and work in another and they have to cope with different national practices and legal systems . they may come across administrative , legal or fiscal obstacles to mobility on a daily basis . eures advisers in these areas provide specific advice and guidance on the rights and obligations of workers living in one country and working in another . the impacts on the performance of health systems are subtle , meaning that they are indirect or hard to discern . moreover , some areas from eastern european countries ( e.g. romanian rural areas ) may be particularly vulnerable , showing some of the highest emigration rates among medical doctors and nurses . for the healthcare systems in source countries emigration can contribute to a major shortage of health professionals . this can be observed in various forms , such as loss of training capacity ( when trainers leave ) , heavier workloads for doctors who decide to stay or disruption of services when a key staff member leaves . in addition , the source country also loses the investment in the education of health professionals as well as the contribution they would have otherwise made for the healthcare system . some source countries may have also some benefits from the emigration of doctors , like reduction in staff surpluses and access to new knowledge and skills , in case the emigration is temporary . in some cases , source countries can benefit from collaborative training programmes , research projects or teaching activities which are initiated by emigrant doctors with their home country . unlike in source countries , in destination countries the benefits tend to be more obvious . for instance , migrant physicians may accept lower salaries than native ones , and they may also accept working in geographic areas avoided by national workers . however , destination countries may also encounter some difficulties related to inflow of medical doctors . cultural differences may hinder communication and lack of familiarity with advanced equipment may lead to higher error rates . for temporary migrants , investment in workplace induction can be relatively high compared to the time of service provided by the migrant physicians . as regards the policy implications of doctor s migration , first , the uncertainties surrounding the impact of the economic crises which forced some countries to drastically reduce their healthcare budgets , while in other countries budgets remained unaffected . a second factor impacting healthcare policies is the uncertainty of the development of healthcare workforce in europe . according to a european commission forecast , a shortage of around 1 million health professionals compensation of health workforce shortages by recruiting from third countries is to an extent restricted by ethical concerns , as stipulated in the who global code of practice for the international recruitment of health personnel adopted in 2010 . for these reasons , the european countries dealing with a high demand for medical doctors will face increasing difficulties in filling their vacancies with doctors from abroad . according to the european observatory on health systems and policies , there are three main sets of policy implications linked with the mobility of medical doctors : the first refers to the amount of data , intelligence and evidence , which are currently not sufficiently developed . therefore , in the absence of inflow and outflow data , policy makers can not take appropriate measures to manage doctors migration.the second set of policy implications refers to the strengthening the general workforce strategies . symptom of underlying domestic workforce aspects , such as working conditions , salaries , and training opportunities.the third policy implication is related to sustaining the re - emerging interest in workforce planning methods and techniques , by taking into account especially the dynamics and the need of healthcare force in future . the first refers to the amount of data , intelligence and evidence , which are currently not sufficiently developed . therefore , in the absence of inflow and outflow data , policy makers can not take appropriate measures to manage doctors migration . symptom of underlying domestic workforce aspects , such as working conditions , salaries , and training opportunities . the third policy implication is related to sustaining the re - emerging interest in workforce planning methods and techniques , by taking into account especially the dynamics and the need of healthcare force in future . in responding to the inflow and outflow of medical doctors , today and in future , countries may take a series of measures aiming at an appropriate management of their healthcare workforce . for instance , they may sign bi - lateral agreements or facilitate the recognition of diplomas from non - european countries or they may consider developing twinning schemes and joint training programmes . although these policy responses may facilitate a better management of doctors mobility , the european countries should seek to solve mainly the domestic problems liked to doctors migration . thus , they should seek to strengthen existing healthcare strategies by improving retention , increasing salaries and providing more advanced training opportunities . in addition , countries could develop new healthcare strategies which can better respond to the current and future mobility trends .

this chapter aims at providing an insight into some major aspects linked to migration of medical doctors within europe . the article describes main factors which contribute to doctors migration . further , the current and future mobility trends in europe are discussed . a major part of this chapter is dedicated to an overview of the eu legal framework impacting healthcare professionals mobility , followed by some useful information related to the procedures for recognition of professional qualifications and offices in charge of mobility . finally , the impacts on healthcare systems and the policy implications of doctor s mobility are described in context of personalised medicine .

Introduction Driving forces for physicians mobility in Europe Current trends in inflows and outflows Legal framework for cross-border professional mobility within Europe Offices in charge of professional mobility and recognition of professional qualifications Impact on healthcare systems and policy implications

medical doctors move across borders being motivated by higher salaries , better working conditions , new professional experience , and training and career opportunities . in europe , migration of medical doctors has been observed since the 1940s and has shown various dynamics over the years . european integration has offered new possibilities for medical doctors to improve their skills , to study or to work in other countries . however , outflows from eastern europe started before accession , due to the political transitions of the late 1980s and the last 1990s . the eu enlargement , first in 2004 and then in 2007 , has generated increased mobility , especially from east to west , namely from eu-12 to eu-15 . studies have shown that migration of physicians from new member states has been lower than the leaving intentions . given the increasing trends in health professionals mobility , the european union has established a legal framework to regulate both the recognition of professional qualification and the free mobility of doctors and patients within europe . however , a legal framework for the recognition of professional qualification for physicians willing to work outside europe and for those coming from non - european countries is still lacking . migration of health professionals in generally and of medical doctors in particular has its roots in current problems of healthcare systems . medical doctors decide to move from one country to another not only because of higher incomes , but in search of better working environments , career opportunities and social recognition . thus , mobility of medical doctors is seen as a symptom of more fundamental health systems problems . while some european countries have to deal with major shortages of medical doctors , other are confronted with increasing pressures to manage maldistribution , both geographically and in terms of specialities needed . usually the positive effects occur when mobility is temporary and with the purpose of achieving new experiences , new specialities and training , followed by return in home country . in addition , mobility of doctors impacts positively the patients access to medical treatment and medical service , because patients can benefit of the knowledge and training achieved by medical doctors in other countries . if mobility is for long - term and outflows occur in countries struggling with shortages of medical doctors , then the negative impacts on the healthcare systems are felt both at macro level - financial loss for the country that has paid for the education of the physicians , national health system has to be adapted to the new situation , and at micro level lack of sufficient medical doctors or maldistribution will impact patients safety and access to care , and finally patients health . especially since the eu became more diverse in socio - economic terms , with larger salary differentials , incentives to seek employment in another member state have increased . in 2009 , a 25% cut in the salaries of health professionals in romania has led to an increase in the number of doctors seeking work abroad . a reverse effect has been observed in poland , lithuania and slovenia , where annual salary increases have helped to diminish the outflow of medical doctors . therefore , many doctors from western european countries ( eu-15 ) may be motivated to seek work overseas . the economic situation of a country has a major impact on the quality and standards of healthcare facilities and on the social benefits offered for health professionals . this is usually the case of new member states , which had to pass through a difficult transition period and are affected by economic problems that do not leave room for further development of the existent healthcare facilities . therefore , when medical doctors are forced to deal with a lower number of hospital beds than the number of patients , with old diagnostic tools , with shortage of medicines and instruments available in hospital pharmacies , or with inappropriate conditions for carrying out complex surgeries , they often decide to work in an environment that can offer them better conditions for doing their job . again , the available training opportunities vary considerably across europe and there are also differences between europe and countries like us or japan . but doctors may seek training and career opportunities in other countries not only because such opportunities lack in their home country , but because the experience and the know - how achieved abroad is often very enriching and acts as a boost for their career . finally , some medical doctors also wish to migrate because they simply want to make a change in their lifestyle . according to a who report on healthcare workforce migration in europe , there are also other factors associated with migration flows that can stimulate migration and affect the choice of a destination country : organizational factors , such as heavy workload , occupational risks , poor management , favouritism or lack of due process , lack of recognition;healthcare system factors , such as the absence or inadequacy of human resource policies , insufficient funding of health services , and centralised decision - making;general environmental factors , such as poor economic conditions and lack of security . organizational factors , such as heavy workload , occupational risks , poor management , favouritism or lack of due process , lack of recognition ; healthcare system factors , such as the absence or inadequacy of human resource policies , insufficient funding of health services , and centralised decision - making ; general environmental factors , such as poor economic conditions and lack of security . the mobility of doctors within the european region was to a large extent influenced by the eu enlargement in 2004 and 2007 . the eu enlargement triggered east west asymmetries in terms of inflows and outflows of health professionals , with migrants from new member states moving to countries from eu-15 . however , many of the eu-15 countries have outflows of the same magnitude as the eu-12 , but unlike the eu-15 , the eu-12 countries have only negligible inflows . in europe , among the major destination countries are germany , france , italy , uk and spain . they receive doctors from countries like poland , greece , romania , switzerland and the czech republic . germany is at the same time a big source and destination country , german doctors choosing to migrate mainly to the united kingdom and italy , followed by switzerland and us . outflows have also been observed in the uk , mainly toward neighbouring countries like ireland and france , but also to spain and the us . according to a who study on health personnel migration , the flow of migrant doctors is very dynamic in the european region . this situation is illustrated in the fig . 1migration of physicians in the who european region ( red arrows indicate two - way flows ) , taken from migration of physicians in the who european region ( red arrows indicate two - way flows ) , taken from in fig . thus , the flow of migrant doctors among belgium , france and the netherlands is also multidirectional . although there have been several recent studies examining the migration of healthcare workforce in europe , data are still limited . policy makers , workforce planners and healthcare managers need to better understand the mobility of trends in order to react with the right measures . due to the european integration and eu enlargement , which brought more flexibility for travelling and working in europe , it became obvious that an eu framework for professional mobility is needed . in response to this situation , the european parliament and the european council adopted the directive 2005/36/ec on recognition of professional qualifications , which affects more than 800 different professions regulated by member states across the eu , including medical doctors , nurses , midwives , and dentists . the directive sets the european legal framework for the recognition of professional qualifications obtained in a member state other than the one where the person wishes to work . with directive 2005/36/ec the eu has reformed the system for recognition of professional qualifications , in order to help make labour markets more flexible , further liberalise the provision of services , encourage more automatic recognition of qualifications , and simplify administrative procedures . the most recent consolidated version of the directive was made available on 24 march 2011 . until 20 october 2007 when the transposition period ended , this directive has replaced 15 existing directives in the field of recognition of professional qualifications , providing the first comprehensive modernisation of the eu system since its introduction over 40 years ago . according to the european commission , only 22 out of 27 member states fully transposed the directive within the given timeframe . considering the complexity of directive and the degree it impacts national legislation , some member states had to adapt a significant number of measures in order to complete the transposition . directive 2005/36/ec facilitates temporary provision of services ( tps ) by replacing the previous system of prior check of qualifications by the simpler , optional , system of prior declaration . however , due to the need to protect consumers , a prior check of qualifications may be maintained for professions having public health or safety implications ( article 7(4 ) of the directive ) . according to the european commission , 26 member states have implemented article 7(4 ) of the directive by april 2010 , apart from greece where the situation was still unclear . , professionals can in principle work on the basis of a declaration made in advance . the directive also applies to professionals wishing to establish themselves in an eu country other than that in which they obtained their professional qualifications , either as an employed or self - employed person , either on a permanent basis . directive 2005/36/ec sets out three systems for the recognition of qualifications : automatic recognition for professions for which the minimum training conditions have been harmonised ( health professionals , architects , veterinary surgeons).basic medical training and general practitioner qualifications ( annex v.5.1.1 and v.5.1.4)specialist doctors qualifications are automatically recognised in certain eu countries ( annex v.5.1)the general system for other regulated professions ( including non - automatic recognition - art . automatic recognition for professions for which the minimum training conditions have been harmonised ( health professionals , architects , veterinary surgeons).basic medical training and general practitioner qualifications ( annex v.5.1.1 and v.5.1.4)specialist doctors qualifications are automatically recognised in certain eu countries ( annex v.5.1 ) basic medical training and general practitioner qualifications ( annex v.5.1.1 and v.5.1.4 ) specialist doctors qualifications are automatically recognised in certain eu countries ( annex v.5.1 ) the general system for other regulated professions ( including non - automatic recognition - art . 10 to 15 ) . recognition on the basis of professional experience for certain professional activities . title ii of directive 2005/36/ec governs the recognition of professional qualifications in the context of a temporary move to the territory of another eu country . the temporary and occasional nature of the activities of a self - employed or employed person is assessed on a case - by - case basis , in light of the duration of the activity , its frequency , regularity and continuity . but the current system must be evaluated in order to verify whether full use has been made of all the opportunities offered by directive 2005/36/ec . the system must also take account of the considerable changes that have occurred in the member states educational and training systems . for this reason , the commission has begun working on evaluating the 2005 directive which culminated in a green paper modernising the professional qualifications directive in june 2011 . specifically the mobility of health professionals , the european commission issued a green paper on european workforce for health in december 2008 and invited all interested organisation to a public consultation , which closed on 31 march 2009 . as regards the impact of directive 2011/24/eu on the mobility of health professionals , the directive stipulates that member states should facilitate cooperation between healthcare providers , purchasers and regulators of different member states at national , regional or local level in order to ensure safe , high - quality and efficient cross - border healthcare . according to directive 2011/24/eu , cooperation between member states may concern practical mechanisms to ensure continuity of care or practical facilitating of cross - border provision of healthcare by health professionals on a temporary or occasional basis . however , in directive 2011/24/eu it is mentioned that this directive should be without prejudice to directive 2005/36/ec on the recognition of professional qualifications . this means that free provision of services of a temporary or occasional nature , including services provided by health professionals in another member state is not subject to specific provisions of union law and is to be restricted for any reason relating to professional qualifications . although the mobility of doctors within the european union is to an extent regulated by eu law , it is also necessary to take into consideration the national legislation from european member states . as mentioned in previous paragraphs , the existing legal framework addresses only the mobility of health professionals ( and other ) when they cross borders within europe . but thus , if a german physician would like to work in china he / she may encounter difficulties in acquiring the recognition of his / her qualifications allowing working in this country . or , if a physician from georgia is seeking work in france , again , there is to date no european legal act which can help in such cases . therefore , the european union should consider introducing further regulations or developing legal instruments aiming at solving this challenge healthcare workforce migration beyond europe s borders . besides the legislative framework aiming at regulating the mobility of doctors in europe , the world health organisation developed a non - regulatory instrument on the international recruitment of health personnel - who global code of practice on the international recruitment of health personnel . the who code of practice was adopted at the sixty - third world health assembly in geneva on 21 may 2010 and its key components are : in destination countries : ethical recruitment practices;protection of the rights of foreign healthcare workers;increased education and training for health sector students;pairing of needs and supply.in source countries : improved conditions for healthcare professionals;continued medical training and increased opportunities;incentives to retain physicians and nurses in countries and regions with human resource shortages . in destination countries : ethical recruitment practices;protection of the rights of foreign healthcare workers;increased education and training for health sector students;pairing of needs and supply . ethical recruitment practices ; protection of the rights of foreign healthcare workers ; increased education and training for health sector students ; pairing of needs and supply . improved conditions for healthcare professionals ; continued medical training and increased opportunities ; incentives to retain physicians and nurses in countries and regions with human resource shortages . this initiative of who comes also in response to the consequences of the financial crisis on labour markets and addresses the need to mitigate the negative effects of migration on health systems in developing countries and to ensure equitable access to health care services while minimizing the need to rely on the immigration of health personnel from other countries . in order to assist the medical doctors ( and other professionals ) who intend to work across borders , national contact points have been established at the initiative of the european commission s service free movement of professionals , directorate general internal market . there are contact points in every eu country that can give information on the recognition of professional qualifications according to the national law and procedures to be followed . the contact points also serve as a guide for the applicants , helping them to complete the required administrative formalities . but contact points can only assist the applicants with their requests and are not in charge for deciding whether the recognition of a certain profession should be granted . the decision - makers are the national competent authorities , who decide whether or not to recognise professional qualifications obtained in other eu countries , in accordance with european and national legislation . the competent authorities in member states are expected to use a set of common rules laid down by a code of conduct . as regards the procedure to be followed for the recognition of professional qualifications , the applicant must apply to the authority that oversees the doctor profession in that country and provide the authority with the proof of qualifications . the competent authority must acknowledge the application within 1 month of receiving it , and ask for missing but necessary documents to process the application . then the authority assesses the qualifications and decides whether to grant the application within 3 months . if the applicant does not accept the decision of the competent authority , he / she can appeal to the relevant court in that country . with the aim of ensuring better coordination both among member states and between member states and ec , the tasks of this group include helping national authorities and the commission work together better , monitoring policies with a bearing on qualifications for regulated professions , and exchanging experiences and good practices in the recognition of qualifications . further support for workforce mobility within europe was made available by the european commission directorate general on employment , social affairs and equal opportunities , who has established eures the european job mobility portal . the purpose of eures is to provide information , advice and recruitment / placement ( job - matching ) services for the benefit of workers and employers as well as any citizen wishing to benefit from the principle of the free movement of persons . eures plays an important especially in cross - border regions , areas in which there are significant levels of cross - border mobility . the impacts on the performance of health systems are subtle , meaning that they are indirect or hard to discern . romanian rural areas ) may be particularly vulnerable , showing some of the highest emigration rates among medical doctors and nurses . for the healthcare systems in source countries emigration can contribute to a major shortage of health professionals . in addition , the source country also loses the investment in the education of health professionals as well as the contribution they would have otherwise made for the healthcare system . some source countries may have also some benefits from the emigration of doctors , like reduction in staff surpluses and access to new knowledge and skills , in case the emigration is temporary . in some cases , source countries can benefit from collaborative training programmes , research projects or teaching activities which are initiated by emigrant doctors with their home country . for instance , migrant physicians may accept lower salaries than native ones , and they may also accept working in geographic areas avoided by national workers . however , destination countries may also encounter some difficulties related to inflow of medical doctors . for temporary migrants , investment in workplace induction can be relatively high compared to the time of service provided by the migrant physicians . as regards the policy implications of doctor s migration , first , the uncertainties surrounding the impact of the economic crises which forced some countries to drastically reduce their healthcare budgets , while in other countries budgets remained unaffected . a second factor impacting healthcare policies is the uncertainty of the development of healthcare workforce in europe . according to a european commission forecast , a shortage of around 1 million health professionals compensation of health workforce shortages by recruiting from third countries is to an extent restricted by ethical concerns , as stipulated in the who global code of practice for the international recruitment of health personnel adopted in 2010 . for these reasons , the european countries dealing with a high demand for medical doctors will face increasing difficulties in filling their vacancies with doctors from abroad . according to the european observatory on health systems and policies , there are three main sets of policy implications linked with the mobility of medical doctors : the first refers to the amount of data , intelligence and evidence , which are currently not sufficiently developed . therefore , in the absence of inflow and outflow data , policy makers can not take appropriate measures to manage doctors migration.the second set of policy implications refers to the strengthening the general workforce strategies . symptom of underlying domestic workforce aspects , such as working conditions , salaries , and training opportunities.the third policy implication is related to sustaining the re - emerging interest in workforce planning methods and techniques , by taking into account especially the dynamics and the need of healthcare force in future . the first refers to the amount of data , intelligence and evidence , which are currently not sufficiently developed . therefore , in the absence of inflow and outflow data , policy makers can not take appropriate measures to manage doctors migration . the third policy implication is related to sustaining the re - emerging interest in workforce planning methods and techniques , by taking into account especially the dynamics and the need of healthcare force in future . in responding to the inflow and outflow of medical doctors , today and in future , countries may take a series of measures aiming at an appropriate management of their healthcare workforce . for instance , they may sign bi - lateral agreements or facilitate the recognition of diplomas from non - european countries or they may consider developing twinning schemes and joint training programmes . although these policy responses may facilitate a better management of doctors mobility , the european countries should seek to solve mainly the domestic problems liked to doctors migration . in addition , countries could develop new healthcare strategies which can better respond to the current and future mobility trends .

lumiracoxib ( cox-189 ) is a cyclooxygenase type 2 ( cox-2 ) inhibitor and non - steroidal anti - inflammatory drug ( nsaid ) used for the treatment of osteoarthritis and acute pain . as a group , nsaids , including both traditional non - selective nsaids and cox-2 selective inhibitors , have been associated with increased risk of cardiovascular events . possible mechanisms have been debated widely and probably include effects on platelet function , blood pressure and sodium retention . lumiracoxib may have certain advantages over other nsaids in terms of its effects on blood pressure [ 2 , 3 ] . however , lumiracoxib has been associated with severe liver injury in a small number of patients , some of whom required liver transplantation . in many of these cases other risk factors for liver disease were also present and it was not clear whether the liver injury was drug - induced . the result was that lumiracoxib was withdrawn from several worldwide markets from 2007 onwards and it also failed to gain approval in other countries because of the potential for hepatotoxicity . recently , a genome - wide association ( gwa ) study identified hla alleles strongly associated with risk of hepatotoxicity with lumiracoxib , opening up the possibility of pre - treatment pharmacogenetic screening to exclude patients at higher risk of liver injury from lumiracoxib treatment . this paper re - evaluates the cardiovascular safety of lumiracoxib in patients with osteoarthritis in comparison to other nsaids and placebo at a time when it looks possible that lumiracoxib might re - emerge on to the market alongside a pharmacogenomic screening test to target its use more safely . a systematic review of clinical trials of lumiracoxib in patients with osteoarthritis reported up to january 2010 was undertaken . trials were included if they used lumiracoxib daily doses of 100400 mg , were of at least 1 week s duration and if they had a substantial cardiovascular component . both published and unpublished trials were included . novartis granted explicit access to their company studies and the right to use these study reports for the purposes of publication in peer reviewed journals . pubmed searches using predefined search criteria ( lumiracoxib and osteoarthritis , limits : none ; cox-189 and osteoarthritis , limits : none ) were used to obtain the relevant published trials . where both were available , published papers were matched to the relevant company clinical study reports to avoid double inclusion of the same study data . each study was graded according to the quality of evidence using an appropriate validated grading system , the jadad scale . only studies judged to be of sufficient quality ( the list of published trials and clinical study reports with jadad scores > 3 that were included for detailed review and meta - analysis in this report is presented in table 1 . cardiovascular events were the primary outcome for only one study and were reported as adverse events in others . adverse events were reviewed by an independent safety committee in a blinded manner for 4 studies [ 2 , 68 ] . in the other studies aes were reported as recorded by investigators or subjects . all patients had to meet the inclusion criterion of primary osteoarthritis and patients who had secondary osteoarthritis , other connective tissue diseases , or significant medical problems were excluded from the studies . the average age of the study participants in table 1 ranged from 59.5 to 65.5 years old and there were more women than men in the studies ( ranging from 59% to 76% across the studies ) . one study reported a safety assessment lasting 2 weeks after the end of study . the studies that were considered but excluded are listed in table 2 , along with the jadad scores assigned and the reasons for exclusion . the initial search of studies and jadad score assignment were carried out by chameleon communications international under our instruction . we subsequently independently reviewed and approved this work.table 1list of studies included for reviewreference ( published studies)study number ( novartis clinical study reports)total number of patientslumiracoxib dose ( number of patients)comparator nsaids and dose ( number of patients)exposure duration ( days)jadad scorecsr10458350 mg bd ( 98)placebo ( 97)284/5100 mg bd ( 96)diclofenac 75 mg bd ( 94)200 mg bd ( 99)400 mg od ( 99)csr1091,600200 mg od ( 462)placebo ( 231)915400 mg od ( 463)celecoxib 200 mg od ( 444)csr1121,702200 mg od ( 487)placebo ( 243)914/5400 mg od ( 491)celecoxib 200 mg od ( 481)csr112e1,235200 mg od ( 411)celecoxib 200 mg od ( 405)2735400 mg od ( 419)csr1261,042200 mg od ( 264)ibuprofen 800 mg tds ( 260)915400 mg od ( 260)celecoxib 200 mg od ( 258)csr128511400 mg od ( 205)placebo ( 204)915rofecoxib 25 mg od ( 102)csr2301364400 mg od ( 144)placebo ( 75)75celecoxib 200 mg bd ( 145)csr2303408200 mg od ( 105)placebo ( 103)75400 mg od ( 99)celecoxib 200 mg bd ( 101)csr2307309400 mg od ( 154)rofecoxib 25 mg od ( 155)425/5csr2316244100 mg od ( 122)placebo ( 122)285csr2319594200 mg od ( 205)placebo ( 196)285400 mg od ( 193)csr23601,551100 mg od ( 391)placebo ( 382)915/5200 mg od ( 385)celecoxib 200 mg od ( 393)csr23611,684100 mg od ( 420)placebo ( 424)915/5200 mg od ( 420)celecoxib 200 mg od ( 420)csr2361e1,310100 mg od ( 853)celecoxib 200 mg od ( 457)2735csr2364703200 mg od ( 352)celecoxib 200 mg od ( 351)425csr23671,262100 mg od ( 427)placebo ( 416)915celecoxib 200 mg od ( 419)csr23693,036100 mg od ( 757)celecoxib 200 mg od ( 759)3645100 mg bd ( 1,520)csr2428787100 mg od ( 394)ibuprofen 600 mg tds ( 393)285/5 target 0117 and 233218,325400 mg od ( 9,156)ibuprofen 800 mg tds ( 4,415)3655/5naproxen 500 mg bd ( 4,754)bd = twice daily ; nsaid = non - steroidal anti - inflammatory drug ; od = once daily ; tds = three times dailyjadad score assigned to published papertable 2list of all studies considered and excluded with jadad scores and reasons for exclusionreferencejadad scorereason for exclusion ( if applicable)published studies fleischmann , r et al . j clin hypertens ( greenwich ) 2008;10:592not applicablepost - hoc analysis of data in farkouh et al .2004 farkouh , me et al . clin gastroenterol hepatol 2006;4:57not applicablestudy in patients with ra nielsen , oh et al . int j clin pract 2004;58:10333study in patients with ra kivitz , aj et al . eur j clin pharmacol 2008;64:2330review article aust nurs j 2007;15:8 [ no authors listed]0commentary burton , b et al . expert opin investig drugs 2005;14:5210review article z orthop ihre grenzgeb 2005;143:158 [ no authors listed]not applicablearticle not in english rordorf , cm et al . acp j club 2005;142:460commentary health news 2004;10:13 [ no authors listed]0commentary kiefer , w et al . eular 2003 poster fri02220not full publication pavelka , k. eular 2005 poster fri03190not full publicationclinical study reports csr1055study in patients with ra csr1105study in patients with ra csr1115study in patients with ra csr 1145study in patients with ra csr 23125study in patients with ra csr 23355study in patients with ra csr 2360e1no comparator csr 23651no comparator csr 24255study in healthy subjects csr 24275study in patients undergoing knee surgeryra = rheumatoid arthritis ; cv = cardiovascular ; eular = european league against rheumatism ; csr = clinical study report list of studies included for review bd = twice daily ; nsaid = non - steroidal anti - inflammatory drug ; od = once daily ; tds = three times daily jadad score assigned to published paper list of all studies considered and excluded with jadad scores and reasons for exclusion ra = rheumatoid arthritis ; cv = cardiovascular ; eular = european league against rheumatism ; csr = clinical study report cardiovascular endpoints of relevance to the present paper primarily included the antiplatelet trialists collaboration ( aptc ) events ( non - fatal myocardial infarction [ mi ] , non - fatal stroke or cardiovascular death ) . however , each of the aptc components was also considered separately where data were available . subject level data were generally not available , although some descriptions of individual serious adverse events and deaths were provided in clinical study reports . data extraction from the published studies and clinical study reports was performed by one author and checked by the other authors . although in many cases , there was clear reporting of numbers of events , in a few cases , a value judgement had to be applied as to whether to include events or not , for example , when events occurred soon after completion of the study . the odds ratio ( or ) and 95% confidence intervals ( cis ) were calculated for each trial based on the total number of patients and total number of events in each group . the fixed effects model was used to obtain pooled ors after a heterogeneity test among the trials . publication bias was assessed by egger s test and begg s funnel plots for each of the endpoints studied by meta - analysis and no bias was found . data extraction from the published studies and clinical study reports was performed by one author and checked by the other authors . although in many cases , there was clear reporting of numbers of events , in a few cases , a value judgement had to be applied as to whether to include events or not , for example , when events occurred soon after completion of the study . the odds ratio ( or ) and 95% confidence intervals ( cis ) were calculated for each trial based on the total number of patients and total number of events in each group . the fixed effects model was used to obtain pooled ors after a heterogeneity test among the trials . publication bias was assessed by egger s test and begg s funnel plots for each of the endpoints studied by meta - analysis and no bias was found . all studies were of lumiracoxib versus placebo or lumiracoxib versus one or more active nsaid comparators ( or both placebo and nsaid comparator ) . doses of lumiracoxib were between 50 mg twice daily and 400 mg once daily ( total daily doses 100400 mg ) . comparator nsaids in the studies included ibuprofen , celecoxib , naproxen , rofecoxib and diclofenac . the duration of drug therapy in the studies was from 1 week to 1 year . all the studies included were of sufficient quality to be graded with a jadad score of > 3 . of the 19 studies included , 7 were reported as both published journal articles and clinical study reports [ 2 , 69 , 1012 ] and the remainder were reported only in unpublished novartis clinical study reports . where both the published journal article and the original clinical study reports were available pertaining to the same data , of the studies included , 11 out of 19 compared lumiracoxib with placebo ; only 6 of these reported the occurrence of any aptc events and event numbers were very small , providing limited data for further analysis . other cardiovascular endpoints were not reported consistently among studies and again , numbers of events were very small . therefore , meta - analysis was limited to the aptc endpoints for this group of trials and was not conducted for any other individual cardiovascular outcomes . the 6 studies comparing lumiracoxib versus placebo and reporting any aptc events are listed in table 3 . only 6 aptc events were recorded in 4,122 lumiracoxib users and 1 aptc event in 1,680 placebo users . in total , there were 959 person - years exposure to lumiracoxib and 385 person - years exposure to placebo within this group of studies . the meta - analysis from the 6 trials revealed no significant difference in the incidence of aptc endpoints between lumiracoxib and placebo ( pooled or 1.10 , 95% ci 0.31 , 3.94 ) . however , the wide confidence intervals did not exclude lumiracoxib being almost 70% better than placebo or 394% worse than placebo.table 3antiplatelet trialists collaboration ( aptc ) events in the lumiracoxib and placebo groupsnovartis study numberreference ( if published)number of patients in the lumiracoxib groupnumber of patients in the placebo groupnumber of aptc events in the lumiracoxib groupnumber of aptc events in the placebo groupcsr10992523110csr12820520411csr231939819610csr236184042410csr236077638210csr11297824310total4,1221,68061 antiplatelet trialists collaboration ( aptc ) events in the lumiracoxib and placebo groups there were 17 trials comparing lumiracoxib with other nsaid comparators . there were sufficient data to perform meta - analysis for aptc events , mi , stroke and cardiovascular death . in total , there were 13,256 person - years exposure to lumiracoxib and 10,964 person - years exposure to other nsaid comparators within this group of studies . 78 events were reported in the lumiracoxib group ( n = 17,434 ) and 58 events in the active nsaid comparator groups ( n = 13,606 ) . the pooled or for the likelihood of aptc endpoints with lumiracoxib versus other nsaids was 1.16 ( 95% ci 0.82 , 1.63 ; fig . 1 ) . a sensitivity analysis , excluding 8 trials in which there were no events in either one of the lumiracoxib or comparator nsaids arms , showed a similar result ( pooled or 1.21 , 95% ci 0.84 , 1.73).fig . 1meta - analysis of aptc endpoints in randomized controlled trials comparing lumiracoxib with other non - steroidal anti - inflammatory drugs ( nsaids ) meta - analysis of aptc endpoints in randomized controlled trials comparing lumiracoxib with other non - steroidal anti - inflammatory drugs ( nsaids ) data for the mi endpoint were available in 7 trials with a total of 33 mi events reported in 24,422 patients ( 12,909 in the lumiracoxib group and 11,513 in the other nsaids group ) . overall , there was no significantly increased risk of mi for lumiracoxib users ( pooled or 1.66 , 95% ci 0.84 , 3.29 ; fig . 2 ) . six trials had only one mi event in either the lumiracoxib group or the other nsaid group . 2meta - analysis of myocardial infarction ( mi ) events in randomized controlled trials comparing lumiracoxib with other nsaids meta - analysis of myocardial infarction ( mi ) events in randomized controlled trials comparing lumiracoxib with other nsaids stroke occurrence was rare within the trials studied . 3meta - analysis of stroke events in randomized controlled trials comparing lumiracoxib with other nsaids meta - analysis of stroke events in randomized controlled trials comparing lumiracoxib with other nsaids eight trials reported cardiovascular death events and meta - analysis found no increased risk of cardiovascular death in the lumiracoxib group compared with the other nsaid group ( fig . 4 ; or 1.04 , 95% ci 0.60 , 1.80 ) . a sensitivity analysis excluding 5 trials with no events occurring in either group resulted in a slightly higher estimate of the pooled or ( or 1.11 , 95% ci 0.61 , 2.01 ) , but this remained statistically non-significant.fig . 4meta - analysis of cardiovascular death events in randomized controlled trials comparing lumiracoxib with other nsaids meta - analysis of cardiovascular death events in randomized controlled trials comparing lumiracoxib with other nsaids all studies were of lumiracoxib versus placebo or lumiracoxib versus one or more active nsaid comparators ( or both placebo and nsaid comparator ) . doses of lumiracoxib were between 50 mg twice daily and 400 mg once daily ( total daily doses 100400 mg ) . comparator nsaids in the studies included ibuprofen , celecoxib , naproxen , rofecoxib and diclofenac . the duration of drug therapy in the studies was from 1 week to 1 year . all the studies included were of sufficient quality to be graded with a jadad score of > 3 . of the 19 studies included , 7 were reported as both published journal articles and clinical study reports [ 2 , 69 , 1012 ] and the remainder were reported only in unpublished novartis clinical study reports . where both the published journal article and the original clinical study reports were available pertaining to the same data , of the studies included , 11 out of 19 compared lumiracoxib with placebo ; only 6 of these reported the occurrence of any aptc events and event numbers were very small , providing limited data for further analysis . other cardiovascular endpoints were not reported consistently among studies and again , numbers of events were very small . therefore , meta - analysis was limited to the aptc endpoints for this group of trials and was not conducted for any other individual cardiovascular outcomes . the 6 studies comparing lumiracoxib versus placebo and reporting any aptc events are listed in table 3 . only 6 aptc events were recorded in 4,122 lumiracoxib users and 1 aptc event in 1,680 placebo users . in total , there were 959 person - years exposure to lumiracoxib and 385 person - years exposure to placebo within this group of studies . the meta - analysis from the 6 trials revealed no significant difference in the incidence of aptc endpoints between lumiracoxib and placebo ( pooled or 1.10 , 95% ci 0.31 , 3.94 ) . however , the wide confidence intervals did not exclude lumiracoxib being almost 70% better than placebo or 394% worse than placebo.table 3antiplatelet trialists collaboration ( aptc ) events in the lumiracoxib and placebo groupsnovartis study numberreference ( if published)number of patients in the lumiracoxib groupnumber of patients in the placebo groupnumber of aptc events in the lumiracoxib groupnumber of aptc events in the placebo groupcsr10992523110csr12820520411csr231939819610csr236184042410csr236077638210csr11297824310total4,1221,68061 antiplatelet trialists collaboration ( aptc ) events in the lumiracoxib and placebo groups there were sufficient data to perform meta - analysis for aptc events , mi , stroke and cardiovascular death . in total , there were 13,256 person - years exposure to lumiracoxib and 10,964 person - years exposure to other nsaid comparators within this group of studies . 78 events were reported in the lumiracoxib group ( n = 17,434 ) and 58 events in the active nsaid comparator groups ( n = 13,606 ) . the pooled or for the likelihood of aptc endpoints with lumiracoxib versus other nsaids was 1.16 ( 95% ci 0.82 , 1.63 ; fig . 1 ) . a sensitivity analysis , excluding 8 trials in which there were no events in either one of the lumiracoxib or comparator nsaids arms , showed a similar result ( pooled or 1.21 , 95% ci 0.84 , 1.73).fig . 1meta - analysis of aptc endpoints in randomized controlled trials comparing lumiracoxib with other non - steroidal anti - inflammatory drugs ( nsaids ) meta - analysis of aptc endpoints in randomized controlled trials comparing lumiracoxib with other non - steroidal anti - inflammatory drugs ( nsaids ) data for the mi endpoint were available in 7 trials with a total of 33 mi events reported in 24,422 patients ( 12,909 in the lumiracoxib group and 11,513 in the other nsaids group ) . overall , there was no significantly increased risk of mi for lumiracoxib users ( pooled or 1.66 , 95% ci 0.84 , 3.29 ; fig . 2 ) . six trials had only one mi event in either the lumiracoxib group or the other nsaid group . 2meta - analysis of myocardial infarction ( mi ) events in randomized controlled trials comparing lumiracoxib with other nsaids meta - analysis of myocardial infarction ( mi ) events in randomized controlled trials comparing lumiracoxib with other nsaids stroke occurrence was rare within the trials studied . 3meta - analysis of stroke events in randomized controlled trials comparing lumiracoxib with other nsaids meta - analysis of stroke events in randomized controlled trials comparing lumiracoxib with other nsaids eight trials reported cardiovascular death events and meta - analysis found no increased risk of cardiovascular death in the lumiracoxib group compared with the other nsaid group ( fig . 4 ; or 1.04 , 95% ci 0.60 , 1.80 ) . a sensitivity analysis excluding 5 trials with no events occurring in either group resulted in a slightly higher estimate of the pooled or ( or 1.11 , 95% ci 0.61 , 2.01 ) , but this remained statistically non-significant.fig . 4meta - analysis of cardiovascular death events in randomized controlled trials comparing lumiracoxib with other nsaids meta - analysis of cardiovascular death events in randomized controlled trials comparing lumiracoxib with other nsaids having reviewed the published and unpublished data available , it is apparent that while several clinical trials have been performed , we still have a paucity of data to judge the cardiovascular safety of lumiracoxib versus placebo or other nsaids , because cardiovascular events were rare in most of the studies . as far as we are able to judge from the data available , there is no evidence of increased risk of cardiovascular events with lumiracoxib versus placebo or versus other nsaids . however , for example for the mi endpoint , because of the wide confidence intervals , the results are compatible with a 16% lower or 229% higher risk of mi with lumiracoxib versus other nsaids . our results are in agreement with a previous meta - analysis by matchaba et al . , which examined the cardiovascular safety of lumiracoxib in randomised controlled trials in patients with osteoarthritis or rheumatoid arthritis . it found no evidence that lumiracoxib was associated with a significant increase in cardiovascular risk compared with naproxen , placebo or all comparators ( placebo , diclofenac , ibuprofen , celecoxib , rofecoxib and naproxen ) . although the studies we used in our analysis included several different nsaid comparators , the total number of events occurring for each individual nsaid was small . therefore , we were not able to draw conclusions about the relative safety of individual nsaid comparators and lumiracoxib , but rather grouped the comparator nsaids together for our analysis . similarly , there was insufficient event data to allow analysis of dose effects within either the lumiracoxib or nsaid comparator groups . while in several studies there is a trend towards some nsaids being safer , e.g. naproxen , and some being more harmful , e.g. rofecoxib , the mechanisms of increased risk are likely to be mixed and to include vascular , platelet and blood pressure effects . in patients with osteoarthritis , a risk benefit judgement must be made to balance pain control and quality of life with any potential side effects of nsaids . at least some of the blood pressure effects of nsaids are likely to be due to sodium retention . interestingly , while modest dietary salt restriction leads to significant reductions in systolic and diastolic blood pressures ( average of up to 5 and 3 mmhg respectively ) in patients with hypertension [ 15 , 16 ] , a low salt diet leads to a much more striking reduction in blood pressure in patients with resistant hypertension ( average 22.7 and 9.1 mmhg reductions in office systolic and diastolic pressures respectively ) . similarly , the potential benefits of lumiracoxib compared with other nsaids on blood pressure could be greatest in those patients with resistant hypertension . therefore , lumiracoxib may be most beneficial for patients with osteoarthritis requiring nsaid therapy for pain control , but who also have resistant hypertension . limitations of our meta - analysis include the small number of cardiovascular events occurring in most of the trials . from the data , we calculated that the estimated incidences of aptc events for the active comparator group and the lumiracoxib group were 5.29 and 5.88 per 1,000 person - years respectively . it would require 29,486 person - years exposure and 195 events to detect a 50% difference in the incidence of aptc events at a 5% significance level and 80% power between lumiracoxib and other nsaids . clearly our meta - analysis is not powered to detect such a difference . by including only higher quality trials ( jadad score > 3 ) we may have excluded some other data , although one could argue that lower quality trials should not be included anyway . our meta - analysis was limited to patients with osteoarthritis , who are generally thought to be at lower cardiovascular risk than patients with rheumatoid arthritis . a recent network meta - analysis including trials in patients taking nsaids for any medical condition found a statistically significant increase with lumiracoxib versus placebo in the rate ratio of stroke rr 2.81 ( 95% ci 1.057.48 ) and of the aptc endpoint rr 2.04 ( 1.134.24 ) , but no increased risk of mi , cardiovascular death or death of any cause . using the network meta - analysis technique to allow indirect comparisons between different nsaids , no statistically significant increase in risk of any of the above endpoints was found with lumiracoxib versus naproxen , etoricoxib , celecoxib , rofecoxib , diclofenac or ibuprofen . the results suggest that the cardiovascular risk with lumiracoxib was not significantly different from that with placebo or with other nsaids . wide confidence intervals mean that further research is needed in this area to confirm these findings . tmm has acted as a consultant for novartis in relation to lumiracoxib and has received honoraria for giving educational lectures . tmm holds research grants from pfizer and tmm and ism hold research grants from pfizer , menarini and ipsen on behalf of the university sponsor . tmm , ism and lw hold research grants from novartis in a different therapeutic area which post - dates this work . ism , lw and tmm were all involved in concept / design , data analysis and interpretation , drafting , critical revision and approval of the article . this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .

purposeto re - evaluate the cardiovascular risk of lumiracoxib compared with other non - steroidal anti - inflammatory drugs ( nsaids ) or placebo in patients with osteoarthritis.methodswe conducted a meta - analysis of randomised controlled trials of lumiracoxib versus placebo or other nsaids in patients with osteoarthritis reported up to january 2010 . both published and unpublished trials were included . pubmed searches using predefined search criteria ( lumiracoxib and osteoarthritis , limits : none ; cox-189 and osteoarthritis , limits : none ) were used to obtain the relevant published trials . novartis granted explicit access to their company studies and the right to use these study reports for the purposes of publication in peer reviewed journals . endpoints were the antiplatelet trialists collaboration ( aptc ) endpoint and individual cardiovascular endpoints.resultsmeta-analysis of 6 trials of lumiracoxib versus placebo revealed no difference in cardiovascular outcomes . meta - analysis of 12 trials of lumiracoxib versus other nsaids also revealed no difference . the pooled odds ratios were : 1.16 ( 95% ci 0.82 , 1.63 ) ; 1.66 ( 95% ci 0.84 , 3.29 ) ; 0.95 ( 95% ci 0.52 , 1.76 ) and 1.04 ( 95% ci 0.60 , 1.80 ) for the aptc endpoint , myocardial infarction , stroke and cardiovascular death respectively.conclusionsthe results suggest that there were no significant differences in cardiovascular outcomes between lumiracoxib and placebo or between lumiracoxib and other nsaids in patients with osteoarthritis . wide confidence intervals mean that further research is needed in this area to confirm these findings .

Introduction Materials and methods Data extraction Statistical analysis Results Characteristics of the studies included in the review Randomised controlled trials of lumiracoxib versus placebo Randomised controlled trials of lumiracoxib versus other NSAIDs Discussion Conclusions Funding Disclosures Author contributions Open Access

lumiracoxib ( cox-189 ) is a cyclooxygenase type 2 ( cox-2 ) inhibitor and non - steroidal anti - inflammatory drug ( nsaid ) used for the treatment of osteoarthritis and acute pain . lumiracoxib may have certain advantages over other nsaids in terms of its effects on blood pressure [ 2 , 3 ] . this paper re - evaluates the cardiovascular safety of lumiracoxib in patients with osteoarthritis in comparison to other nsaids and placebo at a time when it looks possible that lumiracoxib might re - emerge on to the market alongside a pharmacogenomic screening test to target its use more safely . a systematic review of clinical trials of lumiracoxib in patients with osteoarthritis reported up to january 2010 was undertaken . trials were included if they used lumiracoxib daily doses of 100400 mg , were of at least 1 week s duration and if they had a substantial cardiovascular component . both published and unpublished trials were included . novartis granted explicit access to their company studies and the right to use these study reports for the purposes of publication in peer reviewed journals . pubmed searches using predefined search criteria ( lumiracoxib and osteoarthritis , limits : none ; cox-189 and osteoarthritis , limits : none ) were used to obtain the relevant published trials . only studies judged to be of sufficient quality ( the list of published trials and clinical study reports with jadad scores > 3 that were included for detailed review and meta - analysis in this report is presented in table 1 . we subsequently independently reviewed and approved this work.table 1list of studies included for reviewreference ( published studies)study number ( novartis clinical study reports)total number of patientslumiracoxib dose ( number of patients)comparator nsaids and dose ( number of patients)exposure duration ( days)jadad scorecsr10458350 mg bd ( 98)placebo ( 97)284/5100 mg bd ( 96)diclofenac 75 mg bd ( 94)200 mg bd ( 99)400 mg od ( 99)csr1091,600200 mg od ( 462)placebo ( 231)915400 mg od ( 463)celecoxib 200 mg od ( 444)csr1121,702200 mg od ( 487)placebo ( 243)914/5400 mg od ( 491)celecoxib 200 mg od ( 481)csr112e1,235200 mg od ( 411)celecoxib 200 mg od ( 405)2735400 mg od ( 419)csr1261,042200 mg od ( 264)ibuprofen 800 mg tds ( 260)915400 mg od ( 260)celecoxib 200 mg od ( 258)csr128511400 mg od ( 205)placebo ( 204)915rofecoxib 25 mg od ( 102)csr2301364400 mg od ( 144)placebo ( 75)75celecoxib 200 mg bd ( 145)csr2303408200 mg od ( 105)placebo ( 103)75400 mg od ( 99)celecoxib 200 mg bd ( 101)csr2307309400 mg od ( 154)rofecoxib 25 mg od ( 155)425/5csr2316244100 mg od ( 122)placebo ( 122)285csr2319594200 mg od ( 205)placebo ( 196)285400 mg od ( 193)csr23601,551100 mg od ( 391)placebo ( 382)915/5200 mg od ( 385)celecoxib 200 mg od ( 393)csr23611,684100 mg od ( 420)placebo ( 424)915/5200 mg od ( 420)celecoxib 200 mg od ( 420)csr2361e1,310100 mg od ( 853)celecoxib 200 mg od ( 457)2735csr2364703200 mg od ( 352)celecoxib 200 mg od ( 351)425csr23671,262100 mg od ( 427)placebo ( 416)915celecoxib 200 mg od ( 419)csr23693,036100 mg od ( 757)celecoxib 200 mg od ( 759)3645100 mg bd ( 1,520)csr2428787100 mg od ( 394)ibuprofen 600 mg tds ( 393)285/5 target 0117 and 233218,325400 mg od ( 9,156)ibuprofen 800 mg tds ( 4,415)3655/5naproxen 500 mg bd ( 4,754)bd = twice daily ; nsaid = non - steroidal anti - inflammatory drug ; od = once daily ; tds = three times dailyjadad score assigned to published papertable 2list of all studies considered and excluded with jadad scores and reasons for exclusionreferencejadad scorereason for exclusion ( if applicable)published studies fleischmann , r et al . eular 2003 poster fri02220not full publication pavelka , k. eular 2005 poster fri03190not full publicationclinical study reports csr1055study in patients with ra csr1105study in patients with ra csr1115study in patients with ra csr 1145study in patients with ra csr 23125study in patients with ra csr 23355study in patients with ra csr 2360e1no comparator csr 23651no comparator csr 24255study in healthy subjects csr 24275study in patients undergoing knee surgeryra = rheumatoid arthritis ; cv = cardiovascular ; eular = european league against rheumatism ; csr = clinical study report list of studies included for review bd = twice daily ; nsaid = non - steroidal anti - inflammatory drug ; od = once daily ; tds = three times daily jadad score assigned to published paper list of all studies considered and excluded with jadad scores and reasons for exclusion ra = rheumatoid arthritis ; cv = cardiovascular ; eular = european league against rheumatism ; csr = clinical study report cardiovascular endpoints of relevance to the present paper primarily included the antiplatelet trialists collaboration ( aptc ) events ( non - fatal myocardial infarction [ mi ] , non - fatal stroke or cardiovascular death ) . the odds ratio ( or ) and 95% confidence intervals ( cis ) were calculated for each trial based on the total number of patients and total number of events in each group . the odds ratio ( or ) and 95% confidence intervals ( cis ) were calculated for each trial based on the total number of patients and total number of events in each group . publication bias was assessed by egger s test and begg s funnel plots for each of the endpoints studied by meta - analysis and no bias was found . all studies were of lumiracoxib versus placebo or lumiracoxib versus one or more active nsaid comparators ( or both placebo and nsaid comparator ) . of the 19 studies included , 7 were reported as both published journal articles and clinical study reports [ 2 , 69 , 1012 ] and the remainder were reported only in unpublished novartis clinical study reports . where both the published journal article and the original clinical study reports were available pertaining to the same data , of the studies included , 11 out of 19 compared lumiracoxib with placebo ; only 6 of these reported the occurrence of any aptc events and event numbers were very small , providing limited data for further analysis . other cardiovascular endpoints were not reported consistently among studies and again , numbers of events were very small . therefore , meta - analysis was limited to the aptc endpoints for this group of trials and was not conducted for any other individual cardiovascular outcomes . the 6 studies comparing lumiracoxib versus placebo and reporting any aptc events are listed in table 3 . the meta - analysis from the 6 trials revealed no significant difference in the incidence of aptc endpoints between lumiracoxib and placebo ( pooled or 1.10 , 95% ci 0.31 , 3.94 ) . however , the wide confidence intervals did not exclude lumiracoxib being almost 70% better than placebo or 394% worse than placebo.table 3antiplatelet trialists collaboration ( aptc ) events in the lumiracoxib and placebo groupsnovartis study numberreference ( if published)number of patients in the lumiracoxib groupnumber of patients in the placebo groupnumber of aptc events in the lumiracoxib groupnumber of aptc events in the placebo groupcsr10992523110csr12820520411csr231939819610csr236184042410csr236077638210csr11297824310total4,1221,68061 antiplatelet trialists collaboration ( aptc ) events in the lumiracoxib and placebo groups there were 17 trials comparing lumiracoxib with other nsaid comparators . there were sufficient data to perform meta - analysis for aptc events , mi , stroke and cardiovascular death . the pooled or for the likelihood of aptc endpoints with lumiracoxib versus other nsaids was 1.16 ( 95% ci 0.82 , 1.63 ; fig . a sensitivity analysis , excluding 8 trials in which there were no events in either one of the lumiracoxib or comparator nsaids arms , showed a similar result ( pooled or 1.21 , 95% ci 0.84 , 1.73).fig . 1meta - analysis of aptc endpoints in randomized controlled trials comparing lumiracoxib with other non - steroidal anti - inflammatory drugs ( nsaids ) meta - analysis of aptc endpoints in randomized controlled trials comparing lumiracoxib with other non - steroidal anti - inflammatory drugs ( nsaids ) data for the mi endpoint were available in 7 trials with a total of 33 mi events reported in 24,422 patients ( 12,909 in the lumiracoxib group and 11,513 in the other nsaids group ) . overall , there was no significantly increased risk of mi for lumiracoxib users ( pooled or 1.66 , 95% ci 0.84 , 3.29 ; fig . 2meta - analysis of myocardial infarction ( mi ) events in randomized controlled trials comparing lumiracoxib with other nsaids meta - analysis of myocardial infarction ( mi ) events in randomized controlled trials comparing lumiracoxib with other nsaids stroke occurrence was rare within the trials studied . 3meta - analysis of stroke events in randomized controlled trials comparing lumiracoxib with other nsaids meta - analysis of stroke events in randomized controlled trials comparing lumiracoxib with other nsaids eight trials reported cardiovascular death events and meta - analysis found no increased risk of cardiovascular death in the lumiracoxib group compared with the other nsaid group ( fig . 4 ; or 1.04 , 95% ci 0.60 , 1.80 ) . a sensitivity analysis excluding 5 trials with no events occurring in either group resulted in a slightly higher estimate of the pooled or ( or 1.11 , 95% ci 0.61 , 2.01 ) , but this remained statistically non-significant.fig . 4meta - analysis of cardiovascular death events in randomized controlled trials comparing lumiracoxib with other nsaids meta - analysis of cardiovascular death events in randomized controlled trials comparing lumiracoxib with other nsaids all studies were of lumiracoxib versus placebo or lumiracoxib versus one or more active nsaid comparators ( or both placebo and nsaid comparator ) . of the 19 studies included , 7 were reported as both published journal articles and clinical study reports [ 2 , 69 , 1012 ] and the remainder were reported only in unpublished novartis clinical study reports . other cardiovascular endpoints were not reported consistently among studies and again , numbers of events were very small . therefore , meta - analysis was limited to the aptc endpoints for this group of trials and was not conducted for any other individual cardiovascular outcomes . the meta - analysis from the 6 trials revealed no significant difference in the incidence of aptc endpoints between lumiracoxib and placebo ( pooled or 1.10 , 95% ci 0.31 , 3.94 ) . however , the wide confidence intervals did not exclude lumiracoxib being almost 70% better than placebo or 394% worse than placebo.table 3antiplatelet trialists collaboration ( aptc ) events in the lumiracoxib and placebo groupsnovartis study numberreference ( if published)number of patients in the lumiracoxib groupnumber of patients in the placebo groupnumber of aptc events in the lumiracoxib groupnumber of aptc events in the placebo groupcsr10992523110csr12820520411csr231939819610csr236184042410csr236077638210csr11297824310total4,1221,68061 antiplatelet trialists collaboration ( aptc ) events in the lumiracoxib and placebo groups there were sufficient data to perform meta - analysis for aptc events , mi , stroke and cardiovascular death . the pooled or for the likelihood of aptc endpoints with lumiracoxib versus other nsaids was 1.16 ( 95% ci 0.82 , 1.63 ; fig . a sensitivity analysis , excluding 8 trials in which there were no events in either one of the lumiracoxib or comparator nsaids arms , showed a similar result ( pooled or 1.21 , 95% ci 0.84 , 1.73).fig . 1meta - analysis of aptc endpoints in randomized controlled trials comparing lumiracoxib with other non - steroidal anti - inflammatory drugs ( nsaids ) meta - analysis of aptc endpoints in randomized controlled trials comparing lumiracoxib with other non - steroidal anti - inflammatory drugs ( nsaids ) data for the mi endpoint were available in 7 trials with a total of 33 mi events reported in 24,422 patients ( 12,909 in the lumiracoxib group and 11,513 in the other nsaids group ) . overall , there was no significantly increased risk of mi for lumiracoxib users ( pooled or 1.66 , 95% ci 0.84 , 3.29 ; fig . 2meta - analysis of myocardial infarction ( mi ) events in randomized controlled trials comparing lumiracoxib with other nsaids meta - analysis of myocardial infarction ( mi ) events in randomized controlled trials comparing lumiracoxib with other nsaids stroke occurrence was rare within the trials studied . 3meta - analysis of stroke events in randomized controlled trials comparing lumiracoxib with other nsaids meta - analysis of stroke events in randomized controlled trials comparing lumiracoxib with other nsaids eight trials reported cardiovascular death events and meta - analysis found no increased risk of cardiovascular death in the lumiracoxib group compared with the other nsaid group ( fig . 4 ; or 1.04 , 95% ci 0.60 , 1.80 ) . a sensitivity analysis excluding 5 trials with no events occurring in either group resulted in a slightly higher estimate of the pooled or ( or 1.11 , 95% ci 0.61 , 2.01 ) , but this remained statistically non-significant.fig . 4meta - analysis of cardiovascular death events in randomized controlled trials comparing lumiracoxib with other nsaids meta - analysis of cardiovascular death events in randomized controlled trials comparing lumiracoxib with other nsaids having reviewed the published and unpublished data available , it is apparent that while several clinical trials have been performed , we still have a paucity of data to judge the cardiovascular safety of lumiracoxib versus placebo or other nsaids , because cardiovascular events were rare in most of the studies . as far as we are able to judge from the data available , there is no evidence of increased risk of cardiovascular events with lumiracoxib versus placebo or versus other nsaids . however , for example for the mi endpoint , because of the wide confidence intervals , the results are compatible with a 16% lower or 229% higher risk of mi with lumiracoxib versus other nsaids . , which examined the cardiovascular safety of lumiracoxib in randomised controlled trials in patients with osteoarthritis or rheumatoid arthritis . it found no evidence that lumiracoxib was associated with a significant increase in cardiovascular risk compared with naproxen , placebo or all comparators ( placebo , diclofenac , ibuprofen , celecoxib , rofecoxib and naproxen ) . in patients with osteoarthritis , a risk benefit judgement must be made to balance pain control and quality of life with any potential side effects of nsaids . interestingly , while modest dietary salt restriction leads to significant reductions in systolic and diastolic blood pressures ( average of up to 5 and 3 mmhg respectively ) in patients with hypertension [ 15 , 16 ] , a low salt diet leads to a much more striking reduction in blood pressure in patients with resistant hypertension ( average 22.7 and 9.1 mmhg reductions in office systolic and diastolic pressures respectively ) . similarly , the potential benefits of lumiracoxib compared with other nsaids on blood pressure could be greatest in those patients with resistant hypertension . limitations of our meta - analysis include the small number of cardiovascular events occurring in most of the trials . it would require 29,486 person - years exposure and 195 events to detect a 50% difference in the incidence of aptc events at a 5% significance level and 80% power between lumiracoxib and other nsaids . clearly our meta - analysis is not powered to detect such a difference . our meta - analysis was limited to patients with osteoarthritis , who are generally thought to be at lower cardiovascular risk than patients with rheumatoid arthritis . a recent network meta - analysis including trials in patients taking nsaids for any medical condition found a statistically significant increase with lumiracoxib versus placebo in the rate ratio of stroke rr 2.81 ( 95% ci 1.057.48 ) and of the aptc endpoint rr 2.04 ( 1.134.24 ) , but no increased risk of mi , cardiovascular death or death of any cause . using the network meta - analysis technique to allow indirect comparisons between different nsaids , no statistically significant increase in risk of any of the above endpoints was found with lumiracoxib versus naproxen , etoricoxib , celecoxib , rofecoxib , diclofenac or ibuprofen . the results suggest that the cardiovascular risk with lumiracoxib was not significantly different from that with placebo or with other nsaids . wide confidence intervals mean that further research is needed in this area to confirm these findings .

the advent of next - generation sequencing and metagenomics has resulted in increasing numbers of ever - larger datasets describing the community structure and function of a variety of different environments , from the human gut ( arumugam et al . 2011 ; david et al . 2014 ) to arctic peat soils ( lipson et al . 2013 ) and deep - sea vents ( xie et al . next - generation sequencing technologies have greatly reduced sequencing costs and speed , and researchers can now affordably study whole microbial communities and functions . prior to this , the focus was on community species composition , studied using 16s rrna targeted amplicon sequencing . amplicon sequencing does not require the dna coverage that metagenomic studies require and can accurately identify which species are present in a sample ( woese and fox 1977 ; lane et al . 1985 ; hugenholtz 2002 ) , but it does not provide the depth of information , such as gene function , that full metagenome sequencing and annotation provides . cost is no longer the primary limiting factor for undertaking metagenomic studies , rather it is now bioinformatics and processing power required to process the data produced . illumina 's hiseq platform , for example , can affordably sequence the most complex of microbial communities , and the challenge now is to interpret the data produced . ( 2014 ) provide an extensive directory of tools available for different tasks involved in a metagenomic project pipeline , related to a range of omics studies . megan ( huson et al . 2007 ) is a popular graphical user interface program for analyzing and visualizing blast results to study the taxonomy of microbial communities . while megan typically analyses blast results in a few minutes , running blast searches against reference sequences in a database is computationally intensive and slow for metagenomes ( desai et al . 2012 ; hunter et al . 2012 ; thomas , gilbert and meyer 2012 ) . web - based servers are increasingly popular for processing large amounts of data . with an intuitive web interface and a variety of analytical tools to choose from , mg - rast ( meyer et al . mg - rast allows users to upload raw sequence files that are processed through quality filters and annotated using a selection of user - defined parameters , such as reference databases , minimum identity cut - off values , maximum e - values or expect - values , and minimum alignment lengths . details of the processing procedure can be found in the mg - rast technical report ( wilke et al . rapsearch2 ( zhao , tang and ye 2012 ) translates nucleotide sequences and aligns them with annotated protein sequences , reporting to be c. 100-fold faster than blastx with only a 1.3%3.2% reduction in sensitivity ( the proportion of sequences annotated ) . with pauda ( huson and xie 2014 ) uses a similar approach and claims to be 10 000-fold faster than blastx , although with a significant reduction in sensitivity . diamond ( buchfink , xie and huson 2015 ) purports to be both fast and accurate , with a 20 000-fold increase in processing speed compared to blastx . in sensitive mode , 99% of sequences are aligned , with a speed increase of 2000-fold compared to blastx . like blast with megablast , rapsearch2 and diamond offer fast and sensitive modes , each coming at the cost of the other . one codex is a web - based program that uses a different technique to blast and mg - rast to classify sequences ( https://onecodex.com/ ) . the program designers report that it runs 900 times faster than blast while maintaining similar genus - level sensitivity and precision ( the proportion of annotated sequences that are correctly identified ) , taking hours rather than days to classify most metagenomes . one codex works by comparing k - mers ( sequences of a set length ) from a sequence to a reference database of k - mers ; the greatest number of 100% k - mer matches determines the classification . blast and mg - rast classify sequences by matching them with the most similar sequences in a database . unlike mg - rast , one codex does not annotate genes for function . the choice of database , minimum identity cut - off value ( i.e. sequence match stringency ) , minimum alignment length cut - off value and minimum e - value limit ( the probability a match has occurred by chance ) all influence sequence annotation accuracy , which , in turn , affect the reproducibility and interpretation of the data . an inherent issue with metagenomic studies is that establishing the accuracy of sequence annotation for environmental samples is practically impossible , given that the quantities of organisms and genes are unknown . therefore , determining the most effective annotation method is fundamental to investigating environmental communities with confidence . there are a variety of different reference nucleotide and amino acid databases available for annotating gene or protein sequences ( table s1 , supporting information ) . the m5nr database ( wilke et al . 2012 ) incorporates information from a selection of different databases ( see table s1 , supporting information ) , increasing the amount of reference data available for annotation . using a single reference database may be the best option in some cases , for example , where 16s rrna amplicons are used as a method to identify taxa , rather than other genes . whereas taxonomic nomenclature is universal , governed by international conventions , there are multiple approaches for functional classification . two popular methods include clusters of orthologous groups ( cogs ) ( tatusov , koonin and lipman 1997 ) and the kyoto encyclopedia of genes and genomes ( kegg ) ( kanehisa and goto 2000 ) . cogs comprise orthologous functions that allow for functional description of poorly characterized genomes based on protein orthologs . cog descriptions are characterized under cellular processes , information storage and processing , metabolism and poorly characterized . kegg descriptions are characterized under cellular processes , environmental information processing , genetic information processing , human diseases and metabolism . due to the differences in characterization approaches , kegg operates on a subscription basis , and mg - rast uses the latest freely available version ( updated in 2008 ) . selecting a minimum identity cut - off value for metagenome analysis is challenging because interspecific sequence identity varies among genes . too high a value will accurately identify genes with highly conserved regions , such as 16s rrna or highly conserved coding genes with little synonymous substitution , but may fail to identify genes or non - coding regions that are highly variable . conversely , a value too low will allow for highly variable genes to be identified , but may also incorrectly identify an organism / function , thus providing false community / function profiles . the optimum identity cut - off point for species identification using the 16s rrna gene is widely accepted as 97% ( stackebrandt and goebel 1994 ; rossell - mora and amann 2001 ; chun et al . 2007 ; richter and rossell - mra 2009 ; mende et al . 2013 ; vtrovsk and baldrian 2013 ) , although this value has its limitations . some species , such as certain rickettsia spp . , have a 16s rrna gene similarity greater than 97% , thus a cut - off value at this level would not differentiate between the species ( fournier et al . 2003 ) . stackebrandt and goebel ( 1994 ) suggest that a higher value may be more appropriate , but fewer sequences would be annotated due to sequencing errors and sequence mutations . typically , lower cut - off values are suitable for metagenomic studies as the multitude of genes that contain varying degrees of conservation are sequenced . the default value used by mg - rast , and used in many metagenomic studies ( e.g. tatusov , koonin and lipman 1997 ; lipson et al . 2013 ) , is 60% , as this allows for identification using less conserved genes and non - coding regions . a lower value allows shorter sequences to be annotated , although the chance of incorrectly annotating a shorter sequence is higher . a higher value will reduce this chance , but may also reduce the number of annotations overall . combining a low minimum alignment length with a strict minimum identity cut - off value allows shorter sequences to be annotated but with a high match criteria . setting maximum e - values and minimum alignment lengths allows stringency of annotations to be controlled . e - values denote the maximum probability that a sequence annotation has occurred by chance . lower maximum e - values will reduce the number of possible incorrect annotations , although this also reduces number of annotations retained for analysis . the aim of this study is to evaluate the accuracy of megan , mg - rast and one codex annotation methods while investigating how using different databases and parameters impact the annotation of metagenomes . to do this , a novel simulated metagenome was generated using the ncbi whole bacterial genome database and annotated using each pipeline and , for mg - rast , with different reference databases , minimum identity cut - off values , minimum alignment lengths and maximum e - values . using a simulated metagenome comprising known genome abundances allows the accuracy of annotation to be quantified . the simulated metagenome was also annotated using megablast , a faster variation of blast , to provide a control and so that megan , mg - rast and one codex could be compared to a standard in sequence annotation . comparing the megan , mg - rast and one codex annotations to the megablast annotations will quantify the accuracy of these programs for annotating sequences from organisms whose genomes are stored in the ncbi databases . a simulated metagenome , hereafter simmet , was created using nessm ( jia et al . 2013 ) , comprising the complete ncbi bacterial genome database ( ftp://ftp.ncbi.nlm.nih.gov/genomes/bacteria/all.fna.tar.gz , may 2013 collection , accessed on 29/04/14 ) . nessm creates synthetic metagenomes from input genomes based on user - defined parameters ( e.g. sequence count , length and abundance distributions ) that aim to simulate real sequencing data , including expected sequencing errors ( i.e. substitutions , insertions and deletions ) based on the chosen sequencing technology simulated ( see step ii : error models and sequencing coverage bias estimation in jia et al . a total of 2400 000 sequences with a read length of 450 base pairs were designated for simulation , based on 454 pyrosequencing . one strain for each of the 1505 species in the ncbi bacterial genome database was randomly selected to be included in the simulation because certain species , e.g. model organisms and human pathogens such as escherichia coli , salmonella enterica , mycobacterium tuberculosis , bacillus cereus and staphylococcus aureus , have been extensively studied and are overrepresented in the databases . the species abundance distribution used for simulation was derived from the abundance distribution of a pasture soil metagenome ( sequence count : 2378 586 , mg - rast i d : 4554767.3 ) ( see equation 1 ) . ( 1)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{upgreek } \usepackage{mathrsfs } \setlength{\oddsidemargin}{-69pt } \begin{document } } { } \begin{equation * } y = - 2490\ln \left ( x \right ) + \ 19748 \end{equation*}\end{document}where x is the randomly selected species rank . the sequences were processed with sickle ( joshi and fass 2011 ) to trim low - quality ends , with the average threshold phred score set at 20 ( a base call error rate of 1% ) . the simmet metagenome file was annotated with megablast ( available from : http://blast.ncbi.nlm.nih.gov/blast.cgi?page_type = blastdocs&doc_type = download ) as a control , using a reference database of the genomes used to create simmet . the ncbi nucleotide database ( updated 17/11/2014 ) ( ftp://ftp.ncbi.nlm.nih.gov/blast/db/ ) was also used to assess the annotation performance of megablast . the maximum e - value selected was 1-e and the minimum alignment length , 15 bases . megablast annotations using the simmet database will be referred to as control and those using the ncbi nucleotide database will be referred to as megablast. the blast results were uploaded to megan ( version 5.2.3 ) and analyzed using the same parameters used in the blast . the databases investigated within mg - rast were genbank , greengenes , rdp , refseq , seed , swissprot and trembl . the m5nr and m5rna databases were excluded from individual sequence analysis , as individual sequence annotations were not available for download from mg - rast for these databases . for both the megablast and mg - rast annotations , which use a minimum sequence alignment match to annotated sequences , the minimum identity cut - off values tested were as follows : 40% , 50% , 60% , 70% , 80% , 90% , 95% and 97% . the minimum alignment lengths tested were as follows : 10 , 15 , 20 , 25 , 30 , 25 , 40 , 45 , 50 , 55 and 60 bp . the maximum e - values tested were as follows : 1-e , 1-e , 1-e and 1-e . aside from testing , default parameters were used : 60% , 15 bp and 1-e , respectively , for minimum identify cut - off , minimum alignment length and maximum e - value . the sequence ids and annotations were extracted from the megablast results ( https://github.com/sandyjmacdonald/blast_parser ) , and full taxonomic lineages were generated for each sequence using the ncbi taxonomy database ( available from : ftp://ftp.ncbi.nlm.nih.gov/pub/taxonomy/ ; ncbi database version generated 13/05/2013 ) . species level was excluded from analysis due to the high variation in annotated species nomenclature and the accepted caveats associated with microbial species classification ( gevers et al . 2005 ; achtman and wagner 2008 ) , e.g. horizontal gene transfer ( gogarten and townsend 2005 ; bapteste and boucher 2009 ) . discrepancies identified between databases for organism names were corrected for , such as ncbi using the old name chloroflexia ( as of 17/11/14 ) and mg - rast using the new name chloroflexi for the same class . unidentified and those that were annotated but were either ambiguously annotated or not annotated at all taxonomic levels had the corresponding levels in the lineage replaced with unclassified. for megan and one codex , ncbi taxa ids were used to generate the lineages . the taxonomic lineage for each annotated sequence was compared to the lineage for the corresponding source sequence in simmet to determine the annotation sensitivity and precision at each taxonomic level . the effect that minimum identity cut - off values , minimum alignment lengths and maximum e - values had on annotation sensitivity and precision were established using megablast and mg - rast . the correlations between the relative abundances for each taxon in simmet and in the annotations were calculated using pearson 's product moment correlation coefficient . the natural logarithms of the relative abundance values were calculated for plotting , as the original distributions would not visually convey the variations in low abundance taxa . the taxa richness values for each taxonomic level were calculated . unlike investigating taxa , correct functional annotations can not be ascertained with 100% confidence . to investigate functional annotation performance , protein sequences associated with the sequences in simmet were extracted from genbank records and annotated using the kegg automatic annotation server ( kaas ) ( moriya et al . both are web - based functional annotation tools independent of those investigated in this study . they did not contain sequencing errors and thus provided the best possible indication of the functional annotation accuracy , although the caveats associated with sequence annotation ( e.g. possibly incorrectly assigning a function ) are present . kegg orthology and cog ids were extracted from the kass and webmga results , respectively , for each sequence annotated and compared with the ids assigned by mg - rast . the parameters set for the taxonomic investigation were used , and the minimum identity cut - off values investigated were as follows : 40% , 50% , 60% , 70% , 80% , 90% and 95% . the minimum alignment lengths tested were as follows : 10 , 15 , 20 , 25 , 30 , 25 , 40 , 45 , 50 , 55 and 60 base pairs . the maximum e - values tested were as follows : 1-e , 1-e , 1-e and 1-e . nessm produced 2399 077 sequences ( length range : 195459 bp , median length 377 bp ) . s1 , supporting information ) and 98.7% of sequences are between 300 and 400 base pairs long ( fig . s2 , supporting information ) . of the 2399 077 sequences , kass annotated 1 341 362 ( 55.9% ) sequences and webmga annotated 1945 674 sequences ( 81.1% ) . more stringent parameter values resulted in fewer sequence annotations but had a greater precision ; lower values resulted in more annotations being made , but these comprised increases in both correct and incorrect annotations . for example , with cut - off values of 95% and 40% , mg - rast refseq annotated 40.2% and 90.3% sequences , respectively , with incorrect annotation rates of 2.9% and 34.5% at the genus level . this was observed for all parameters tested and for both taxonomic and functional annotations ( figs 16 ) . as the taxonomic level moved up the taxonomic hierarchy , more sequences were correctly annotated ( e.g. 0.5% and 10.2% for mg - rast refseq with cut - off values of 95% and 40% , respectively , at the class level ) . the effect of changing minimum identity cut - off value on the number of sequences correctly and incorrectly annotated across the taxonomic levels . the effect of changing minimum identity cut - off value on the number of sequences correctly and incorrectly annotated for functions . the effect of changing minimum alignment length on the number of sequences correctly and incorrectly annotated across the taxonomic levels . the effect of changing minimum alignment length on the number of sequences correctly and incorrectly annotated for functions . the effect of changing maximum e - value on the number of sequences correctly and incorrectly annotated across the taxonomic levels . the effect of changing maximum e - value value on the number of sequences correctly and incorrectly annotated for functions . the correlation coefficients between the taxa relative abundances in simmet and in the annotations decreased as parameter stringency increased ( fig . 7 , associated scatter plots in figs s3s5 , supporting information ) . most databases achieved maximum correlations with a minimum identity cut - off value of 50% , a minimum alignment length of 30 bp and a maximum e - value of 1-e . greater decreases in correlation coefficients occurred with a minimum identity cut - off value above 70% and a minimum alignment length greater than 40 bp . the pearson 's product - moment correlation coefficients for the correlations between the genus relative abundances from simmet and those from various annotation methods using different ( a ) minimum identity cut - off values , ( b ) minimum alignment lengths and ( c ) maximum e - values . this produced the greatest number of correct annotations ( 99.4% ) ( table 1 , fig . 8 , table s2 , supporting information , for all taxonomic levels ) . one codex annotated all of the sequences , but incorrectly annotated more sequences ( 5.8% ) than megan ( 2.9% ) , megablast ( 2.5% ) and the control ( 0.5% ) . megablast , megan and one codex correctly annotated 97.3% , 95.7% and 94.2% sequences respectively , significantly more than the next most successful methods : mg - rast refseq ( 55.9% ) , mg - rast trembl ( 54.9% ) and mg - rast genbank ( 52.7% ) . mg - rast rdp and mg - rast greengenes , both rrna databases , annotated less than 1% of the sequences . this is consistent with the expected frequency of rrna genes within bacterial genomes ( vtrovsk and baldrian 2013 ) . as the taxonomic level increases , precision increases and becomes more similar across the different databases . the annotation sensitivity and number of sequences correctly annotated from a variety of methods and databases across the taxonomic levels investigated . the simmet taxonomic annotation statistics for each method and database at the genus level using default parameters . mg - rast kegg annotated 63.3% of the sequences and had a precision of 71.7% , with 45.4% of sequences correctly assigned a function and 17.9% incorrectly assigned a function . mg - rast cog annotated 50.5% of the sequences and had a precision of 91.1% , resulting in 46.0% of sequence being correctly assigned a function and 4.5% being incorrectly assigned a function ( tables s3s5 , supporting information ) . the portions of sequences correctly annotated by both methods were 81.5% for mg - rast kegg and 55.4% for mg - rast cog . megablast had the greatest genus - level correlation with simmet after the control ( r = 0.95 ) , while mg - rast seed had the weakest ( r = 0.49 ) . megan and one codex had genus - level correlations of r = 0.90 and r = 0.93 , respectively . the greatest correlation achieved , aside from the control , was by megablast at the phylum level ( r > 0.99 ) ( fig . the pearson 's product - moment correlation coefficients for the correlations between the relative abundances from simmet and those from the annotation methods . mg - rast m5nr and mg - rast refseq generated 87 and 56 false positive class identifications , respectively ( table 2 ) . megan had only two false positive class identifications ( unidentified and insecta ) and one false negative identification ( solibacteres ) . one codex also had a low abundance of false positive class identifications ( eight ) and no false negative class identifications . classes with many false positive identifications include eukaryotes , particularly fungi and bacteria such as spartobacteria . the greatest fold differences for classes can be found in table s6 ( supporting information ) . false positive and negative class abundances . the false positive and negative classes from the mg - rast m5nr , refseq , one codex and megan annotations . six of the annotation methods underestimated the genus richness and six overestimated it ( table 3 , fig . the next closest estimate was achieved by megan ( 97.7% ) , followed by mg - rast swissprot ( 95.5% ) , mg - rast m5rna ( 95.2% ) , megablast ( 110.2% ) and mg - rast refseq ( 118.2% ) . mg - rast m5nr produced the most incorrect richness value at 1244 genera ( 180.8% ) . the methods were inconsistent in response to the taxonomic level . with increasing taxonomic level , some estimates increased in accuracy while others decreased ( fig . 10 , table s7 , supporting information ) . excluding the control and the domain level , where the number of taxa is low , megan achieved the most accurate richness value ( 101.2% ) at the family level . mg - rast m5nr achieved the most inaccurate richness value ( 253.3% ) at the order level . megablast and one codex achieved accurate results relative to other methods , but they still overstated taxa richness at every taxonomic level . the differences between annotated richness values and the actual richness value ( dashed line ) for each taxonomic level . the genus richness estimates and the differences from simmet for each annotation method . due to the low numbers richness values at all taxonomic levels can be found in table s7 ( supporting information ) . in the study , we evaluated the performances of megan , mg - rast , one codex and megablast by determining their sequence annotation accuracies . all common taxonomic levels above species are studied , building on the work by lindgreen , adair and gardner ( 2016 ) who study several tools at the genus and phylum levels . by studying a range of taxonomic levels , we provide a guideline for researchers to establish the annotation accuracy costs of investigating lower taxonomic levels , allowing them to optimize their investigations depending on their requirements for taxonomic resolution . mg - rast and megablast use a selection of parameters to determine the stringency of matching a sequence with a reference sequence in a database . less stringent parameters ( i.e. lower minimum identity cut - off values , lower minimum alignment lengths and higher maximum e - values ) annotate more sequences , but more incorrect annotations are made , thus producing an incorrect community profile . more stringent parameters reduce the number of incorrect annotations , but many fewer annotations are made , resulting in much of the data being rejected . decreases in sensitivity generally occur from minimum identify cut - off values above 60% , a minimum alignment lengths greater than 30 bp or a maximum e - value below 1-e ; therefore , the default values used by mg - rast maximize sensitivity . according to carr and borenstein ( 2014 ) , the impact of parameters such as e - value will vary depending on read length , something that should be considered in future evaluations as newer sequencing technologies produce longer reads ( e.g. nanopore sequencing ; branton et al . the sensitivities and the number of sequences correctly annotated are relatively low for mg - rast at the genus and family levels . at the order level , the values are higher , suggesting that this would be the optimum taxonomic level to study , which maximizes the amount of data used without producing too many incorrect annotations . ultimately , there is a trade - off between taxonomic resolution and annotation accuracy , and this must be considered when determining methods for metagenomic studies . a marginal number of sequences were not annotated by the control and an even smaller number were incorrectly annotated . we can therefore conclude that 0.5% of intersample difference at the genus level may be attributed to sequencing error , an important consideration when interpreting data obtained from environmental samples using these methods . this is supported by hoff ( 2009 ) and carr and borenstein ( 2014 ) , who found that increasing error rates decrease gene prediction accuracy . as the error rates of next - generation sequencing technologies improve , this effect will reduce . this is likely to be due to a combination of the kmer - based annotation method that it uses and that the simulated metagenome was created using the ncbi genome database , the primary reference source for one codex . other than the control , megablast correctly annotated the most sequences at the genus level ( 97.3% ) , although the sensitivity of this method was 0.2% less than one codex . megan had the second highest precision , annotating 98.6% of sequences , with 95.7% correct annotations . this suggests that megablast is the most reliable method for annotating sequences , and indicates that it is more conservative than one codex when assigning a sequence hit but also less likely to misidentify a sequence . megan 's performance was similar to megablast , which is expected as megan processed the megablast output . mg - rast refseq had the fifth greatest annotation sensitivity and the greatest of the mg - rast annotations ( excluding mg - rast m5nr , for which sequence - specific annotation data were unavailable ) , although it also achieved the greatest number of misidentifications . at the genus level , 33.7% of sequences were misidentified and 55.9% were correctly identified , leaving the remainder unassigned despite the fact that all taxa in simmet are fully sequenced . this would suggest that investigating metagenomes at the genus level would be unreliable , generating many false positives and implying an incorrect community structure and composition . this supports garcia - etxebarria , garcia - garcer and calafell ( 2014 ) , who found that more annotations are made at higher taxonomic levels and that discrepancies between known frequencies and annotations increase at lower taxonomic levels , and lindgreen , adair and gardner ( 2016 ) , who report decreases in community annotation accuracy at the genus level compared to phylum . at the class level , the proportion of incorrect annotations is reduced to fewer than 10% for mg - rast refseq , with 80% being annotated correctly . while taxonomic resolution is reduced , it ensures that the confidence in the annotations remains high . mg - rast kegg correctly annotated a similar number of sequences to mg - rast cog , but incorrectly annotated many more . kegg offers a more descriptive annotation as it comprises specific gene and pathway annotations , whereas cog provides descriptions based on orthologous sequences . however , the specificity of kegg classifications may be the cause of the incorrect annotations as there are more annotations to be selected from and there may be more closely related functions , increasing the chance of misidentification . because kegg is now subscription based , and mg - rast uses the last free version ( 2008 ) , it will not contain information added after that date . our results are in line with those produced by lindgreen , adair and gardner ( 2016 ) , who also conclude that mg - rast 's functional annotation was accurate . the control , one codex , megablast and megan achieved the greatest correlation coefficients between simmet and annotation abundances at the genus level , all above 0.9 . for all mg - rast annotations , the greatest correlation of all abundances was achieved at the order level by the m5nr database , closely followed by trembl and refseq . these correlations inform us about community - wide analyses , but they are not as sequence sensitivity and precision as correlating abundances values may occur from coincidental incorrect annotations . mg - rast overannotated many more classes than megan and one codex , for which the most abundant feature was the unidentified group . this supports the sensitivity and precision data in suggesting that one codex is more likely to categorize unknown sequences as unidentified , rather than incorrectly identifying them . the genus richness estimated by mg - rast m5nr was 81.0% greater that simmet 's actual richness , the highest overstatement , while megan achieved the most accurate genus richness value ( 2.3% lower ) after the control ( 100.2% ) . this overstatement could be due to the greater number of sequences present in mg - rast m5nr . mg - rast m5rna produced a relatively accurate estimate of genus richness ( 95.2% ) ; as m5rna is a 16s rrna database , it is unlikely to annotate non-16s rrna sequences , reducing the number of incorrect identifications . however , the taxa abundance correlations show that mg - rast m5rna achieved the second lowest correlation with simmet at the genus level , and the lowest at all other taxonomic levels . mg - rast refseq generated the fifth most accurate richness value , greater than one codex , although not as accurate as megablast and megan . combined with its high abundance correlation with simmet , this suggests that mg - rast refseq provides a relatively accurate representation of both the richness of a community and the abundance of organisms present . megan and one codex achieve more accurate taxa richness values and taxa abundance correlations than mg - rast refseq at the family level and above , suggesting they would be a viable alternative to mg - rast refseq . one limitation with evaluating annotations using organism nomenclature , rather than taxon ids ( which were unavailable for mg - rast sequence - specific annotation data ) , is the lack of taxonomic metadata curation in some databases . some genomes in the ncbi database are stored with the abbreviated species name rather than complete name , thus amycolatopsis mediterranei would not automatically be identified as an amycolatopsis species . for example , the class chloroflexia has been renamed to chloroflexi , and is called this by mg - rast . however , ncbi is using the old name chloroflexia ( as of 17/11/14 ) , thus sequences identified as chloroflexi would not be correctly matched in simmet . these issues were corrected for during data processing ; however , there may be other cases of disparities in the plethora of organisms present in the analysis . a solution to this would be to use the taxon ids instead ; however , these were not available for sequence - specific annotations downloaded from mg - rast . in conclusion , we found that one codex , megablast and megan are suitable methods for annotating dna sequences that are located in the reference databases that they use for annotation , with one codex offering fast , web - based analyses and megan providing a user - friendly graphical user interface to analyze blast results . results appear to vary significantly depending on the program and parameters used , a conclusion also drawn by lindgreen , adair and gardner ( 2016 ) . while mg - rast appears to have a greater rate of incorrect assignments , this is reduced when investigating higher taxonomic levels ( e.g. with refseq : over 33% at the genus level compared to less than 15% and 10% at the order and class levels ) . the correlations between the annotated taxa abundances are greatest for mg - rast at the order level , using m5nr , trembl or refseq . in many of the tests , mg - rast m5nr proved to be a reliable database , but the diversity indices suggest that it is less reliable than mg - rast refseq ; at the class , order and family levels , mg - rast m5nr estimates more the double the actual richness values . therefore , we hypothesize that mg - rast m5nr would generate more false positive sequence annotations than mg - rast refseq . ( 2007 ) , who evaluated different metagenomic processing methods using simulated metagenome developed from 113 isolated genomes , and by pignatelli and moya ( 2011 ) , who used simulated data to study the performances of de novo short - read assembly programs . it should be noted that the performances of the methods discussed in this study are likely to differ from the reported results when annotating environmental sequence data ; a greater number of sequences are likely to be unidentified due to the multitude of uncultured microorganisms ( streit and schmitz 2004 ) and non - sequenced microbial genomes ( tringe et al . 2005 ) that are currently absent from the ncbi whole bacterial genome database . while this research focused on a selection of annotation methods , the overall conclusions drawn should be considered for any pipeline . in this study , we highlight and quantify the annotation errors for a selection of parameters and databases . we show that analysis pipelines are not equivalent and certain parameters can significantly reduce the confidence in results . these findings should be used as a guideline when determining methods for annotating metagenomic sequences and considered when interpreting metagenomic results . ultimately , the most appropriate balance between taxonomic resolution , annotation sensitivity and annotation precision needs to be identified for each study conducted .

the advent of next - generation sequencing has allowed huge amounts of dna sequence data to be produced , advancing the capabilities of microbial ecosystem studies . the current challenge is to identify from which microorganisms and genes the dna originated . several tools and databases are available for annotating dna sequences . the tools , databases and parameters used can have a significant impact on the results : nave choice of these factors can result in a false representation of community composition and function . we use a simulated metagenome to show how different parameters affect annotation accuracy by evaluating the sequence annotation performances of megan , mg - rast , one codex and megablast . this simulated metagenome allowed the recovery of known organism and function abundances to be quantitatively evaluated , which is not possible for environmental metagenomes . the performance of each program and database varied , e.g. one codex correctly annotated many sequences at the genus level , whereas mg - rast refseq produced many false positive annotations . this effect decreased as the taxonomic level investigated increased . selecting more stringent parameters decreases the annotation sensitivity , but increases precision . ultimately , there is a trade - off between taxonomic resolution and annotation accuracy . these results should be considered when annotating metagenomes and interpreting results from previous studies .

INTRODUCTION METHODOLOGY RESULTS DISCUSSION SUPPLEMENTARY DATA FUNDING

the advent of next - generation sequencing and metagenomics has resulted in increasing numbers of ever - larger datasets describing the community structure and function of a variety of different environments , from the human gut ( arumugam et al . next - generation sequencing technologies have greatly reduced sequencing costs and speed , and researchers can now affordably study whole microbial communities and functions . amplicon sequencing does not require the dna coverage that metagenomic studies require and can accurately identify which species are present in a sample ( woese and fox 1977 ; lane et al . 1985 ; hugenholtz 2002 ) , but it does not provide the depth of information , such as gene function , that full metagenome sequencing and annotation provides . illumina 's hiseq platform , for example , can affordably sequence the most complex of microbial communities , and the challenge now is to interpret the data produced . ( 2014 ) provide an extensive directory of tools available for different tasks involved in a metagenomic project pipeline , related to a range of omics studies . 2007 ) is a popular graphical user interface program for analyzing and visualizing blast results to study the taxonomy of microbial communities . with an intuitive web interface and a variety of analytical tools to choose from , mg - rast ( meyer et al . mg - rast allows users to upload raw sequence files that are processed through quality filters and annotated using a selection of user - defined parameters , such as reference databases , minimum identity cut - off values , maximum e - values or expect - values , and minimum alignment lengths . with pauda ( huson and xie 2014 ) uses a similar approach and claims to be 10 000-fold faster than blastx , although with a significant reduction in sensitivity . one codex is a web - based program that uses a different technique to blast and mg - rast to classify sequences ( https://onecodex.com/ ) . blast and mg - rast classify sequences by matching them with the most similar sequences in a database . unlike mg - rast , one codex does not annotate genes for function . the choice of database , minimum identity cut - off value ( i.e. sequence match stringency ) , minimum alignment length cut - off value and minimum e - value limit ( the probability a match has occurred by chance ) all influence sequence annotation accuracy , which , in turn , affect the reproducibility and interpretation of the data . an inherent issue with metagenomic studies is that establishing the accuracy of sequence annotation for environmental samples is practically impossible , given that the quantities of organisms and genes are unknown . there are a variety of different reference nucleotide and amino acid databases available for annotating gene or protein sequences ( table s1 , supporting information ) . due to the differences in characterization approaches , kegg operates on a subscription basis , and mg - rast uses the latest freely available version ( updated in 2008 ) . too high a value will accurately identify genes with highly conserved regions , such as 16s rrna or highly conserved coding genes with little synonymous substitution , but may fail to identify genes or non - coding regions that are highly variable . conversely , a value too low will allow for highly variable genes to be identified , but may also incorrectly identify an organism / function , thus providing false community / function profiles . typically , lower cut - off values are suitable for metagenomic studies as the multitude of genes that contain varying degrees of conservation are sequenced . the default value used by mg - rast , and used in many metagenomic studies ( e.g. combining a low minimum alignment length with a strict minimum identity cut - off value allows shorter sequences to be annotated but with a high match criteria . the aim of this study is to evaluate the accuracy of megan , mg - rast and one codex annotation methods while investigating how using different databases and parameters impact the annotation of metagenomes . to do this , a novel simulated metagenome was generated using the ncbi whole bacterial genome database and annotated using each pipeline and , for mg - rast , with different reference databases , minimum identity cut - off values , minimum alignment lengths and maximum e - values . using a simulated metagenome comprising known genome abundances allows the accuracy of annotation to be quantified . the simulated metagenome was also annotated using megablast , a faster variation of blast , to provide a control and so that megan , mg - rast and one codex could be compared to a standard in sequence annotation . comparing the megan , mg - rast and one codex annotations to the megablast annotations will quantify the accuracy of these programs for annotating sequences from organisms whose genomes are stored in the ncbi databases . a simulated metagenome , hereafter simmet , was created using nessm ( jia et al . one strain for each of the 1505 species in the ncbi bacterial genome database was randomly selected to be included in the simulation because certain species , e.g. the species abundance distribution used for simulation was derived from the abundance distribution of a pasture soil metagenome ( sequence count : 2378 586 , mg - rast i d : 4554767.3 ) ( see equation 1 ) . the ncbi nucleotide database ( updated 17/11/2014 ) ( ftp://ftp.ncbi.nlm.nih.gov/blast/db/ ) was also used to assess the annotation performance of megablast . the m5nr and m5rna databases were excluded from individual sequence analysis , as individual sequence annotations were not available for download from mg - rast for these databases . for both the megablast and mg - rast annotations , which use a minimum sequence alignment match to annotated sequences , the minimum identity cut - off values tested were as follows : 40% , 50% , 60% , 70% , 80% , 90% , 95% and 97% . 2005 ; achtman and wagner 2008 ) , e.g. discrepancies identified between databases for organism names were corrected for , such as ncbi using the old name chloroflexia ( as of 17/11/14 ) and mg - rast using the new name chloroflexi for the same class . the taxonomic lineage for each annotated sequence was compared to the lineage for the corresponding source sequence in simmet to determine the annotation sensitivity and precision at each taxonomic level . the effect that minimum identity cut - off values , minimum alignment lengths and maximum e - values had on annotation sensitivity and precision were established using megablast and mg - rast . they did not contain sequencing errors and thus provided the best possible indication of the functional annotation accuracy , although the caveats associated with sequence annotation ( e.g. kegg orthology and cog ids were extracted from the kass and webmga results , respectively , for each sequence annotated and compared with the ids assigned by mg - rast . more stringent parameter values resulted in fewer sequence annotations but had a greater precision ; lower values resulted in more annotations being made , but these comprised increases in both correct and incorrect annotations . for example , with cut - off values of 95% and 40% , mg - rast refseq annotated 40.2% and 90.3% sequences , respectively , with incorrect annotation rates of 2.9% and 34.5% at the genus level . as the taxonomic level moved up the taxonomic hierarchy , more sequences were correctly annotated ( e.g. 0.5% and 10.2% for mg - rast refseq with cut - off values of 95% and 40% , respectively , at the class level ) . the effect of changing minimum identity cut - off value on the number of sequences correctly and incorrectly annotated across the taxonomic levels . the effect of changing minimum identity cut - off value on the number of sequences correctly and incorrectly annotated for functions . the effect of changing minimum alignment length on the number of sequences correctly and incorrectly annotated across the taxonomic levels . the effect of changing maximum e - value on the number of sequences correctly and incorrectly annotated across the taxonomic levels . one codex annotated all of the sequences , but incorrectly annotated more sequences ( 5.8% ) than megan ( 2.9% ) , megablast ( 2.5% ) and the control ( 0.5% ) . megablast , megan and one codex correctly annotated 97.3% , 95.7% and 94.2% sequences respectively , significantly more than the next most successful methods : mg - rast refseq ( 55.9% ) , mg - rast trembl ( 54.9% ) and mg - rast genbank ( 52.7% ) . as the taxonomic level increases , precision increases and becomes more similar across the different databases . the annotation sensitivity and number of sequences correctly annotated from a variety of methods and databases across the taxonomic levels investigated . the simmet taxonomic annotation statistics for each method and database at the genus level using default parameters . mg - rast kegg annotated 63.3% of the sequences and had a precision of 71.7% , with 45.4% of sequences correctly assigned a function and 17.9% incorrectly assigned a function . the portions of sequences correctly annotated by both methods were 81.5% for mg - rast kegg and 55.4% for mg - rast cog . megablast had the greatest genus - level correlation with simmet after the control ( r = 0.95 ) , while mg - rast seed had the weakest ( r = 0.49 ) . mg - rast m5nr and mg - rast refseq generated 87 and 56 false positive class identifications , respectively ( table 2 ) . the false positive and negative classes from the mg - rast m5nr , refseq , one codex and megan annotations . six of the annotation methods underestimated the genus richness and six overestimated it ( table 3 , fig . the next closest estimate was achieved by megan ( 97.7% ) , followed by mg - rast swissprot ( 95.5% ) , mg - rast m5rna ( 95.2% ) , megablast ( 110.2% ) and mg - rast refseq ( 118.2% ) . the methods were inconsistent in response to the taxonomic level . with increasing taxonomic level , some estimates increased in accuracy while others decreased ( fig . excluding the control and the domain level , where the number of taxa is low , megan achieved the most accurate richness value ( 101.2% ) at the family level . mg - rast m5nr achieved the most inaccurate richness value ( 253.3% ) at the order level . megablast and one codex achieved accurate results relative to other methods , but they still overstated taxa richness at every taxonomic level . in the study , we evaluated the performances of megan , mg - rast , one codex and megablast by determining their sequence annotation accuracies . all common taxonomic levels above species are studied , building on the work by lindgreen , adair and gardner ( 2016 ) who study several tools at the genus and phylum levels . by studying a range of taxonomic levels , we provide a guideline for researchers to establish the annotation accuracy costs of investigating lower taxonomic levels , allowing them to optimize their investigations depending on their requirements for taxonomic resolution . mg - rast and megablast use a selection of parameters to determine the stringency of matching a sequence with a reference sequence in a database . lower minimum identity cut - off values , lower minimum alignment lengths and higher maximum e - values ) annotate more sequences , but more incorrect annotations are made , thus producing an incorrect community profile . more stringent parameters reduce the number of incorrect annotations , but many fewer annotations are made , resulting in much of the data being rejected . decreases in sensitivity generally occur from minimum identify cut - off values above 60% , a minimum alignment lengths greater than 30 bp or a maximum e - value below 1-e ; therefore , the default values used by mg - rast maximize sensitivity . according to carr and borenstein ( 2014 ) , the impact of parameters such as e - value will vary depending on read length , something that should be considered in future evaluations as newer sequencing technologies produce longer reads ( e.g. the sensitivities and the number of sequences correctly annotated are relatively low for mg - rast at the genus and family levels . at the order level , the values are higher , suggesting that this would be the optimum taxonomic level to study , which maximizes the amount of data used without producing too many incorrect annotations . ultimately , there is a trade - off between taxonomic resolution and annotation accuracy , and this must be considered when determining methods for metagenomic studies . we can therefore conclude that 0.5% of intersample difference at the genus level may be attributed to sequencing error , an important consideration when interpreting data obtained from environmental samples using these methods . as the error rates of next - generation sequencing technologies improve , this effect will reduce . this is likely to be due to a combination of the kmer - based annotation method that it uses and that the simulated metagenome was created using the ncbi genome database , the primary reference source for one codex . other than the control , megablast correctly annotated the most sequences at the genus level ( 97.3% ) , although the sensitivity of this method was 0.2% less than one codex . mg - rast refseq had the fifth greatest annotation sensitivity and the greatest of the mg - rast annotations ( excluding mg - rast m5nr , for which sequence - specific annotation data were unavailable ) , although it also achieved the greatest number of misidentifications . at the genus level , 33.7% of sequences were misidentified and 55.9% were correctly identified , leaving the remainder unassigned despite the fact that all taxa in simmet are fully sequenced . this would suggest that investigating metagenomes at the genus level would be unreliable , generating many false positives and implying an incorrect community structure and composition . this supports garcia - etxebarria , garcia - garcer and calafell ( 2014 ) , who found that more annotations are made at higher taxonomic levels and that discrepancies between known frequencies and annotations increase at lower taxonomic levels , and lindgreen , adair and gardner ( 2016 ) , who report decreases in community annotation accuracy at the genus level compared to phylum . at the class level , the proportion of incorrect annotations is reduced to fewer than 10% for mg - rast refseq , with 80% being annotated correctly . mg - rast kegg correctly annotated a similar number of sequences to mg - rast cog , but incorrectly annotated many more . our results are in line with those produced by lindgreen , adair and gardner ( 2016 ) , who also conclude that mg - rast 's functional annotation was accurate . the control , one codex , megablast and megan achieved the greatest correlation coefficients between simmet and annotation abundances at the genus level , all above 0.9 . for all mg - rast annotations , the greatest correlation of all abundances was achieved at the order level by the m5nr database , closely followed by trembl and refseq . mg - rast overannotated many more classes than megan and one codex , for which the most abundant feature was the unidentified group . the genus richness estimated by mg - rast m5nr was 81.0% greater that simmet 's actual richness , the highest overstatement , while megan achieved the most accurate genus richness value ( 2.3% lower ) after the control ( 100.2% ) . this overstatement could be due to the greater number of sequences present in mg - rast m5nr . mg - rast m5rna produced a relatively accurate estimate of genus richness ( 95.2% ) ; as m5rna is a 16s rrna database , it is unlikely to annotate non-16s rrna sequences , reducing the number of incorrect identifications . however , the taxa abundance correlations show that mg - rast m5rna achieved the second lowest correlation with simmet at the genus level , and the lowest at all other taxonomic levels . mg - rast refseq generated the fifth most accurate richness value , greater than one codex , although not as accurate as megablast and megan . combined with its high abundance correlation with simmet , this suggests that mg - rast refseq provides a relatively accurate representation of both the richness of a community and the abundance of organisms present . megan and one codex achieve more accurate taxa richness values and taxa abundance correlations than mg - rast refseq at the family level and above , suggesting they would be a viable alternative to mg - rast refseq . one limitation with evaluating annotations using organism nomenclature , rather than taxon ids ( which were unavailable for mg - rast sequence - specific annotation data ) , is the lack of taxonomic metadata curation in some databases . for example , the class chloroflexia has been renamed to chloroflexi , and is called this by mg - rast . a solution to this would be to use the taxon ids instead ; however , these were not available for sequence - specific annotations downloaded from mg - rast . in conclusion , we found that one codex , megablast and megan are suitable methods for annotating dna sequences that are located in the reference databases that they use for annotation , with one codex offering fast , web - based analyses and megan providing a user - friendly graphical user interface to analyze blast results . results appear to vary significantly depending on the program and parameters used , a conclusion also drawn by lindgreen , adair and gardner ( 2016 ) . while mg - rast appears to have a greater rate of incorrect assignments , this is reduced when investigating higher taxonomic levels ( e.g. with refseq : over 33% at the genus level compared to less than 15% and 10% at the order and class levels ) . the correlations between the annotated taxa abundances are greatest for mg - rast at the order level , using m5nr , trembl or refseq . in many of the tests , mg - rast m5nr proved to be a reliable database , but the diversity indices suggest that it is less reliable than mg - rast refseq ; at the class , order and family levels , mg - rast m5nr estimates more the double the actual richness values . therefore , we hypothesize that mg - rast m5nr would generate more false positive sequence annotations than mg - rast refseq . ( 2007 ) , who evaluated different metagenomic processing methods using simulated metagenome developed from 113 isolated genomes , and by pignatelli and moya ( 2011 ) , who used simulated data to study the performances of de novo short - read assembly programs . it should be noted that the performances of the methods discussed in this study are likely to differ from the reported results when annotating environmental sequence data ; a greater number of sequences are likely to be unidentified due to the multitude of uncultured microorganisms ( streit and schmitz 2004 ) and non - sequenced microbial genomes ( tringe et al . in this study , we highlight and quantify the annotation errors for a selection of parameters and databases . these findings should be used as a guideline when determining methods for annotating metagenomic sequences and considered when interpreting metagenomic results . ultimately , the most appropriate balance between taxonomic resolution , annotation sensitivity and annotation precision needs to be identified for each study conducted .

congestive heart failure is a leading cause of morbidity and mortality in developed countries , with an estimated prevalence of 1% to 2% in the general population , reaching 7% to 8% in individuals aged > 75 years.1 elevated blood pressure appears to play an important role in the pathogenesis of both left ventricular ( lv ) systolic and diastolic dysfunction , at least partly by increasing cardiac afterload.2 this has led to the therapeutic concept of reducing cardiac afterload , which has proven highly beneficial in heart failure with reduced ejection fraction but not in heart failure with a preserved ejection fraction.3 thus , broadening the understanding of the role of central hemodynamics in the pathogenesis of systolic and diastolic lv dysfunction is critical to inform the development of novel therapeutic approaches . blood pressure is routinely measured at the brachial artery , whereas the actual cardiac pressure load is determined by the hemodynamics in the proximal aorta . this is a relevant distinction , as central pulse pressure may differ from brachial pulse pressure , particularly in younger adults , due to agerelated differences in central pressure augmentation . the extent to which central pressure augmentation is explained by peripheral wave reflection or by the windkessel function of the proximal aorta is the subject of ongoing debate.4 , 5 limited data suggest that central pressure may be more closely related to cardiovascular disease than peripheral blood pressure.6 furthermore , variable relations between aortic stiffness ( which increases early systolic load on the heart ) and wave reflection ( which increases late systolic load ) may have differing implications for systolic and diastolic lv structure and function.7 in contrast , the steady component of blood pressure , ie , the mean arterial pressure , is fairly constant throughout large arteries.8 in this context , the relations of central hemodynamics with cardiac structure and function have not been fully delineated . we hypothesized that the pulsatile component ( and its determinants aortic stiffness and pressure augmentation ) and the steady component of central blood pressure may have different relations with lv structure and function . thus , we investigated the crosssectional relations of central hemodynamics and aortic stiffness to echocardiographic measures of lv structure and systolic and diastolic function in a large communitybased sample . individuals were derived from the framingham offspring and the framingham third generation cohorts , which have been described.9 , 10 the framingham offspring cohort was recruited in 19711974 and includes individuals who are children of the framingham original cohort or the children 's spouses . the framingham third generation cohort , recruited in 20022005 , comprises children of the framingham offspring cohort . participants of both cohorts are evaluated approximately every 4 to 8 years in our study clinic during a visit that includes a medical history , physical examination , and phlebotomy . the present investigation is based on examination cycle 8 of the framingham offspring cohort ( 20052008 ) and examination cycle 1 of the third generation cohort ( 20022005 ) . of 3021 participants who attended offspring examination cycle 8 and 4095 participants recruited into the third generation cohort , we excluded 252 individuals with atrial fibrillation , 703 for missing echocardiographic measurements , 334 for missing tonometry , and 28 for missing covariates ; that left 5799 ( 2184 offspring , 3615 third generation ) individuals for this investigation . excluded individuals were generally older and had a higher morbidity ( higher prevalence of diabetes , hypertension , and prevalent cardiovascular disease ) than those included in the analyses . the study protocols were approved by the boston university medical center institutional review board , and participants signed informed consent . supine brachial systolic and diastolic blood pressures were obtained using an auscultatory device.11 arterial tonometry measurements were performed as previously described.11 , 12 , 13 briefly , arterial tonometry ( using a standard applanation tonometry device ) with simultaneous ecg was performed on the brachial , femoral , and carotid arteries . transit distances were assessed by body surface measurements from the suprasternal notch to the pulserecording site . mean arterial pressure ( map ) was derived from integration of the brachial waveform calibrated with bp at the time of tonometry . direct measurement of carotid pressure , as compared to transfer function based estimates , is associated with a smaller difference between central and peripheral pulse pressure.14 details of signal analyses and data processing have been published elsewhere.11 , 12 , 13 we primarily assessed 4 measures of arterial stiffness and central hemodynamics : ( 1 ) carotidfemoral pulse wave velocity ( cfpwv ) , the current reference standard for aortic stiffness , ( 2 ) central pulse pressure , ie , the blood pressure amplitude in the proximal aorta , ( 3 ) augmentation index , ie , the fraction of central pulse pressure attributable to late systolic pressure augmentation ( expressed as percentage ) , and ( 4 ) mean arterial pressure . secondary analyses assessed additional measures of central pulse wave form and peripheral reflection , in particular forward wave amplitude , reflected wave amplitude , and the global reflection coefficient . all echocardiograms were evaluated by an experienced sonographer or cardiologist based on a standardized reading protocol . cardiac dimensions were quantified using the leadingedge technique as recommended by the american society of echocardiography ( ase ) . lv mass was calculated according to ase guidelines , applying the method of devereux et al.15 the sum of the diastolic thicknesses of the septum and posterior wall was used as an estimate of lv wall thickness . early systolic mitral annulus velocity ( e ) was measured at the lateral mitral annulus using tissue doppler imaging and transmitral doppler flow velocities recorded using a standardized protocol . repeated analysis of diastolic function measures ( mitral e and a peak velocity , tissue doppler e and a peak velocity ) yielded interobserver correlation coefficients of > 0.97 . lv dimension , lv mass , and left atrial ( la ) diameter distributions were skewed and therefore natural logarithmically transformed for all analyses . our primary analyses focused on cfpwv , central pulse pressure , mean arterial pressure , and augmentation index . analyses of lv dimensions ( lv mass , lv wall thickness , and diastolic dimension ) were performed in 3 stages : ( 1 ) adjusting only for age , age , sex , height , and study cohort ( offspring vs third generation ) ; ( 2 ) additionally adjusting for clinical risk factors ( excluding blood pressure ) and antihypertensive medication , ie , weight , heart rate , diabetes , serum total cholesterol , highdensity lipoprotein cholesterol ( hdlc ) , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , intake of angiotensinconverting enzyme inhibitors , angiotensin receptor blockers , blockers , diuretics , calcium channel blockers ( binary variable for each class ) ; and ( 3 ) additionally adjusting for brachial map . analyses evaluating lv systolic and diastolic function ( fractional shortening , e , e / e ) were constructed similarly in a staged design : ( 1 ) adjusting only for age , age , sex , height , and study cohort ; ( 2 ) additionally adjusting for clinical risk factors ( excluding blood pressure ) and antihypertensive medication , ie , weight , heart rate , diabetes , total cholesterol , hdlc , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , intake of angiotensinconverting enzyme inhibitors , angiotensin receptor blockers , blockers , diuretics , calcium channel blockers ; ( 3 ) additionally adjusting for lv mass ; and ( 4 ) further adjusting for either map ( in models investigating pulsatile blood pressure traits ) or central pulse pressure ( in models investigating map ) . for figure construction we also estimated leastsquares means based on regression models with lv structure or function traits as dependent variable and tertiles of cfpwv as predictor variable , adjusting for the covariates of stage 2 . logarithmically transformed lv diastolic diameter was then backtransformed to original scale ; thus , means represent geometric means . given that our primary analyses assessed the relation of 4 tonometry traits ( central pulse pressure , map , cfpwv , augmentation index ) to 3 lv structure traits ( lv mass , lv diastolic diameter , lv wall thickness ) in 3 statistical models , as well as the relation of the same 4 tonometry traits to 3 lv function traits ( lv fractional shortening , e , e / e ) in 4 statistical models , we introduced a bonferroni correction for ( 433)+(434)=84 statistical tests and regarded a pvalue of 0.0006 ( 0.05/84 ) as statistically significant . individuals were derived from the framingham offspring and the framingham third generation cohorts , which have been described.9 , 10 the framingham offspring cohort was recruited in 19711974 and includes individuals who are children of the framingham original cohort or the children 's spouses . the framingham third generation cohort , recruited in 20022005 , comprises children of the framingham offspring cohort . participants of both cohorts are evaluated approximately every 4 to 8 years in our study clinic during a visit that includes a medical history , physical examination , and phlebotomy . the present investigation is based on examination cycle 8 of the framingham offspring cohort ( 20052008 ) and examination cycle 1 of the third generation cohort ( 20022005 ) . of 3021 participants who attended offspring examination cycle 8 and 4095 participants recruited into the third generation cohort , we excluded 252 individuals with atrial fibrillation , 703 for missing echocardiographic measurements , 334 for missing tonometry , and 28 for missing covariates ; that left 5799 ( 2184 offspring , 3615 third generation ) individuals for this investigation . excluded individuals were generally older and had a higher morbidity ( higher prevalence of diabetes , hypertension , and prevalent cardiovascular disease ) than those included in the analyses . the study protocols were approved by the boston university medical center institutional review board , and participants signed informed consent . supine brachial systolic and diastolic blood pressures were obtained using an auscultatory device.11 arterial tonometry measurements were performed as previously described.11 , 12 , 13 briefly , arterial tonometry ( using a standard applanation tonometry device ) with simultaneous ecg was performed on the brachial , femoral , and carotid arteries . transit distances were assessed by body surface measurements from the suprasternal notch to the pulserecording site . mean arterial pressure ( map ) was derived from integration of the brachial waveform calibrated with bp at the time of tonometry . diastolic blood pressure and integrated map were used to calibrate carotid pressure tracings . calibrated carotid pressure direct measurement of carotid pressure , as compared to transfer function based estimates , is associated with a smaller difference between central and peripheral pulse pressure.14 details of signal analyses and data processing have been published elsewhere.11 , 12 , 13 we primarily assessed 4 measures of arterial stiffness and central hemodynamics : ( 1 ) carotidfemoral pulse wave velocity ( cfpwv ) , the current reference standard for aortic stiffness , ( 2 ) central pulse pressure , ie , the blood pressure amplitude in the proximal aorta , ( 3 ) augmentation index , ie , the fraction of central pulse pressure attributable to late systolic pressure augmentation ( expressed as percentage ) , and ( 4 ) mean arterial pressure . secondary analyses assessed additional measures of central pulse wave form and peripheral reflection , in particular forward wave amplitude , reflected wave amplitude , and the global reflection coefficient . all echocardiograms were evaluated by an experienced sonographer or cardiologist based on a standardized reading protocol . cardiac dimensions were quantified using the leadingedge technique as recommended by the american society of echocardiography ( ase ) . lv mass was calculated according to ase guidelines , applying the method of devereux et al.15 the sum of the diastolic thicknesses of the septum and posterior wall was used as an estimate of lv wall thickness . early systolic mitral annulus velocity ( e ) was measured at the lateral mitral annulus using tissue doppler imaging and transmitral doppler flow velocities recorded using a standardized protocol . repeated analysis of diastolic function measures ( mitral e and a peak velocity , tissue doppler e and a peak velocity ) yielded interobserver correlation coefficients of > 0.97 . lv dimension , lv mass , and left atrial ( la ) diameter distributions were skewed and therefore natural logarithmically transformed for all analyses . our primary analyses focused on cfpwv , central pulse pressure , mean arterial pressure , and augmentation index . analyses of lv dimensions ( lv mass , lv wall thickness , and diastolic dimension ) were performed in 3 stages : ( 1 ) adjusting only for age , age , sex , height , and study cohort ( offspring vs third generation ) ; ( 2 ) additionally adjusting for clinical risk factors ( excluding blood pressure ) and antihypertensive medication , ie , weight , heart rate , diabetes , serum total cholesterol , highdensity lipoprotein cholesterol ( hdlc ) , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , intake of angiotensinconverting enzyme inhibitors , angiotensin receptor blockers , blockers , diuretics , calcium channel blockers ( binary variable for each class ) ; and ( 3 ) additionally adjusting for brachial map . analyses evaluating lv systolic and diastolic function ( fractional shortening , e , e / e ) were constructed similarly in a staged design : ( 1 ) adjusting only for age , age , sex , height , and study cohort ; ( 2 ) additionally adjusting for clinical risk factors ( excluding blood pressure ) and antihypertensive medication , ie , weight , heart rate , diabetes , total cholesterol , hdlc , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , intake of angiotensinconverting enzyme inhibitors , angiotensin receptor blockers , blockers , diuretics , calcium channel blockers ; ( 3 ) additionally adjusting for lv mass ; and ( 4 ) further adjusting for either map ( in models investigating pulsatile blood pressure traits ) or central pulse pressure ( in models investigating map ) . for figure construction we also estimated leastsquares means based on regression models with lv structure or function traits as dependent variable and tertiles of cfpwv as predictor variable , adjusting for the covariates of stage 2 . logarithmically transformed lv diastolic diameter was then backtransformed to original scale ; thus , means represent geometric means . given that our primary analyses assessed the relation of 4 tonometry traits ( central pulse pressure , map , cfpwv , augmentation index ) to 3 lv structure traits ( lv mass , lv diastolic diameter , lv wall thickness ) in 3 statistical models , as well as the relation of the same 4 tonometry traits to 3 lv function traits ( lv fractional shortening , e , e / e ) in 4 statistical models , we introduced a bonferroni correction for ( 433)+(434)=84 statistical tests and regarded a pvalue of 0.0006 ( 0.05/84 ) as statistically significant . characteristics of the entire framingham offspring and third generation cohort are given in the left part of table 1 ; the characteristics of the final study sample after exclusions ( see methods for details ) are shown in the right part of table 1 . lv hypertrophy was present in 1574 individuals ( 27% ) of the study sample ; impaired systolic lv function was present in 46 individuals ( 1% ) . the unadjusted pairwise correlations between our primary tonometry and blood pressure traits are provided in table 2 . clinical , echocardiographic , and hemodynamic characteristics available n values were as follows : n=7032 , n= 6995 , n=7096 , n=7115 , n=6986 , n=6987 , n=6984 , n=7116 , n=7094 , n=6976 , n=6497 , n=6500 , n=6751 , n=6494 , n=6823 , n=6761 , n=6587 , n= 6918 , n=6878 , n=6797 . acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin receptor blocker ; e , carotidfemoral pulse wave velocity ; e , peak early diastolic mitral inflow velocity ; e , peak early diastolic mitral annulus velocity ; lv , left ventricular . ai indicates augmentation index ; cfpwv , carotidfemoral pulse wave velocity ; dbp , diastolic blood pressure ; map , mean arterial pressure ; pp , pulse pressure ; sbp , systolic blood pressure . unadjusted correlations between the tonometry measures and echocardiographic traits reflecting cardiac dimensions are presented in table 3 . adjusted relations of central hemodynamics and aortic stiffness with lv dimensions are displayed in table 4 . mean arterial pressure was positively associated with lv mass , even after multivariable adjustment ( p<0.0001 ) . results for lv mass index and lv hypertrophy were essentially similar ( data not shown ) . when we separately investigated the 2 components of lv mass , ie , lv wall thickness and lv diastolic diameter , we observed that the association of map with lv wall thickness was statistically robust and persisted after additional adjustment for central pulse pressure ( p<0.0001 for all models ) , whereas the association of map with lv diastolic diameter was weaker and no longer statistically significant ( p=0.15 ) after adjustment for central pulse pressure . central hemodynamics , aortic stiffness , and cardiac geometry ( n=5799 ) data are partial pearson correlation coefficients and the respective pvalues . multivariable model additionally adjusted for weight , heart rate , diabetes , total cholesterol , hdl cholesterol , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , and intake of angiotensinconverting enzyme inhibitors / angiotensin receptor blockers , blockers , diuretics , or calcium channel blockers . cpp indicates central pulse pressure ; lv , left ventricular ; map , mean arterial pressure . central pulse pressure was positively correlated with higher lv mass ( p<0.0001 ) , which was attributable to comparable relations with lv diastolic diameter and lv wall thickness ( both p<0.0001 ) . relations remained statistically significant after adjustment for multiple clinical covariates and also persisted on additional adjustment for map ( all p<0.0001 ) . figure 1 depicts multivariableadjusted means of lv diastolic diameter ( figure 1a ) and lv wall thickness ( figure 1b ) , stratified by tertiles of mean arterial pressure or central pulse pressure . multivariable adjusted means of left ventricular ( lv ) diastolic diameter ( a ) and lv anterior+posterior wall thickness ( b ) , plotted by tertiles of mean arterial pressure ( map ; squares ) and central pulse pressure ( cpp ; triangles ) . higher aortic stiffness , as assessed by cfpwv , was associated with greater lv mass and greater lv wall thickness , but the associations were attenuated on multivariable adjustment and rendered statistically nonsignificant on additional adjustment for mean arterial pressure . higher augmentation index correlated with higher lv mass and higher lv diastolic diameter ( all p<0.0001 ) ; however , the associations were no longer significant in multivariable ( including map)adjusted analyses ( all p>0.003 ) . in secondary analyses , we related additional subphenotypes of the central pressure waveform , ie , forward wave , reflected wave , and reflection factor , to lv structure ( table 5 ) . in multivariable ( including map)adjusted analyses , forward and reflected waves generally showed similar associations with lv mass , lv diastolic diameter , and lv wall thickness ( r=0.0530.089 , p<0.0001 ) , except for a nonsignificant association of the reflected wave with lv wall thickness ( r=0.034 , p=0.01 ) . in mutually adjusted analyses , forward wave relations with lv mass persisted after adjusting for reflected wave ( table 5 , p<0.0001 ) , whereas reflected wave was not related to lv mass after adjusting for forward wave ( p=0.63 ) . the reflection factor was not associated with lv structure after accounting for multiple statistical testing ( all p0.003 ) . secondary analyses : relations of central pressure waveform components with left ventricular structure data are partial pearson correlation coefficients . model 1 adjusted for age , age , sex , height , study cohort , weight , heart rate , diabetes , total cholesterol , hdlc , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , intake of angiotensinconverting enzyme inhibitors , angiotensin receptor blockers , blockers , diuretics , or calcium channel blockers , and mean arterial pressure . unadjusted correlations between the assessed tonometry measures and echocardiographic measures of lv systolic and diastolic function are presented in table 6 . the adjusted relations of central hemodynamics and aortic stiffness with systolic and diastolic lv function are given in table 7 . mean arterial pressure was not associated with lv fractional shortening ( p>0.05 in all models ) . in contrast , higher map was associated with lower e and higher e / e ( all p<0.0001 ) . correlations between map and lv filling measures persisted after adjustment for clinical covariates and also after additional adjustment for lv mass ( all p<0.0001 ) . cfpwv indicates carotidfemoral pulse wave velocity ; e , maximum early diastolic mitral inflow velocity ; e , maximum early diastolic mitral annulus velocity . central hemodynamics , aortic stiffness , and cardiac function ( n=5799 ) data are partial pearson correlation coefficients and the respective pvalues . base model : adjusted for age , age , sex , height , and study cohort . multivariable model : additionally adjusted for weight , heart rate , diabetes , total cholesterol , hdlcholesterol , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , intake of angiotensinconverting enzyme inhibitors , angiotensin receptor blockers , blockers , diuretics , calcium channel blockers . cpp indicates central pulse pressure ; e , maximum early diastolic mitral inflow velocity ; e , maximum early diastolic mitral annulus velocity ; lvm , left ventricular mass ; map , mean arterial pressure . central pulse pressure was moderately and positively correlated with fractional shortening ( p<0.0001 ) , even after multivariable adjustment ( p<0.0001 , table 7 ) . central pulse pressure also correlated very modestly and inversely with e ( a measure of diastolic relaxation ) , whereas we observed a stronger direct correlation of central pulse pressure with e / e ( p<0.0001 ) , a surrogate for lv filling pressure . correlations between central pulse pressure and lv filling measures were only slightly attenuated by adjustment for multiple potential clinical confounders including lv mass . after additional adjustment for mean arterial pressure , the association of central pulse pressure with e / e was maintained , whereas the directionality of the modest negative association with e was reversed . higher cfpwv was not related to fractional shortening but correlated with worse diastolic function ( as assessed by e and e / e , all p<0.0001 , table 3 ) . the correlations of cfpwv with measures of slowed relaxation and higher lv filling pressure persisted after multivariable adjustment , including adjustment for map and lv mass ( all p<0.0001 ) . figure 2 depicts multivariable adjusted means of lv fractional shortening ( figure 2a ) and e ( figure 2b ) , stratified by tertiles of cfpwv . multivariable adjusted means of left ventricular ( lv ) fractional shortening ( a ) and early mitral valve annulus diastolic velocity ( e , b ) , plotted by tertiles of carotidfemoral pulse wave velocity ( cfpwv ) . error bars represent 95% confidence intervals . in a base model , higher augmentation index correlated modestly with greater fractional shortening . however , in multivariableadjusted analyses , higher augmentation index correlated modestly and inversely with lv systolic function , even after adjustment for lv mass and mean arterial pressure ( p<0.0001 , table 7 ) . we found similar reversal of directionality of very modest associations between augmentation index and e following multivariable adjustment . in contrast , augmentation index was associated positively with e / e in our base model ( p>0.0001 ) , and the association and directionality persisted with multivariable adjustment . in secondary analyses ( see table 8) , forward wave amplitude correlated with fractional shortening ( r=0.086 , p<0.0001 ) , whereas reflected wave amplitude did not ( p=0.06 ) . forward and reflected waves similarly correlated with measures of diastolic lv function ( r=0.0530.066 , all p<0.0001 ) . reflection factor correlated inversely with fractional shortening ( r=0.077 , p<0.0001 ) but was not related to diastolic function traits ( all p>0.05 ) . secondary analyses : relations of central pressure waveform components with left ventricular function data are partial pearson correlation coefficients , adjusted for age , age , sex , height , study cohort , weight , heart rate , diabetes , total cholesterol , hdlc , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , intake of angiotensinconverting enzyme inhibitors , angiotensin receptor blockers , blockers , diuretics , calcium channel blockers , mean arterial pressure and left ventricular mass . e , maximum early diastolic mitral annulus velocity ; e , maximum early diastolic mitral inflow velocity . additionally adjusted for characteristics of the entire framingham offspring and third generation cohort are given in the left part of table 1 ; the characteristics of the final study sample after exclusions ( see methods for details ) are shown in the right part of table 1 . lv hypertrophy was present in 1574 individuals ( 27% ) of the study sample ; impaired systolic lv function was present in 46 individuals ( 1% ) . the unadjusted pairwise correlations between our primary tonometry and blood pressure traits are provided in table 2 . clinical , echocardiographic , and hemodynamic characteristics available n values were as follows : n=7032 , n= 6995 , n=7096 , n=7115 , n=6986 , n=6987 , n=6984 , n=7116 , n=7094 , n=6976 , n=6497 , n=6500 , n=6751 , n=6494 , n=6823 , n=6761 , n=6587 , n= 6918 , n=6878 , n=6797 . acei indicates angiotensinconverting enzyme inhibitor ; arb , angiotensin receptor blocker ; e , carotidfemoral pulse wave velocity ; e , peak early diastolic mitral inflow velocity ; e , peak early diastolic mitral annulus velocity ; lv , left ventricular . ai indicates augmentation index ; cfpwv , carotidfemoral pulse wave velocity ; dbp , diastolic blood pressure ; map , mean arterial pressure ; pp , pulse pressure ; sbp , systolic blood pressure . unadjusted correlations between the tonometry measures and echocardiographic traits reflecting cardiac dimensions are presented in table 3 . adjusted relations of central hemodynamics and aortic stiffness with lv dimensions are displayed in table 4 . mean arterial pressure was positively associated with lv mass , even after multivariable adjustment ( p<0.0001 ) . results for lv mass index and lv hypertrophy were essentially similar ( data not shown ) . when we separately investigated the 2 components of lv mass , ie , lv wall thickness and lv diastolic diameter , we observed that the association of map with lv wall thickness was statistically robust and persisted after additional adjustment for central pulse pressure ( p<0.0001 for all models ) , whereas the association of map with lv diastolic diameter was weaker and no longer statistically significant ( p=0.15 ) after adjustment for central pulse pressure . central hemodynamics , aortic stiffness , and cardiac geometry ( n=5799 ) data are partial pearson correlation coefficients and the respective pvalues . base model adjusted for age , age , sex , height , study cohort . multivariable model additionally adjusted for weight , heart rate , diabetes , total cholesterol , hdl cholesterol , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , and intake of angiotensinconverting enzyme inhibitors / angiotensin receptor blockers , blockers , diuretics , or calcium channel blockers . cpp indicates central pulse pressure ; lv , left ventricular ; map , mean arterial pressure . central pulse pressure was positively correlated with higher lv mass ( p<0.0001 ) , which was attributable to comparable relations with lv diastolic diameter and lv wall thickness ( both p<0.0001 ) . relations remained statistically significant after adjustment for multiple clinical covariates and also persisted on additional adjustment for map ( all p<0.0001 ) . figure 1 depicts multivariableadjusted means of lv diastolic diameter ( figure 1a ) and lv wall thickness ( figure 1b ) , stratified by tertiles of mean arterial pressure or central pulse pressure . multivariable adjusted means of left ventricular ( lv ) diastolic diameter ( a ) and lv anterior+posterior wall thickness ( b ) , plotted by tertiles of mean arterial pressure ( map ; squares ) and central pulse pressure ( cpp ; triangles ) . higher aortic stiffness , as assessed by cfpwv , was associated with greater lv mass and greater lv wall thickness , but the associations were attenuated on multivariable adjustment and rendered statistically nonsignificant on additional adjustment for mean arterial pressure . higher augmentation index correlated with higher lv mass and higher lv diastolic diameter ( all p<0.0001 ) ; however , the associations were no longer significant in multivariable ( including map)adjusted analyses ( all p>0.003 ) . in secondary analyses , we related additional subphenotypes of the central pressure waveform , ie , forward wave , reflected wave , and reflection factor , to lv structure ( table 5 ) . in multivariable ( including map)adjusted analyses , forward and reflected waves generally showed similar associations with lv mass , lv diastolic diameter , and lv wall thickness ( r=0.0530.089 , p<0.0001 ) , except for a nonsignificant association of the reflected wave with lv wall thickness ( r=0.034 , p=0.01 ) . in mutually adjusted analyses , forward wave relations with lv mass persisted after adjusting for reflected wave ( table 5 , p<0.0001 ) , whereas reflected wave was not related to lv mass after adjusting for forward wave ( p=0.63 ) . the reflection factor was not associated with lv structure after accounting for multiple statistical testing ( all p0.003 ) . secondary analyses : relations of central pressure waveform components with left ventricular structure data are partial pearson correlation coefficients . model 1 adjusted for age , age , sex , height , study cohort , weight , heart rate , diabetes , total cholesterol , hdlc , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , intake of angiotensinconverting enzyme inhibitors , angiotensin receptor blockers , blockers , diuretics , or calcium channel blockers , and mean arterial pressure . unadjusted correlations between the assessed tonometry measures and echocardiographic measures of lv systolic and diastolic function are presented in table 6 . the adjusted relations of central hemodynamics and aortic stiffness with systolic and diastolic lv function are given in table 7 . mean arterial pressure was not associated with lv fractional shortening ( p>0.05 in all models ) . in contrast , higher map was associated with lower e and higher e / e ( all p<0.0001 ) . correlations between map and lv filling measures persisted after adjustment for clinical covariates and also after additional adjustment for lv mass ( all p<0.0001 ) . cfpwv indicates carotidfemoral pulse wave velocity ; e , maximum early diastolic mitral inflow velocity ; e , maximum early diastolic mitral annulus velocity . central hemodynamics , aortic stiffness , and cardiac function ( n=5799 ) data are partial pearson correlation coefficients and the respective pvalues . base model : adjusted for age , age , sex , height , and study cohort . multivariable model : additionally adjusted for weight , heart rate , diabetes , total cholesterol , hdlcholesterol , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , intake of angiotensinconverting enzyme inhibitors , angiotensin receptor blockers , blockers , diuretics , calcium channel blockers . cpp indicates central pulse pressure ; e , maximum early diastolic mitral inflow velocity ; e , maximum early diastolic mitral annulus velocity ; lvm , left ventricular mass ; map , mean arterial pressure . inversetransformed for normality and multiplied by 1 to restore directionality . central pulse pressure was moderately and positively correlated with fractional shortening ( p<0.0001 ) , even after multivariable adjustment ( p<0.0001 , table 7 ) . central pulse pressure also correlated very modestly and inversely with e ( a measure of diastolic relaxation ) , whereas we observed a stronger direct correlation of central pulse pressure with e / e ( p<0.0001 ) , a surrogate for lv filling pressure . correlations between central pulse pressure and lv filling measures were only slightly attenuated by adjustment for multiple potential clinical confounders including lv mass . after additional adjustment for mean arterial pressure , the association of central pulse pressure with e / e was maintained , whereas the directionality of the modest negative association with e was reversed . higher cfpwv was not related to fractional shortening but correlated with worse diastolic function ( as assessed by e and e / e , all p<0.0001 , table 3 ) . the correlations of cfpwv with measures of slowed relaxation and higher lv filling pressure persisted after multivariable adjustment , including adjustment for map and lv mass ( all p<0.0001 ) . figure 2 depicts multivariable adjusted means of lv fractional shortening ( figure 2a ) and e ( figure 2b ) , stratified by tertiles of cfpwv . multivariable adjusted means of left ventricular ( lv ) fractional shortening ( a ) and early mitral valve annulus diastolic velocity ( e , b ) , plotted by tertiles of carotidfemoral pulse wave velocity ( cfpwv ) . error bars represent 95% confidence intervals . in a base model , higher augmentation index correlated modestly with greater fractional shortening . however , in multivariableadjusted analyses , higher augmentation index correlated modestly and inversely with lv systolic function , even after adjustment for lv mass and mean arterial pressure ( p<0.0001 , table 7 ) . we found similar reversal of directionality of very modest associations between augmentation index and e following multivariable adjustment . in contrast , augmentation index was associated positively with e / e in our base model ( p>0.0001 ) , and the association and directionality persisted with multivariable adjustment . in secondary analyses ( see table 8) , forward wave amplitude correlated with fractional shortening ( r=0.086 , p<0.0001 ) , whereas reflected wave amplitude did not ( p=0.06 ) . forward and reflected waves similarly correlated with measures of diastolic lv function ( r=0.0530.066 , all p<0.0001 ) . reflection factor correlated inversely with fractional shortening ( r=0.077 , p<0.0001 ) but was not related to diastolic function traits ( all p>0.05 ) . secondary analyses : relations of central pressure waveform components with left ventricular function data are partial pearson correlation coefficients , adjusted for age , age , sex , height , study cohort , weight , heart rate , diabetes , total cholesterol , hdlc , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , intake of angiotensinconverting enzyme inhibitors , angiotensin receptor blockers , blockers , diuretics , calcium channel blockers , mean arterial pressure and left ventricular mass . e , maximum early diastolic mitral annulus velocity ; e , maximum early diastolic mitral inflow velocity . additionally adjusted for in the present investigation we examined differing associations of pulsatile and steady components of central blood pressure and aortic stiffness with left ventricular structure and function . first , both steady ( ie , map ) and pulsatile ( ie , central pulse pressure ) components of central blood pressure were correlated positively with lv mass . however , whereas higher map was primarily associated with higher lv wall thickness , central pulse pressure was positively associated with both lv diameter and lv wall thickness . second , higher map and greater aortic stiffness were correlated inversely with lv diastolic function , and these correlations at least partly persisted in models adjusting of lv mass . third , aortic stiffness , as assessed by cfpwv , was associated inversely with lv diastolic function but showed no independent correlation with lv dimensions or circumferential lv systolic function . whereas the relation between peripheral blood pressure and cardiac structure has been well documented,16 few studies have assessed relations among cardiac structure , central hemodynamics , and vascular stiffness using detailed tonometry measures . these prior studies were limited by small sample size17 or an indirect assessment of central hemodynamics,18 restricted to noneuropean ancestry individuals18 , 19 , 20 or relied on electrocardiographic lv hypertrophy.21 in a sample of 1272 chinese indiviudals , wang et al reported that central pulse pressure was associated with lv mass.19 however , their study did not separately assess associations with lv wall thickness versus lv dimensions . an independent association of proximal ( ascending ) aortic stiffness with lv mass was recently reported in 347 elderly participants of the age , gene / environment susceptibility ( ages)reykjavik study cohort.22 that study investigated the hypothesis of a direct coupling between lv and a stiffened proximal aorta as a novel type of mechanical , rather than hemodynamic , load on the left ventricle and thus did not evaluate relations with cfpwv . to our knowledge , the present analysis is the largest investigation of the relation of central hemodynamics and cfpwv ( the reference measure of aortic stiffness ) with cardiac structure and function in a sample of european ancestry . we observed that higher central pulse pressure is associated with both higher lv diameter and greater wall thickness , whereas higher map ( steady component of central pressure ) is primarily associated with higher lv wall thickness ( rather than with lv diameter ) . thus , the steady and pulsatile components of central blood pressure may conjointly yet variably contribute to differences in lv mass . of note the fact that neither aortic stiffness nor wave reflection is independently associated with lv mass in our analyses suggests that the relation between lv mass and pulse pressure may be largely attributable to mismatch between ventricular outflow and the ability of the aorta to accommodate that flow , as recently described by torjesen et al.23 existing literature on the relations among central hemodynamics , vascular stiffness , and cardiac function is sparse , and most studies were of limited size,24 , 25 , 26 , 27 , 28 restricted to certain patient populations,27 or did not include tissue doppler assessment of lv diastolic function.20 abhayaratna et al investigated the relation of arterial stiffness to lv diastolic dysfunction in a sample of 188 elderly individuals and observed a significant correlation between central pulse pressure and severity of diastolic dysfunction.29 however , cfpwv was not associated with diastolic dysfunction in their study . russo et al reported in 983 individuals that several tonometryderived measures of central hemodynamics , greater arterial stiffness , and more wave reflection were all associated with worse lv diastolic function.30 however , after multivariable adjustment , only the ratio of central pulse pressure to stroke volume index ( a measure of global arterial stiffness ) remained associated with lv diastolic dysfunction . kang et al measured brachialankle pulse wave velocity in 1929 individuals in shanghai and reported an association with diastolic heart failure.31 notably , cfpwv was not measured in the latter studies . our considerably larger analysis demonstrates that higher mean arterial pressure and aortic stiffness are associated inversely with measures of lv diastolic function . the fact that these associations persisted after adjustment for lv mass is consistent with the notion that diastolic dysfunction may be only partly dependent on lv hypertrophy.32 aortic and cardiac stiffness ( hence lv diastolic dysfunction ) may share etiologic mechanisms such as excessive tissue fibrosis . the extent to which aortic stiffness may contribute to lv diastolic dysfunction or that common pathophysiological mechanisms may contribute to parallel increases in both aortic and cardiac stiffness if greater aortic stiffness is indeed shown to contribute to lv diastolic dysfunction in additional studies , therapeutic interventions aimed at decreasing macrovascular stiffness may be a promising tool for the prevention and mitigation of diastolic dysfunction , a premise that warrants further study . the relations of central pulse pressure and augmentation index to lv diastolic dysfunction were not straightforward in our analyses . whereas we observed a consistent association of higher central pulse pressure with higher e / e ( a surrogate measure of elevated lv filling pressure ) , the association of central pulse pressure with the fillingphase measure of diastolic relaxation ( ie , e ) was weak and changed directionality after adjustment for potential confounding by map . hence , central pulse pressure and augmentation index appear to be primarily associated with lv filling pressures rather than with lv diastolic relaxation . interestingly , we observed a positive correlation between higher central pulse pressure and lv systolic function , which may seem surprising . the most likely explanation for this finding is that a greater fractional shortening corresponds to higher stroke volume and peak flow rate in the proximal aorta and thus may be a cause rather than a consequence of higher central pulse pressure . similarly , the lack of an association between map and lv systolic function in our crosssectional analysis may possibly be explained by various opposing effects of map on lv function . an acute increase in map reduces lv systolic function ( inverse relation ) but may promote lv hypertrophy and remodeling , which restore lv wall stress and systolic function to normal levels ( thereby resulting in a null relation ) . first , our study sample is community based and predominantly comprised of middleaged adults of european ancestry . the applicability of our findings to younger individuals , to other ethnicities or certain patient groups , remains to be explored in future studies . second , our study is crosssectional and observational ; thus , causal inferences can not be drawn . also , we would like to emphasize that our study was focused on the physiological relations of central hemodynamics with cardiac structure and function . we did not investigate whether central blood pressure may be more strongly related to certain echocardiography traits than to arm blood pressure . in fact , brachial and central pulse pressure were highly correlated ( r=0.93 ) in our data . consequently , the findings for brachial pulse pressure were similar to those observed for central pulse pressure ( see table 9).14 similarly , we did not study aggregate measures of pulsatile and steady pressure components , eg , central systolic blood pressure . brachial versus central pressure in relation to echocardiographic traits e indicates maximum early diastolic mitral inflow velocity ; e ' , maximum early diastolic mitral annulus velocity ; lv , left ventricular.data are partial pearson correlations , adjusted for age , age , sex , height and study cohort . however , we would like to underscore that our main analyses were adjusted for multiple potential confounders . last , we have performed multiple statistical tests , potentially inflating the type 1 error rate . however , we accounted for multiple testing using a bonferroni correction . of note , most of our findings were highly statistically significant ( p<0.0001 ) and hence likely to be true associations . whereas the relation between peripheral blood pressure and cardiac structure has been well documented,16 few studies have assessed relations among cardiac structure , central hemodynamics , and vascular stiffness using detailed tonometry measures . these prior studies were limited by small sample size17 or an indirect assessment of central hemodynamics,18 restricted to noneuropean ancestry individuals18 , 19 , 20 or relied on electrocardiographic lv hypertrophy.21 in a sample of 1272 chinese indiviudals , wang et al reported that central pulse pressure was associated with lv mass.19 however , their study did not separately assess associations with lv wall thickness versus lv dimensions . an independent association of proximal ( ascending ) aortic stiffness with lv mass was recently reported in 347 elderly participants of the age , gene / environment susceptibility ( ages)reykjavik study cohort.22 that study investigated the hypothesis of a direct coupling between lv and a stiffened proximal aorta as a novel type of mechanical , rather than hemodynamic , load on the left ventricle and thus did not evaluate relations with cfpwv . to our knowledge , the present analysis is the largest investigation of the relation of central hemodynamics and cfpwv ( the reference measure of aortic stiffness ) with cardiac structure and function in a sample of european ancestry . we observed that higher central pulse pressure is associated with both higher lv diameter and greater wall thickness , whereas higher map ( steady component of central pressure ) is primarily associated with higher lv wall thickness ( rather than with lv diameter ) . thus , the steady and pulsatile components of central blood pressure may conjointly yet variably contribute to differences in lv mass . of note , however , the relation between lv mass and pulse pressure is likely bidirectional . the fact that neither aortic stiffness nor wave reflection is independently associated with lv mass in our analyses suggests that the relation between lv mass and pulse pressure may be largely attributable to mismatch between ventricular outflow and the ability of the aorta to accommodate that flow , as recently described by torjesen et al.23 existing literature on the relations among central hemodynamics , vascular stiffness , and cardiac function is sparse , and most studies were of limited size,24 , 25 , 26 , 27 , 28 restricted to certain patient populations,27 or did not include tissue doppler assessment of lv diastolic function.20 abhayaratna et al investigated the relation of arterial stiffness to lv diastolic dysfunction in a sample of 188 elderly individuals and observed a significant correlation between central pulse pressure and severity of diastolic dysfunction.29 however , cfpwv was not associated with diastolic dysfunction in their study . russo et al reported in 983 individuals that several tonometryderived measures of central hemodynamics , greater arterial stiffness , and more wave reflection were all associated with worse lv diastolic function.30 however , after multivariable adjustment , only the ratio of central pulse pressure to stroke volume index ( a measure of global arterial stiffness ) remained associated with lv diastolic dysfunction . kang et al measured brachialankle pulse wave velocity in 1929 individuals in shanghai and reported an association with diastolic heart failure.31 notably , cfpwv was not measured in the latter studies . our considerably larger analysis demonstrates that higher mean arterial pressure and aortic stiffness are associated inversely with measures of lv diastolic function . the fact that these associations persisted after adjustment for lv mass is consistent with the notion that diastolic dysfunction may be only partly dependent on lv hypertrophy.32 aortic and cardiac stiffness ( hence lv diastolic dysfunction ) may share etiologic mechanisms such as excessive tissue fibrosis . the extent to which aortic stiffness may contribute to lv diastolic dysfunction or that common pathophysiological mechanisms may contribute to parallel increases in both aortic and cardiac stiffness can not be assessed in our crosssectional analyses and therefore remains to be elucidated . if greater aortic stiffness is indeed shown to contribute to lv diastolic dysfunction in additional studies , therapeutic interventions aimed at decreasing macrovascular stiffness may be a promising tool for the prevention and mitigation of diastolic dysfunction , a premise that warrants further study . the relations of central pulse pressure and augmentation index to lv diastolic dysfunction were not straightforward in our analyses . whereas we observed a consistent association of higher central pulse pressure with higher e / e ( a surrogate measure of elevated lv filling pressure ) , the association of central pulse pressure with the fillingphase measure of diastolic relaxation ( ie , e ) was weak and changed directionality after adjustment for potential confounding by map . hence , central pulse pressure and augmentation index appear to be primarily associated with lv filling pressures rather than with lv diastolic relaxation . interestingly , we observed a positive correlation between higher central pulse pressure and lv systolic function , which may seem surprising . the most likely explanation for this finding is that a greater fractional shortening corresponds to higher stroke volume and peak flow rate in the proximal aorta and thus may be a cause rather than a consequence of higher central pulse pressure . similarly , the lack of an association between map and lv systolic function in our crosssectional analysis may possibly be explained by various opposing effects of map on lv function . an acute increase in map reduces lv systolic function ( inverse relation ) but may promote lv hypertrophy and remodeling , which restore lv wall stress and systolic function to normal levels ( thereby resulting in a null relation ) . first , our study sample is community based and predominantly comprised of middleaged adults of european ancestry . the applicability of our findings to younger individuals , to other ethnicities or certain patient groups , remains to be explored in future studies . second , our study is crosssectional and observational ; thus , causal inferences can not be drawn . this is particularly relevant as some of the observed correlations are likely bidirectional . in addition , the potential for residual confounding can not be eliminated . also , we would like to emphasize that our study was focused on the physiological relations of central hemodynamics with cardiac structure and function . we did not investigate whether central blood pressure may be more strongly related to certain echocardiography traits than to arm blood pressure . in fact , brachial and central pulse pressure were highly correlated ( r=0.93 ) in our data . consequently , the findings for brachial pulse pressure were similar to those observed for central pulse pressure ( see table 9).14 similarly , we did not study aggregate measures of pulsatile and steady pressure components , eg , central systolic blood pressure . brachial versus central pressure in relation to echocardiographic traits e indicates maximum early diastolic mitral inflow velocity ; e ' , maximum early diastolic mitral annulus velocity ; lv , left ventricular.data are partial pearson correlations , adjusted for age , age , sex , height and study cohort . however , we would like to underscore that our main analyses were adjusted for multiple potential confounders . in unadjusted analyses , last , we have performed multiple statistical tests , potentially inflating the type 1 error rate . however , we accounted for multiple testing using a bonferroni correction . of note , most of our findings were highly statistically significant ( p<0.0001 ) and hence likely to be true associations . in the present investigation , we assessed relations of steady and pulsatile components of central hemodynamics and aortic stiffness to cardiac structure and lv systolic and diastolic function in a large communitybased sample with a broad age range . steady and pulsatile components of central blood pressure were jointly and variably related to components of lv structure . aortic stiffness and map were associated inversely with measures of lv diastolic function but not with lv systolic function . this work was supported by the nhlbi , framingham heart study ( nhlbi / nih contracts n01hc25195 and hhsn268201500001i ) , the boston university school of medicine , and by hl076784 , g028321 , hl070100 , hl060040 , hl080124 , hl071039 , hl077447 , hl107385 , 2k24hl04334 , and r01hl126136 . dr mitchell is owner of cardiovascular engineering inc ( a company that develops and manufactures devices to measure vascular stiffness ) and serves as a consultant to novartis , merck , and servier .

backgroundthe differing relations of steady and pulsatile components of central hemodynamics and aortic stiffness with cardiac dimensions and function have not been fully elucidated.methods and resultscentral hemodynamics and carotidfemoral pulse wave velocity ( cfpwv , a measure of aortic stiffness ) were measured by arterial tonometry in 5799 participants of the framingham heart study ( mean age 51 years , 54% women ) and related to echocardiographic left ventricular ( lv ) dimensions and systolic and diastolic function using multivariableadjusted partial pearson correlations . mean arterial pressure ( map , steady component of central blood pressure ) was associated positively with lv wall thickness ( r=0.168 ; p<0.0001 ) but showed only a weak direct association with lv diastolic dimension ( r=0.035 , p=0.006 ) . central pulse pressure ( pulsatile component of central blood pressure ) showed a direct correlation with both lv diastolic dimension and lv wall thickness ( r=0.08 and 0.044 , both p<0.0001 in multivariable models that included map ) . cfpwv was not associated with lv structure ( all p0.27 ) in mapadjusted models ) . both map and cfpwv were associated inversely with lv diastolic function ( e ; r=0.140 and 0.153 , respectively ; both p<0.0001 ) , and these associations persisted after additional adjustment for lv mass and central pulse pressure ( r=0.142 and 0.108 , both p<0.0001 ) . map and cfpwv were not associated with lv fractional shortening ( p0.10 ) , whereas central pulse pressure was positively related ( r=0.064 , p<0.0001).conclusionspulsatile and steady components of central pressure are conjointly yet variably related to lv structure . cfpwv is related to lv diastolic function but not to systolic function . additional studies are warranted to confirm these observations .

Introduction Methods Study Sample Blood Pressure and Arterial Tonometry Echocardiography Statistical Analyses Results Study Sample Central Hemodynamics, Aortic Stiffness, and LV Mass, Dimensions, and Wall Thickness Central Hemodynamics, Aortic Stiffness, and LV Systolic and Diastolic Function Discussion Central Hemodynamics, Aortic Stiffness, and Cardiac Structure Central Hemodynamics, Aortic Stiffness, and LV Systolic and Diastolic Function Limitations Conclusions Sources of Funding Disclosures

congestive heart failure is a leading cause of morbidity and mortality in developed countries , with an estimated prevalence of 1% to 2% in the general population , reaching 7% to 8% in individuals aged > 75 years.1 elevated blood pressure appears to play an important role in the pathogenesis of both left ventricular ( lv ) systolic and diastolic dysfunction , at least partly by increasing cardiac afterload.2 this has led to the therapeutic concept of reducing cardiac afterload , which has proven highly beneficial in heart failure with reduced ejection fraction but not in heart failure with a preserved ejection fraction.3 thus , broadening the understanding of the role of central hemodynamics in the pathogenesis of systolic and diastolic lv dysfunction is critical to inform the development of novel therapeutic approaches . the extent to which central pressure augmentation is explained by peripheral wave reflection or by the windkessel function of the proximal aorta is the subject of ongoing debate.4 , 5 limited data suggest that central pressure may be more closely related to cardiovascular disease than peripheral blood pressure.6 furthermore , variable relations between aortic stiffness ( which increases early systolic load on the heart ) and wave reflection ( which increases late systolic load ) may have differing implications for systolic and diastolic lv structure and function.7 in contrast , the steady component of blood pressure , ie , the mean arterial pressure , is fairly constant throughout large arteries.8 in this context , the relations of central hemodynamics with cardiac structure and function have not been fully delineated . we hypothesized that the pulsatile component ( and its determinants aortic stiffness and pressure augmentation ) and the steady component of central blood pressure may have different relations with lv structure and function . thus , we investigated the crosssectional relations of central hemodynamics and aortic stiffness to echocardiographic measures of lv structure and systolic and diastolic function in a large communitybased sample . direct measurement of carotid pressure , as compared to transfer function based estimates , is associated with a smaller difference between central and peripheral pulse pressure.14 details of signal analyses and data processing have been published elsewhere.11 , 12 , 13 we primarily assessed 4 measures of arterial stiffness and central hemodynamics : ( 1 ) carotidfemoral pulse wave velocity ( cfpwv ) , the current reference standard for aortic stiffness , ( 2 ) central pulse pressure , ie , the blood pressure amplitude in the proximal aorta , ( 3 ) augmentation index , ie , the fraction of central pulse pressure attributable to late systolic pressure augmentation ( expressed as percentage ) , and ( 4 ) mean arterial pressure . analyses of lv dimensions ( lv mass , lv wall thickness , and diastolic dimension ) were performed in 3 stages : ( 1 ) adjusting only for age , age , sex , height , and study cohort ( offspring vs third generation ) ; ( 2 ) additionally adjusting for clinical risk factors ( excluding blood pressure ) and antihypertensive medication , ie , weight , heart rate , diabetes , serum total cholesterol , highdensity lipoprotein cholesterol ( hdlc ) , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , intake of angiotensinconverting enzyme inhibitors , angiotensin receptor blockers , blockers , diuretics , calcium channel blockers ( binary variable for each class ) ; and ( 3 ) additionally adjusting for brachial map . analyses evaluating lv systolic and diastolic function ( fractional shortening , e , e / e ) were constructed similarly in a staged design : ( 1 ) adjusting only for age , age , sex , height , and study cohort ; ( 2 ) additionally adjusting for clinical risk factors ( excluding blood pressure ) and antihypertensive medication , ie , weight , heart rate , diabetes , total cholesterol , hdlc , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , intake of angiotensinconverting enzyme inhibitors , angiotensin receptor blockers , blockers , diuretics , calcium channel blockers ; ( 3 ) additionally adjusting for lv mass ; and ( 4 ) further adjusting for either map ( in models investigating pulsatile blood pressure traits ) or central pulse pressure ( in models investigating map ) . given that our primary analyses assessed the relation of 4 tonometry traits ( central pulse pressure , map , cfpwv , augmentation index ) to 3 lv structure traits ( lv mass , lv diastolic diameter , lv wall thickness ) in 3 statistical models , as well as the relation of the same 4 tonometry traits to 3 lv function traits ( lv fractional shortening , e , e / e ) in 4 statistical models , we introduced a bonferroni correction for ( 433)+(434)=84 statistical tests and regarded a pvalue of 0.0006 ( 0.05/84 ) as statistically significant . calibrated carotid pressure direct measurement of carotid pressure , as compared to transfer function based estimates , is associated with a smaller difference between central and peripheral pulse pressure.14 details of signal analyses and data processing have been published elsewhere.11 , 12 , 13 we primarily assessed 4 measures of arterial stiffness and central hemodynamics : ( 1 ) carotidfemoral pulse wave velocity ( cfpwv ) , the current reference standard for aortic stiffness , ( 2 ) central pulse pressure , ie , the blood pressure amplitude in the proximal aorta , ( 3 ) augmentation index , ie , the fraction of central pulse pressure attributable to late systolic pressure augmentation ( expressed as percentage ) , and ( 4 ) mean arterial pressure . analyses of lv dimensions ( lv mass , lv wall thickness , and diastolic dimension ) were performed in 3 stages : ( 1 ) adjusting only for age , age , sex , height , and study cohort ( offspring vs third generation ) ; ( 2 ) additionally adjusting for clinical risk factors ( excluding blood pressure ) and antihypertensive medication , ie , weight , heart rate , diabetes , serum total cholesterol , highdensity lipoprotein cholesterol ( hdlc ) , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , intake of angiotensinconverting enzyme inhibitors , angiotensin receptor blockers , blockers , diuretics , calcium channel blockers ( binary variable for each class ) ; and ( 3 ) additionally adjusting for brachial map . analyses evaluating lv systolic and diastolic function ( fractional shortening , e , e / e ) were constructed similarly in a staged design : ( 1 ) adjusting only for age , age , sex , height , and study cohort ; ( 2 ) additionally adjusting for clinical risk factors ( excluding blood pressure ) and antihypertensive medication , ie , weight , heart rate , diabetes , total cholesterol , hdlc , triglycerides , fasting glucose , prevalent cardiovascular disease , current smoking , intake of angiotensinconverting enzyme inhibitors , angiotensin receptor blockers , blockers , diuretics , calcium channel blockers ; ( 3 ) additionally adjusting for lv mass ; and ( 4 ) further adjusting for either map ( in models investigating pulsatile blood pressure traits ) or central pulse pressure ( in models investigating map ) . given that our primary analyses assessed the relation of 4 tonometry traits ( central pulse pressure , map , cfpwv , augmentation index ) to 3 lv structure traits ( lv mass , lv diastolic diameter , lv wall thickness ) in 3 statistical models , as well as the relation of the same 4 tonometry traits to 3 lv function traits ( lv fractional shortening , e , e / e ) in 4 statistical models , we introduced a bonferroni correction for ( 433)+(434)=84 statistical tests and regarded a pvalue of 0.0006 ( 0.05/84 ) as statistically significant . when we separately investigated the 2 components of lv mass , ie , lv wall thickness and lv diastolic diameter , we observed that the association of map with lv wall thickness was statistically robust and persisted after additional adjustment for central pulse pressure ( p<0.0001 for all models ) , whereas the association of map with lv diastolic diameter was weaker and no longer statistically significant ( p=0.15 ) after adjustment for central pulse pressure . central pulse pressure was positively correlated with higher lv mass ( p<0.0001 ) , which was attributable to comparable relations with lv diastolic diameter and lv wall thickness ( both p<0.0001 ) . figure 1 depicts multivariableadjusted means of lv diastolic diameter ( figure 1a ) and lv wall thickness ( figure 1b ) , stratified by tertiles of mean arterial pressure or central pulse pressure . multivariable adjusted means of left ventricular ( lv ) diastolic diameter ( a ) and lv anterior+posterior wall thickness ( b ) , plotted by tertiles of mean arterial pressure ( map ; squares ) and central pulse pressure ( cpp ; triangles ) . higher aortic stiffness , as assessed by cfpwv , was associated with greater lv mass and greater lv wall thickness , but the associations were attenuated on multivariable adjustment and rendered statistically nonsignificant on additional adjustment for mean arterial pressure . in multivariable ( including map)adjusted analyses , forward and reflected waves generally showed similar associations with lv mass , lv diastolic diameter , and lv wall thickness ( r=0.0530.089 , p<0.0001 ) , except for a nonsignificant association of the reflected wave with lv wall thickness ( r=0.034 , p=0.01 ) . in mutually adjusted analyses , forward wave relations with lv mass persisted after adjusting for reflected wave ( table 5 , p<0.0001 ) , whereas reflected wave was not related to lv mass after adjusting for forward wave ( p=0.63 ) . mean arterial pressure was not associated with lv fractional shortening ( p>0.05 in all models ) . correlations between map and lv filling measures persisted after adjustment for clinical covariates and also after additional adjustment for lv mass ( all p<0.0001 ) . central pulse pressure also correlated very modestly and inversely with e ( a measure of diastolic relaxation ) , whereas we observed a stronger direct correlation of central pulse pressure with e / e ( p<0.0001 ) , a surrogate for lv filling pressure . after additional adjustment for mean arterial pressure , the association of central pulse pressure with e / e was maintained , whereas the directionality of the modest negative association with e was reversed . multivariable adjusted means of left ventricular ( lv ) fractional shortening ( a ) and early mitral valve annulus diastolic velocity ( e , b ) , plotted by tertiles of carotidfemoral pulse wave velocity ( cfpwv ) . however , in multivariableadjusted analyses , higher augmentation index correlated modestly and inversely with lv systolic function , even after adjustment for lv mass and mean arterial pressure ( p<0.0001 , table 7 ) . reflection factor correlated inversely with fractional shortening ( r=0.077 , p<0.0001 ) but was not related to diastolic function traits ( all p>0.05 ) . when we separately investigated the 2 components of lv mass , ie , lv wall thickness and lv diastolic diameter , we observed that the association of map with lv wall thickness was statistically robust and persisted after additional adjustment for central pulse pressure ( p<0.0001 for all models ) , whereas the association of map with lv diastolic diameter was weaker and no longer statistically significant ( p=0.15 ) after adjustment for central pulse pressure . central pulse pressure was positively correlated with higher lv mass ( p<0.0001 ) , which was attributable to comparable relations with lv diastolic diameter and lv wall thickness ( both p<0.0001 ) . figure 1 depicts multivariableadjusted means of lv diastolic diameter ( figure 1a ) and lv wall thickness ( figure 1b ) , stratified by tertiles of mean arterial pressure or central pulse pressure . multivariable adjusted means of left ventricular ( lv ) diastolic diameter ( a ) and lv anterior+posterior wall thickness ( b ) , plotted by tertiles of mean arterial pressure ( map ; squares ) and central pulse pressure ( cpp ; triangles ) . higher aortic stiffness , as assessed by cfpwv , was associated with greater lv mass and greater lv wall thickness , but the associations were attenuated on multivariable adjustment and rendered statistically nonsignificant on additional adjustment for mean arterial pressure . in multivariable ( including map)adjusted analyses , forward and reflected waves generally showed similar associations with lv mass , lv diastolic diameter , and lv wall thickness ( r=0.0530.089 , p<0.0001 ) , except for a nonsignificant association of the reflected wave with lv wall thickness ( r=0.034 , p=0.01 ) . in mutually adjusted analyses , forward wave relations with lv mass persisted after adjusting for reflected wave ( table 5 , p<0.0001 ) , whereas reflected wave was not related to lv mass after adjusting for forward wave ( p=0.63 ) . mean arterial pressure was not associated with lv fractional shortening ( p>0.05 in all models ) . correlations between map and lv filling measures persisted after adjustment for clinical covariates and also after additional adjustment for lv mass ( all p<0.0001 ) . central pulse pressure also correlated very modestly and inversely with e ( a measure of diastolic relaxation ) , whereas we observed a stronger direct correlation of central pulse pressure with e / e ( p<0.0001 ) , a surrogate for lv filling pressure . after additional adjustment for mean arterial pressure , the association of central pulse pressure with e / e was maintained , whereas the directionality of the modest negative association with e was reversed . multivariable adjusted means of left ventricular ( lv ) fractional shortening ( a ) and early mitral valve annulus diastolic velocity ( e , b ) , plotted by tertiles of carotidfemoral pulse wave velocity ( cfpwv ) . however , in multivariableadjusted analyses , higher augmentation index correlated modestly and inversely with lv systolic function , even after adjustment for lv mass and mean arterial pressure ( p<0.0001 , table 7 ) . reflection factor correlated inversely with fractional shortening ( r=0.077 , p<0.0001 ) but was not related to diastolic function traits ( all p>0.05 ) . additionally adjusted for in the present investigation we examined differing associations of pulsatile and steady components of central blood pressure and aortic stiffness with left ventricular structure and function . first , both steady ( ie , map ) and pulsatile ( ie , central pulse pressure ) components of central blood pressure were correlated positively with lv mass . however , whereas higher map was primarily associated with higher lv wall thickness , central pulse pressure was positively associated with both lv diameter and lv wall thickness . third , aortic stiffness , as assessed by cfpwv , was associated inversely with lv diastolic function but showed no independent correlation with lv dimensions or circumferential lv systolic function . to our knowledge , the present analysis is the largest investigation of the relation of central hemodynamics and cfpwv ( the reference measure of aortic stiffness ) with cardiac structure and function in a sample of european ancestry . we observed that higher central pulse pressure is associated with both higher lv diameter and greater wall thickness , whereas higher map ( steady component of central pressure ) is primarily associated with higher lv wall thickness ( rather than with lv diameter ) . thus , the steady and pulsatile components of central blood pressure may conjointly yet variably contribute to differences in lv mass . of note the fact that neither aortic stiffness nor wave reflection is independently associated with lv mass in our analyses suggests that the relation between lv mass and pulse pressure may be largely attributable to mismatch between ventricular outflow and the ability of the aorta to accommodate that flow , as recently described by torjesen et al.23 existing literature on the relations among central hemodynamics , vascular stiffness , and cardiac function is sparse , and most studies were of limited size,24 , 25 , 26 , 27 , 28 restricted to certain patient populations,27 or did not include tissue doppler assessment of lv diastolic function.20 abhayaratna et al investigated the relation of arterial stiffness to lv diastolic dysfunction in a sample of 188 elderly individuals and observed a significant correlation between central pulse pressure and severity of diastolic dysfunction.29 however , cfpwv was not associated with diastolic dysfunction in their study . russo et al reported in 983 individuals that several tonometryderived measures of central hemodynamics , greater arterial stiffness , and more wave reflection were all associated with worse lv diastolic function.30 however , after multivariable adjustment , only the ratio of central pulse pressure to stroke volume index ( a measure of global arterial stiffness ) remained associated with lv diastolic dysfunction . to our knowledge , the present analysis is the largest investigation of the relation of central hemodynamics and cfpwv ( the reference measure of aortic stiffness ) with cardiac structure and function in a sample of european ancestry . we observed that higher central pulse pressure is associated with both higher lv diameter and greater wall thickness , whereas higher map ( steady component of central pressure ) is primarily associated with higher lv wall thickness ( rather than with lv diameter ) . the fact that neither aortic stiffness nor wave reflection is independently associated with lv mass in our analyses suggests that the relation between lv mass and pulse pressure may be largely attributable to mismatch between ventricular outflow and the ability of the aorta to accommodate that flow , as recently described by torjesen et al.23 existing literature on the relations among central hemodynamics , vascular stiffness , and cardiac function is sparse , and most studies were of limited size,24 , 25 , 26 , 27 , 28 restricted to certain patient populations,27 or did not include tissue doppler assessment of lv diastolic function.20 abhayaratna et al investigated the relation of arterial stiffness to lv diastolic dysfunction in a sample of 188 elderly individuals and observed a significant correlation between central pulse pressure and severity of diastolic dysfunction.29 however , cfpwv was not associated with diastolic dysfunction in their study . russo et al reported in 983 individuals that several tonometryderived measures of central hemodynamics , greater arterial stiffness , and more wave reflection were all associated with worse lv diastolic function.30 however , after multivariable adjustment , only the ratio of central pulse pressure to stroke volume index ( a measure of global arterial stiffness ) remained associated with lv diastolic dysfunction . in the present investigation , we assessed relations of steady and pulsatile components of central hemodynamics and aortic stiffness to cardiac structure and lv systolic and diastolic function in a large communitybased sample with a broad age range .

recent successes in gene transfer ( gt ) for ocular applications have revived long - standing hopes for the field . in the 1990s , gt was hailed as a miracle technology and was misleadingly referred to as gene therapy in spite of its experimental nature . despite high hopes , serious setbacks damaged gt 's reputation and resulted in significant public scrutiny . in 2007 , however , phase i trials for leber congenital amaurosis ( lca ) , an infant - onset retinopathy , displayed short - term safety and efficacy . optimistic clinicians described these early results as a paradigm shift in our management of retinal dystrophies of all types , previously thought to be an untreatable group of human diseases . while adults derived some visual benefits in the lca trials ( improvements in visual acuity , visual field , pupillary reaction , and nystagmus ) , children showed the most dramatic responses in later trials indeed , after gt , an 8-year - old child displayed comparable light sensitivity levels to age - matched controls , indicating that early gt application may lead to greater visual improvements . the results therefore suggested a limited therapeutic window for visual gain , a finding that has fueled patient urgency to access gt , especially as lca trials are now entering phase iii ( clinicaltrials.gov nct00999609 ) . while concerns have recently been raised about the long - term efficacy of the intervention in the lca trials , further early - stage gt trials for retinopathies now include stargardt disease ( clinicaltrials.gov nct01367444 ) , retinitis pigmentosa ( clinicaltrials.gov nct01482195 ) , and usher syndrome ( clinicaltrials.gov nct01505062 ) . in october 2011 , a phase i choroideremia trial was initiated in the united kingdom focused on safety and dosage ( clinicaltrials.gov nct01461213 ) . choroideremia is a sex - linked retinopathy affecting ~1 of 50,000 males . in the absence of a treatment , choroideremia causes progressive vision loss from childhood to legal blindness by middle age . positive media coverage of ocular gt of early evidence from the lca trials combined with optimistic statements by researchers and clinicians have ignited patient hopes for a successful treatment for a range of retinopathies . high hopes based on limited data , however , present challenges for communicating about risks of harm and potential benefits of early - stage trials as well as time frames for clinical application of therapies . of greatest concern , communication deficits may compromise the integrity of informed consent for potential trial participants . participants may misunderstand the safety focus of early - stage trials , expecting therapeutic benefit . termed therapeutic misconception ' , patients commonly conflate the goals of research and those of clinical care . similarly , patients may overestimate therapeutic benefits or underestimate risks of harm associated with research , termed therapeutic misestimation ' . therapeutic misconception and misestimation are not only characteristic of patients , but also affect stakeholders involved directly and indirectly in the informed consent process , such as clinical investigators and research oversight bodies . despite the rapid advances in ocular gt , little research has been directed at exploring the values and priorities of key stakeholders such as patients , patient advocates , and clinicians . similar research gaps are evident in the translation of other novel biotechnologies from bench research to clinical trials . our study , therefore , addresses multistakeholder perspectives about ocular gt trials , focusing on choroideremia . it examines the commonalities and differences in views between stakeholder groups ( patients , clinicians , and patient advocates ) . we specifically address the following issues : ( i ) potential benefits and therapeutic hopes ; ( ii ) risks of harm ; ( iii ) urgency to access gt ; and ( iv ) time frames for clinical implementation . we conclude with recommendations for responsible communications for gt trials that counter the historical sensationalism associated with this field of biotechnology . we interviewed 20 north american choroideremia patients ( p ) ; 15 clinicians from north america and europe ( c ) ; and 6 representatives of north american and european patient advocacy organizations ( advocates / a ) . patients were affected males older than 18 years of age who may be eligible to participate in the upcoming choroideremia trials . five of the 15 clinicians , ophthalmologists and genetic counselors who specialized in ocular genetics , were directly involved in ocular gt trials . advocates were representatives from the boards of patient advocacy organizations involved in fundraising for choroideremia research and patient education . we recruited patients from the regional eye centre , royal alexandra hospital , edmonton , and via notices on patient advocacy organization websites . s.b . conducted 45 min to 1 h semistructured interviews with each participant between june 2011 and june 2012 . the interview guides were informed by research on communication in genetics and were reviewed for depth and breadth of coverage by experts in ocular genetics , risk communication , and bioethics . interview guides were specific to stakeholder groups , but all addressed potential benefits , risks of harm , and time frames of choroideremia gt trials . patient interviews focused on experiences living with choroideremia ; understanding of diagnosis ; awareness of ocular gt research ; sources of communications about gt ; and views about participation in chm trials . clinician interviews focused on diagnosis ; management strategies for visual impairment ; perspectives on patient hopes ; and communication strategies with other stakeholders . interviews with advocates focused on the supports and information they provided for members ; position on gt ; communication with members , including about gt ; advocacy role ; and external stakeholder communications strategies . the health panel of the ethics review board at the university of alberta approved this study . we analyzed verbatim transcripts of digitally recorded interviews using nvivo 9.1 data analysis software ( qsr international 2010 ) . our qualitative analysis of interview transcripts , using the constant comparison method ' , involved a rigorous and iterative process . coded each line of a subset of transcripts , and analyzed these codes for similarities and differences between transcripts . starting with this close examination of the transcripts , and through ongoing discussions with other investigators , s.b . developed a preliminary codebook representative of key perspectives and ideas . analyzed the remaining transcripts and reexamined the already analyzed transcripts as new codes became apparent . our coding method provided a rich analysis of the data as multiple codes could apply to different dimensions of the same text . as explained by illes et al.,17 the goal of such analysis is to identify a broad range of perspectives , not necessarily a consensus among participants , and to deliver a coherent conceptual description of the data that capture thematic patterns and characterize the phenomena of interest while accounting for the individual variations within them . a third coder external to the project reviewed the final codebook and transcripts to provide an objective assessment . the external coder suggested collapsing closely related themes , adding nuanced subthemes to emphasize key ideas , and refining the definitions of several constructs . finally , s.b . revised transcript coding again in light of the final codebook . to ensure that we captured participant views accurately , s.b . , i.m . , and t.b . prepared reports explaining the main themes that emerged from the interviews . we sent these reports to participants , inviting them to ask additional questions or provide feedback . we also reviewed all selected quotes in the context of the original transcripts to ensure that they retained their meaning . our recruitment of patients from advocacy websites may have biased our sample to those more knowledgeable about their disease and gt , which may have influenced their risk perspectives and hopes . choroideremia trials in north america have not yet started , limiting our patient interviews to potential research participants . finally , we interviewed only a small number of advocates , who had the most heterogeneous perspectives of all stakeholders . we interviewed 20 north american choroideremia patients ( p ) ; 15 clinicians from north america and europe ( c ) ; and 6 representatives of north american and european patient advocacy organizations ( advocates / a ) . patients were affected males older than 18 years of age who may be eligible to participate in the upcoming choroideremia trials . five of the 15 clinicians , ophthalmologists and genetic counselors who specialized in ocular genetics , were directly involved in ocular gt trials . advocates were representatives from the boards of patient advocacy organizations involved in fundraising for choroideremia research and patient education . we recruited patients from the regional eye centre , royal alexandra hospital , edmonton , and via notices on patient advocacy organization websites . the interview guides were informed by research on communication in genetics and were reviewed for depth and breadth of coverage by experts in ocular genetics , risk communication , and bioethics . interview guides were specific to stakeholder groups , but all addressed potential benefits , risks of harm , and time frames of choroideremia gt trials . patient interviews focused on experiences living with choroideremia ; understanding of diagnosis ; awareness of ocular gt research ; sources of communications about gt ; and views about participation in chm trials . clinician interviews focused on diagnosis ; management strategies for visual impairment ; perspectives on patient hopes ; and communication strategies with other stakeholders . interviews with advocates focused on the supports and information they provided for members ; position on gt ; communication with members , including about gt ; advocacy role ; and external stakeholder communications strategies . the health panel of the ethics review board at the university of alberta approved this study . we analyzed verbatim transcripts of digitally recorded interviews using nvivo 9.1 data analysis software ( qsr international 2010 ) . our qualitative analysis of interview transcripts , using the constant comparison method ' , involved a rigorous and iterative process . coded each line of a subset of transcripts , and analyzed these codes for similarities and differences between transcripts . starting with this close examination of the transcripts , and through ongoing discussions with other investigators , s.b . developed a preliminary codebook representative of key perspectives and ideas . analyzed the remaining transcripts and reexamined the already analyzed transcripts as new codes became apparent . our coding method provided a rich analysis of the data as multiple codes could apply to different dimensions of the same text . as explained by illes et al.,17 the goal of such analysis is to identify a broad range of perspectives , not necessarily a consensus among participants , and to deliver a coherent conceptual description of the data that capture thematic patterns and characterize the phenomena of interest while accounting for the individual variations within them . a third coder external to the project reviewed the final codebook and transcripts to provide an objective assessment . the external coder suggested collapsing closely related themes , adding nuanced subthemes to emphasize key ideas , and refining the definitions of several constructs . finally , s.b . revised transcript coding again in light of the final codebook . to ensure that we captured participant views accurately , s.b . , i.m . , and t.b . prepared reports explaining the main themes that emerged from the interviews . we sent these reports to participants , inviting them to ask additional questions or provide feedback . we also reviewed all selected quotes in the context of the original transcripts to ensure that they retained their meaning . our recruitment of patients from advocacy websites may have biased our sample to those more knowledgeable about their disease and gt , which may have influenced their risk perspectives and hopes . choroideremia trials in north america have not yet started , limiting our patient interviews to potential research participants . finally , we interviewed only a small number of advocates , who had the most heterogeneous perspectives of all stakeholders . while stakeholders agreed on many potential benefits of gt , key differences arose in their visual outcome hopes along a continuum from no visual benefit to a complete cure . patients described aspirational ( to future patients or society ) , collateral ( arising from research participation that do not depend on the experimental intervention such as empowerment secondary to research contribution ) , and direct benefits ( associated with experimental intervention such as visual improvement ) arising from participation in a gt trial ( table 1 ) . visual benefit was the main motivator for patients , even though most recognized the possibility that gt trials may provide no visual benefit . clinician and advocate visual benefit perspectives , mediated by the outcomes of the lca trials , converged from slowing down vision loss , halting vision loss , to a partial reversal of lost vision . these stakeholders emphasized that gt could provide a treatment but not a cure because gt is unlikely to provide regenerative benefits to retinal cells that have already degenerated . patients , on the other hand , voiced hopes for a cure or a partial reversal of lost vision , but most hoped for a halt in vision loss . some clinicians raised patient difficulties in conceptualizing the meaning of a treatment for choroideremia in light of their understanding of treatment for other diseases : people say therapy like : oh , we can treat the pneumonia , therefore my lungs are normal again . i 'm going to have gene therapy all my vision is going to be restored.c11 . some clinicians also believed that patients lack the tools to distinguish between the potential visual outcomes of choroideremia gt . however , despite such clinician concerns , most patients articulated their hopes for visual outcome in a nuanced manner . much like other stakeholders , most patients hoped for a treatment rather than a cure . patients were focused on gaining access to gt rather than on risks of harm ( table 2 ) . clinicians explained that patients seldom inquired about the safety of gt and felt that patient hope for a treatment diverted attention from the risks of harm : deep down in the heart of any patient for whom there is no effective treatment or cure , clinical trials mean this might be the thing that 's going to help me so safety is , many patients questioned the interviewer about gaining access to cts rather than about risks of harm . a quarter of patients even expressed a no risk perspective : for me there 's no risks . most patients , however , acknowledged the risk of accelerated vision loss , but opinions diverged with respect to the personal relevance of this risk . others , especially those who described their visual field as significantly deteriorated , expressed a willingness to accept the risk of accelerated vision loss : if i lose my sight , it 's going anyway.p2 patients also described factors that attenuated their risk perspectives . many patients normalized the nature of risk as inherent to all trials : accepting the risk would go with [ a gt trial ] and moving the scientific project forward , because you need people to others expressed trust in the clinicians , researchers , or scientific traditions behind gt trials : i have faith in our medical system , that they do make sure that things are safe.p8 patients and patient advocates affected by choroideremia emphasized their urgency to access gt : if [ gt ] came out tomorrow i 'd have that procedure done , absolutely.p2 . but many patients worried that the time frame for clinical implementation of gt may not meet their own therapeutic window : my eyes are degenerating . i think there 's urgency , and [ gt ] would allow me to keep more of my vision the sooner i get it.p8 . others expressed frustration with the slow regulatory approval process : the treatment is not available fast enough i know everybody wants to do it in careful way ; but for me , i 'd rather take the chance and save my eyes.p20 many patients explained that they would do anything to access gt : incur financial burdens ; take time off of work ; or travel . some advocates echoed this perspective , revealing their personal stakes : i will go for the second mortgage on my home if that 's what it takes.a1 . i think anybody faced with the prospect of blindness [ would ] say that i would do anything to undertake clinical trial patients have said to me i 'd rather have a heart attack than lose my vision.c9 . uncertainty about time frames for the clinical implementation of gt was a major patient concern , particularly for patients who wanted to make practical arrangements for the future but did not know whether to accept their current prognosis and to plan for further vision loss or for the possibility of an intervention within a limited therapeutic window : i do n't even know the average time for any given clinical trial to go through the phases ... let 's assume for example that the choroideremia [ trial ] phases are all successful , is it the best case scenario for something within a two - year time frame , five years , ten years , twenty years?p10 . advocates were similarly frustrated with vague time frames presented by clinicians at fundraising venues : it 's difficult for [ clinicians ] because they 're there to generate optimism , because their goal is to generate funds at the same time they have to be cautious about not raising hopes too high , and so you get the vague answer . like , when will a certain thing be standard of care ? it will become the five to eight year time frame.a2 . advocates were further disappointed and confused when projected time frames were not met : time frames in research never seem to be accurate [ patients ask ] when is [ gt ] going to be in the clinic ? so their main question is when are we going to see the fruit of all of the research that has been done over the years that 's going to and they 're blunt about it cure the disease ? pa3 . nevertheless , most clinicians did not communicate effectively about time frames , frustrating patients with vague or dismissive responses . left to interpret vague time frames , patients shared their estimates . however , some , who already had significantly deteriorated visual fields , understood that their therapeutic window had passed . for other patients , including affected advocates , the success of the lca trials fueled hopes that adults , including themselves , will benefit from gt : it 's going to be too late for us then in the blink of an eye [ following the lca trials] that whole mindset did a complete 180 . now there 's a chance for my sight to be saved by a genetic therapy.a1 . in terms of clinical implementation of gt most advocates also believed that gt would be available to children , some displaying a great deal of certainty in this prospect : if a baby is born today , i have absolute full confidence that that baby is going to have a treatment before their eyes get so bad that there 's going to be a noticeable difference in their sight.a1 as clinicians and advocates quantified time frame estimates for the clinical implementation of gt , further ambiguities became apparent , with predictions ranging between 3 and 10 years . while some clinicians quantified their predictions , others made general remarks about the progress in the field or refused to provide estimates altogether . clinicians explained that it is difficult to predict time frames , but some suggested informing patients about the phases of clinical trials and associated average time frames : the one thing that i think would help when they [ patients ] think about research [ is] explaining those different phases of a clinical trialc2 clinicians explained that communications must balance patient hopes for visual benefit with the uncertainties of early - stage gt trials and the current reality of limited clinical care . clinicians identified a two - pronged approach to promote balanced communications about gt , which would enable clinicians to share in patient hope for a future treatment , while emphasizing the experimental nature of gt and associated risks of harm ( table 2 ) . a realistic representation of time frames may prepare patients for a prognosis of continued visual impairment if a treatment does not materialize within a limited therapeutic window . the two - pronged approach could distinguish between the theoretical promise of gt , affirming patient hopes , and the current clinical reality : we can not assume what [ choroideremia gt ] will look like in five years . we can say where we hope to go , but be very clear that that 's a wish , that is not reality right now.c3 . while stakeholders agreed on many potential benefits of gt , key differences arose in their visual outcome hopes along a continuum from no visual benefit to a complete cure . patients described aspirational ( to future patients or society ) , collateral ( arising from research participation that do not depend on the experimental intervention such as empowerment secondary to research contribution ) , and direct benefits ( associated with experimental intervention such as visual improvement ) arising from participation in a gt trial ( table 1 ) . visual benefit was the main motivator for patients , even though most recognized the possibility that gt trials may provide no visual benefit . clinician and advocate visual benefit perspectives , mediated by the outcomes of the lca trials , converged from slowing down vision loss , halting vision loss , to a partial reversal of lost vision . these stakeholders emphasized that gt could provide a treatment but not a cure because gt is unlikely to provide regenerative benefits to retinal cells that have already degenerated . patients , on the other hand , voiced hopes for a cure or a partial reversal of lost vision , but most hoped for a halt in vision loss . some clinicians raised patient difficulties in conceptualizing the meaning of a treatment for choroideremia in light of their understanding of treatment for other diseases : people say therapy like : oh , we can treat the pneumonia , therefore my lungs are normal again . i 'm going to have gene therapy all my vision is going to be restored.c11 . some clinicians also believed that patients lack the tools to distinguish between the potential visual outcomes of choroideremia gt . however , despite such clinician concerns , most patients articulated their hopes for visual outcome in a nuanced manner . much like other stakeholders , most patients hoped for a treatment rather than a cure . patients were focused on gaining access to gt rather than on risks of harm ( table 2 ) . clinicians explained that patients seldom inquired about the safety of gt and felt that patient hope for a treatment diverted attention from the risks of harm : deep down in the heart of any patient for whom there is no effective treatment or cure , clinical trials mean this might be the thing that 's going to help me so safety is not their first concern.c10 . supporting this view , many patients questioned the interviewer about gaining access to cts rather than about risks of harm . a quarter of patients even expressed a no risk perspective : for me there 's no risks . most patients , however , acknowledged the risk of accelerated vision loss , but opinions diverged with respect to the personal relevance of this risk . some patients were hesitant in risking their remaining functional vision . others , especially those who described their visual field as significantly deteriorated , expressed a willingness to accept the risk of accelerated vision loss : if i lose my sight , it 's going anyway.p2 patients also described factors that attenuated their risk perspectives . many patients normalized the nature of risk as inherent to all trials : accepting the risk would go with [ a gt trial ] and moving the scientific project forward , because you need people to others expressed trust in the clinicians , researchers , or scientific traditions behind gt trials : i have faith in our medical system , that they do make sure that things are safe.p8 patients and patient advocates affected by choroideremia emphasized their urgency to access gt : if [ gt ] came out tomorrow i 'd have that procedure done , absolutely.p2 . but many patients worried that the time frame for clinical implementation of gt may not meet their own therapeutic window : my eyes are degenerating . i think there 's urgency , and [ gt ] would allow me to keep more of my vision the sooner i get it.p8 . others expressed frustration with the slow regulatory approval process : the treatment is not available fast enough i know everybody wants to do it in careful way ; but for me , i 'd rather take the chance and save my eyes.p20 many patients explained that they would do anything to access gt : incur financial burdens ; take time off of work ; or travel . some advocates echoed this perspective , revealing their personal stakes : i will go for the second mortgage on my home if that 's what it takes.a1 . i think anybody faced with the prospect of blindness [ would ] say that i would do anything to undertake clinical trial patients have said to me uncertainty about time frames for the clinical implementation of gt was a major patient concern , particularly for patients who wanted to make practical arrangements for the future but did not know whether to accept their current prognosis and to plan for further vision loss or for the possibility of an intervention within a limited therapeutic window : i do n't even know the average time for any given clinical trial to go through the phases ... let 's assume for example that the choroideremia [ trial ] phases are all successful , is it the best case scenario for something within a two - year time frame , five years , ten years , twenty years?p10 . advocates were similarly frustrated with vague time frames presented by clinicians at fundraising venues : it 's difficult for [ clinicians ] because they 're there to generate optimism , because their goal is to generate funds at the same time they have to be cautious about not raising hopes too high , and so you get the vague answer . advocates were further disappointed and confused when projected time frames were not met : time frames in research never seem to be accurate [ patients ask ] when is [ gt ] going to be in the clinic ? so their main question is when are we going to see the fruit of all of the research that has been done over the years that 's going to and they 're blunt about it cure the disease ? pa3 . nevertheless , most clinicians did not communicate effectively about time frames , frustrating patients with vague or dismissive responses . however , some , who already had significantly deteriorated visual fields , understood that their therapeutic window had passed . for other patients , including affected advocates , the success of the lca trials fueled hopes that adults , including themselves , will benefit from gt : it 's going to be too late for us then in the blink of an eye [ following the lca trials] that whole mindset did a complete 180 . now there 's a chance for my sight to be saved by a genetic therapy.a1 . in terms of clinical implementation of gt most advocates also believed that gt would be available to children , some displaying a great deal of certainty in this prospect : if a baby is born today , i have absolute full confidence that that baby is going to have a treatment before their eyes get so bad that there 's going to be a noticeable difference in their sight.a1 as clinicians and advocates quantified time frame estimates for the clinical implementation of gt , further ambiguities became apparent , with predictions ranging between 3 and 10 years . while some clinicians quantified their predictions , others made general remarks about the progress in the field or refused to provide estimates altogether . clinicians explained that it is difficult to predict time frames , but some suggested informing patients about the phases of clinical trials and associated average time frames : the one thing that i think would help when they [ patients ] think about research [ is] explaining those different phases of a clinical trialc2 clinicians explained that communications must balance patient hopes for visual benefit with the uncertainties of early - stage gt trials and the current reality of limited clinical care . clinicians identified a two - pronged approach to promote balanced communications about gt , which would enable clinicians to share in patient hope for a future treatment , while emphasizing the experimental nature of gt and associated risks of harm ( table 2 ) . a realistic representation of time frames may prepare patients for a prognosis of continued visual impairment if a treatment does not materialize within a limited therapeutic window . the two - pronged approach could distinguish between the theoretical promise of gt , affirming patient hopes , and the current clinical reality : we can not assume what [ choroideremia gt ] will look like in five years . we can say where we hope to go , but be very clear that that 's a wish , that is not reality right now.c3 . the prospect of gt for choroideremia has fueled patient hopes for therapeutic benefit ; provided a sense of urgency for its clinical implementation ; and , in the interim , motivated patients to privilege considerations of potential benefits over risks of harm in anticipated clinical trial recruitment . most participants cited qualified hopes for potential benefits rather than confident expectations . in other words , they remained optimistic without being able to express specific likelihoods for benefits and acknowledged the possibility that gt may not generate visual benefits . similar to other studies , patient hope for direct benefits greatly surpassed aspirational and collateral benefits as a motivator for trial participation , a finding indicative of a therapeutic misconception . when stakeholders view cts as both science and a source of succor or an opportunity for patient care , problems arise because there is a less than 1% chance for clinical improvement in phase i gt trials . nevertheless , lazarus - like responses have occurred in phase i trials , generating debate on the appropriateness of discussing direct benefits . in oncology trials , critics raise concerns about the use of surrogate measures ( e.g. , tumor response , stable disease ) , rather than end point measures ( e.g. , increased survival , improved quality of life ) . surrogate measures may not be clinically meaningful and , at best , are suggestive of direct benefit . gt , as a novel and complex intervention , is at the forefront adapted trial design use , such as the use of secondary outcome measures . these may obscure the research - treatment distinction in early - phase trials , further confounding communications about the safety focus of phase i trials . secondary outcome measures in ocular phase i gt trials ( e.g. , microperimetry , fundus imaging ) confirm safety , but also measure potential efficacy . the lca trials employed surrogate end point measures with significant shortcomings : end point measures for long - term safety and efficacy are still under development , as are measures for clinically meaningful improved visual function and quality of life . although disputed by lead researchers of the lca trials , a recent analysis indicates photoreceptor degradation continues after gt despite short - term improvements in visual function , raising concerns about long - term efficacy of gt . other clinical trial design decisions , such as the inclusion of children in a phase i lca trial to accommodate the limited therapeutic window of patients , further blurs the research - treatment distinction . the implications of such adaptations to standard phase i clinical trial designs for the therapeutic hopes of patients and clinical investigators require further investigation . however , our results show that patients overestimated therapeutic benefit compared with other stakeholder groups . gt is not regenerative ; it can not revive degenerated photoreceptor cells and so can not cure adult patients . discrepancies may be due to heterogeneous interpretations of benefit from disease amelioration to cure . therapeutic misestimation engages not only overestimation of potential benefits , but also underestimation risks of harm . as such , patient attention was diverted from risks of harm and directed toward gaining access to trials . trust also mediated patient risk perspectives : patients revealed trust in science and trust in their physicians . trust allows patients to reinterpret uncertainties in phase i trials as opportunities for potential benefits rather than as risks of harm . despite prominent discussion of therapeutic benefits , gt has often been characterized as permanently 5 years away from clinical application . media portrayals of gt as an imminent cure , supported by positive quotations by leading researchers , reinforce this perspective . overly optimistic hopes about time frames may not only lead to disappointment but also undermine the stated trust in researchers and threaten funding support . while predicting the future of ocular gt is impossible , the frustration of patients and advocates about uncertain or delayed time frames necessitates improved communication strategies . research on communicating time frames , however , has focused primarily on communicating patient prognoses . communications of clinicians and advocacy organizations with patients must account for both therapeutic misconception and misestimation . the former demands a detailed explanation of the safety focus of early - stage clinical trials . however , the safety focus of phase i trials does not preclude the prospect of direct benefit . communicators should therefore highlight that , while choroideremia gt could theoretically result in direct visual benefit , clinical equipoise exists given the lack of empirical data . ensuring patient comprehension of the relevant risks of harm and uncertainties , qualifying incremental evidence in cases where medical benefits were observed in the lca trials ( i.e. , measures of long - term efficacy and clinically meaningful improvement in quality of life are still under development ) , and emphasizing that aspirational and collateral benefits are more likely than direct medical benefits , are key strategies in promoting informed hope among patients and informed consent for participation in gt trials . to address therapeutic misestimation , patient communications should be clear about the full spectrum of theoretically feasible visual outcomes from gt , in general , and in light of patient - specific therapeutic windows . clinicians and advocates should be aware of the dominance of curative public discourse in the media and beyond and be prepared to counter such representations . communicators should clearly situate the current state of choroideremia gt within the context of clinical research stages and time frames . as an exemplar , the lca trials began in 2007 , but are only now , in 2013 , beginning to enroll patients in phase iii studies ( clinicaltrials.gov nct00999609 ) . historical evidence suggests that it takes 1014 years to move from novel target to drug approval . novel biotechnologies , such as gt , given their unique risk profile and additional regulatory steps , may take even longer . while grounded in realistic visual benefits and time frames , patient communicators should not downplay the hope raised by promising novel biotechnologies . a two - pronged approach to patient communications balances such hope with pragmatic discussion about strategies for living with progressive visual impairment . patient advocacy organizations , in particular , should carefully balance their communications to raise funds for promising research against more pragmatic information on disease management , peer support , and access to credible information sources . finally , patients demonstrated a more nuanced understanding of potential visual outcomes than suggested by clinicians . it is therefore important for other stakeholder groups to recognize patients as critically thinking experts , while acknowledging patient vulnerabilities . with this understanding , clinicians and patient advocacy organizations should focus on the quality of their communications in terms of presenting a balanced account of risks , benefits , and the state of research advances , rather than attempting to manage patient expectations . with these strategies , communicators may counter the sensationalism historically associated with gt , promote informed consent , and honor patient hope while grounding communications in current clinical realities . communications of clinicians and advocacy organizations with patients must account for both therapeutic misconception and misestimation . the former demands a detailed explanation of the safety focus of early - stage clinical trials . however , the safety focus of phase i trials does not preclude the prospect of direct benefit . communicators should therefore highlight that , while choroideremia gt could theoretically result in direct visual benefit , clinical equipoise exists given the lack of empirical data . ensuring patient comprehension of the relevant risks of harm and uncertainties , qualifying incremental evidence in cases where medical benefits were observed in the lca trials ( i.e. , measures of long - term efficacy and clinically meaningful improvement in quality of life are still under development ) , and emphasizing that aspirational and collateral benefits are more likely than direct medical benefits , are key strategies in promoting informed hope among patients and informed consent for participation in gt trials . to address therapeutic misestimation , patient communications should be clear about the full spectrum of theoretically feasible visual outcomes from gt , in general , and in light of patient - specific therapeutic windows . clinicians and advocates should be aware of the dominance of curative public discourse in the media and beyond and be prepared to counter such representations . communicators should clearly situate the current state of choroideremia gt within the context of clinical research stages and time frames . as an exemplar , the lca trials began in 2007 , but are only now , in 2013 , beginning to enroll patients in phase iii studies ( clinicaltrials.gov nct00999609 ) . historical evidence suggests that it takes 1014 years to move from novel target to drug approval . novel biotechnologies , such as gt , given their unique risk profile and additional regulatory steps , may take even longer . while grounded in realistic visual benefits and time frames , patient communicators should not downplay the hope raised by promising novel biotechnologies . a two - pronged approach to patient communications balances such hope with pragmatic discussion about strategies for living with progressive visual impairment . patient advocacy organizations , in particular , should carefully balance their communications to raise funds for promising research against more pragmatic information on disease management , peer support , and access to credible information sources . finally , patients demonstrated a more nuanced understanding of potential visual outcomes than suggested by clinicians . it is therefore important for other stakeholder groups to recognize patients as critically thinking experts , while acknowledging patient vulnerabilities . with this understanding , clinicians and patient advocacy organizations should focus on the quality of their communications in terms of presenting a balanced account of risks , benefits , and the state of research advances , rather than attempting to manage patient expectations . with these strategies , communicators may counter the sensationalism historically associated with gt , promote informed consent , and honor patient hope while grounding communications in current clinical realities .

purpose : ocular gene transfer clinical trials are raising patient hopes for the treatment of choroideremia a blinding degenerative retinopathy . phase i choroideremia gene transfer trials necessitate communicating about the risks of harm and potential benefits with patients while avoiding the sensationalism that has historically undermined this field of translational medicine.methods:we conducted interviews between june 2011 and june 2012 with 6 choroideremia patient advocates , 20 patients , and 15 clinicians about their hopes for benefits , perceived risks of harm , and hopes for the time frame of clinical implementation of choroideremia gene transfer.results:despite the safety focus of phase i trials , participants hoped for direct visual benefits with evident discrepancies between stakeholder perspectives about the degree of visual benefit . clinicians and patient advocates were concerned by limited patient attention to risks of harm . interviews revealed confusion about the time frames for the clinical implementation of choroideremia gene transfer and patient urgency to access gene transfer within a limited therapeutic window.conclusion:differences in stakeholder perspectives about choroideremia gene transfer necessitate strategies that promote responsible communications about choroideremia gene transfer and aid in its translation . strategies should counter historical sensationalism associated with gene transfer , promote informed consent , and honor patient hope while grounding communications in current clinical realities .

Introduction Materials and Methods Participants Data collection Data analysis Study limitations Results Potential benefits of choroideremia GT Risks of harm of choroideremia GT Urgency: I would do Time frames: I know it's in sight, but will it be in Balancing risks of harm and potential benefits in light of uncertain time frames: the two-pronged approach Discussion Recommendations and conclusion Disclosure

recent successes in gene transfer ( gt ) for ocular applications have revived long - standing hopes for the field . while adults derived some visual benefits in the lca trials ( improvements in visual acuity , visual field , pupillary reaction , and nystagmus ) , children showed the most dramatic responses in later trials indeed , after gt , an 8-year - old child displayed comparable light sensitivity levels to age - matched controls , indicating that early gt application may lead to greater visual improvements . the results therefore suggested a limited therapeutic window for visual gain , a finding that has fueled patient urgency to access gt , especially as lca trials are now entering phase iii ( clinicaltrials.gov nct00999609 ) . while concerns have recently been raised about the long - term efficacy of the intervention in the lca trials , further early - stage gt trials for retinopathies now include stargardt disease ( clinicaltrials.gov nct01367444 ) , retinitis pigmentosa ( clinicaltrials.gov nct01482195 ) , and usher syndrome ( clinicaltrials.gov nct01505062 ) . in october 2011 , a phase i choroideremia trial was initiated in the united kingdom focused on safety and dosage ( clinicaltrials.gov nct01461213 ) . positive media coverage of ocular gt of early evidence from the lca trials combined with optimistic statements by researchers and clinicians have ignited patient hopes for a successful treatment for a range of retinopathies . high hopes based on limited data , however , present challenges for communicating about risks of harm and potential benefits of early - stage trials as well as time frames for clinical application of therapies . participants may misunderstand the safety focus of early - stage trials , expecting therapeutic benefit . similarly , patients may overestimate therapeutic benefits or underestimate risks of harm associated with research , termed therapeutic misestimation ' . therapeutic misconception and misestimation are not only characteristic of patients , but also affect stakeholders involved directly and indirectly in the informed consent process , such as clinical investigators and research oversight bodies . despite the rapid advances in ocular gt , little research has been directed at exploring the values and priorities of key stakeholders such as patients , patient advocates , and clinicians . it examines the commonalities and differences in views between stakeholder groups ( patients , clinicians , and patient advocates ) . we specifically address the following issues : ( i ) potential benefits and therapeutic hopes ; ( ii ) risks of harm ; ( iii ) urgency to access gt ; and ( iv ) time frames for clinical implementation . we conclude with recommendations for responsible communications for gt trials that counter the historical sensationalism associated with this field of biotechnology . conducted 45 min to 1 h semistructured interviews with each participant between june 2011 and june 2012 . interview guides were specific to stakeholder groups , but all addressed potential benefits , risks of harm , and time frames of choroideremia gt trials . as explained by illes et al.,17 the goal of such analysis is to identify a broad range of perspectives , not necessarily a consensus among participants , and to deliver a coherent conceptual description of the data that capture thematic patterns and characterize the phenomena of interest while accounting for the individual variations within them . our recruitment of patients from advocacy websites may have biased our sample to those more knowledgeable about their disease and gt , which may have influenced their risk perspectives and hopes . advocates were representatives from the boards of patient advocacy organizations involved in fundraising for choroideremia research and patient education . interview guides were specific to stakeholder groups , but all addressed potential benefits , risks of harm , and time frames of choroideremia gt trials . as explained by illes et al.,17 the goal of such analysis is to identify a broad range of perspectives , not necessarily a consensus among participants , and to deliver a coherent conceptual description of the data that capture thematic patterns and characterize the phenomena of interest while accounting for the individual variations within them . our recruitment of patients from advocacy websites may have biased our sample to those more knowledgeable about their disease and gt , which may have influenced their risk perspectives and hopes . while stakeholders agreed on many potential benefits of gt , key differences arose in their visual outcome hopes along a continuum from no visual benefit to a complete cure . patients described aspirational ( to future patients or society ) , collateral ( arising from research participation that do not depend on the experimental intervention such as empowerment secondary to research contribution ) , and direct benefits ( associated with experimental intervention such as visual improvement ) arising from participation in a gt trial ( table 1 ) . visual benefit was the main motivator for patients , even though most recognized the possibility that gt trials may provide no visual benefit . patients , on the other hand , voiced hopes for a cure or a partial reversal of lost vision , but most hoped for a halt in vision loss . however , despite such clinician concerns , most patients articulated their hopes for visual outcome in a nuanced manner . patients were focused on gaining access to gt rather than on risks of harm ( table 2 ) . clinicians explained that patients seldom inquired about the safety of gt and felt that patient hope for a treatment diverted attention from the risks of harm : deep down in the heart of any patient for whom there is no effective treatment or cure , clinical trials mean this might be the thing that 's going to help me so safety is , many patients questioned the interviewer about gaining access to cts rather than about risks of harm . many patients normalized the nature of risk as inherent to all trials : accepting the risk would go with [ a gt trial ] and moving the scientific project forward , because you need people to others expressed trust in the clinicians , researchers , or scientific traditions behind gt trials : i have faith in our medical system , that they do make sure that things are safe.p8 patients and patient advocates affected by choroideremia emphasized their urgency to access gt : if [ gt ] came out tomorrow i 'd have that procedure done , absolutely.p2 . but many patients worried that the time frame for clinical implementation of gt may not meet their own therapeutic window : my eyes are degenerating . uncertainty about time frames for the clinical implementation of gt was a major patient concern , particularly for patients who wanted to make practical arrangements for the future but did not know whether to accept their current prognosis and to plan for further vision loss or for the possibility of an intervention within a limited therapeutic window : i do n't even know the average time for any given clinical trial to go through the phases ... let 's assume for example that the choroideremia [ trial ] phases are all successful , is it the best case scenario for something within a two - year time frame , five years , ten years , twenty years?p10 . advocates were similarly frustrated with vague time frames presented by clinicians at fundraising venues : it 's difficult for [ clinicians ] because they 're there to generate optimism , because their goal is to generate funds at the same time they have to be cautious about not raising hopes too high , and so you get the vague answer . advocates were further disappointed and confused when projected time frames were not met : time frames in research never seem to be accurate [ patients ask ] when is [ gt ] going to be in the clinic ? for other patients , including affected advocates , the success of the lca trials fueled hopes that adults , including themselves , will benefit from gt : it 's going to be too late for us then in the blink of an eye [ following the lca trials] that whole mindset did a complete 180 . in terms of clinical implementation of gt most advocates also believed that gt would be available to children , some displaying a great deal of certainty in this prospect : if a baby is born today , i have absolute full confidence that that baby is going to have a treatment before their eyes get so bad that there 's going to be a noticeable difference in their sight.a1 as clinicians and advocates quantified time frame estimates for the clinical implementation of gt , further ambiguities became apparent , with predictions ranging between 3 and 10 years . clinicians explained that it is difficult to predict time frames , but some suggested informing patients about the phases of clinical trials and associated average time frames : the one thing that i think would help when they [ patients ] think about research [ is] explaining those different phases of a clinical trialc2 clinicians explained that communications must balance patient hopes for visual benefit with the uncertainties of early - stage gt trials and the current reality of limited clinical care . clinicians identified a two - pronged approach to promote balanced communications about gt , which would enable clinicians to share in patient hope for a future treatment , while emphasizing the experimental nature of gt and associated risks of harm ( table 2 ) . a realistic representation of time frames may prepare patients for a prognosis of continued visual impairment if a treatment does not materialize within a limited therapeutic window . the two - pronged approach could distinguish between the theoretical promise of gt , affirming patient hopes , and the current clinical reality : we can not assume what [ choroideremia gt ] will look like in five years . while stakeholders agreed on many potential benefits of gt , key differences arose in their visual outcome hopes along a continuum from no visual benefit to a complete cure . patients described aspirational ( to future patients or society ) , collateral ( arising from research participation that do not depend on the experimental intervention such as empowerment secondary to research contribution ) , and direct benefits ( associated with experimental intervention such as visual improvement ) arising from participation in a gt trial ( table 1 ) . visual benefit was the main motivator for patients , even though most recognized the possibility that gt trials may provide no visual benefit . clinician and advocate visual benefit perspectives , mediated by the outcomes of the lca trials , converged from slowing down vision loss , halting vision loss , to a partial reversal of lost vision . patients , on the other hand , voiced hopes for a cure or a partial reversal of lost vision , but most hoped for a halt in vision loss . however , despite such clinician concerns , most patients articulated their hopes for visual outcome in a nuanced manner . clinicians explained that patients seldom inquired about the safety of gt and felt that patient hope for a treatment diverted attention from the risks of harm : deep down in the heart of any patient for whom there is no effective treatment or cure , clinical trials mean this might be the thing that 's going to help me so safety is not their first concern.c10 . supporting this view , many patients questioned the interviewer about gaining access to cts rather than about risks of harm . many patients normalized the nature of risk as inherent to all trials : accepting the risk would go with [ a gt trial ] and moving the scientific project forward , because you need people to others expressed trust in the clinicians , researchers , or scientific traditions behind gt trials : i have faith in our medical system , that they do make sure that things are safe.p8 patients and patient advocates affected by choroideremia emphasized their urgency to access gt : if [ gt ] came out tomorrow i 'd have that procedure done , absolutely.p2 . but many patients worried that the time frame for clinical implementation of gt may not meet their own therapeutic window : my eyes are degenerating . others expressed frustration with the slow regulatory approval process : the treatment is not available fast enough i know everybody wants to do it in careful way ; but for me , i 'd rather take the chance and save my eyes.p20 many patients explained that they would do anything to access gt : incur financial burdens ; take time off of work ; or travel . i think anybody faced with the prospect of blindness [ would ] say that i would do anything to undertake clinical trial patients have said to me uncertainty about time frames for the clinical implementation of gt was a major patient concern , particularly for patients who wanted to make practical arrangements for the future but did not know whether to accept their current prognosis and to plan for further vision loss or for the possibility of an intervention within a limited therapeutic window : i do n't even know the average time for any given clinical trial to go through the phases ... let 's assume for example that the choroideremia [ trial ] phases are all successful , is it the best case scenario for something within a two - year time frame , five years , ten years , twenty years?p10 . advocates were similarly frustrated with vague time frames presented by clinicians at fundraising venues : it 's difficult for [ clinicians ] because they 're there to generate optimism , because their goal is to generate funds at the same time they have to be cautious about not raising hopes too high , and so you get the vague answer . advocates were further disappointed and confused when projected time frames were not met : time frames in research never seem to be accurate [ patients ask ] when is [ gt ] going to be in the clinic ? in terms of clinical implementation of gt most advocates also believed that gt would be available to children , some displaying a great deal of certainty in this prospect : if a baby is born today , i have absolute full confidence that that baby is going to have a treatment before their eyes get so bad that there 's going to be a noticeable difference in their sight.a1 as clinicians and advocates quantified time frame estimates for the clinical implementation of gt , further ambiguities became apparent , with predictions ranging between 3 and 10 years . clinicians explained that it is difficult to predict time frames , but some suggested informing patients about the phases of clinical trials and associated average time frames : the one thing that i think would help when they [ patients ] think about research [ is] explaining those different phases of a clinical trialc2 clinicians explained that communications must balance patient hopes for visual benefit with the uncertainties of early - stage gt trials and the current reality of limited clinical care . clinicians identified a two - pronged approach to promote balanced communications about gt , which would enable clinicians to share in patient hope for a future treatment , while emphasizing the experimental nature of gt and associated risks of harm ( table 2 ) . a realistic representation of time frames may prepare patients for a prognosis of continued visual impairment if a treatment does not materialize within a limited therapeutic window . the two - pronged approach could distinguish between the theoretical promise of gt , affirming patient hopes , and the current clinical reality : we can not assume what [ choroideremia gt ] will look like in five years . the prospect of gt for choroideremia has fueled patient hopes for therapeutic benefit ; provided a sense of urgency for its clinical implementation ; and , in the interim , motivated patients to privilege considerations of potential benefits over risks of harm in anticipated clinical trial recruitment . most participants cited qualified hopes for potential benefits rather than confident expectations . in other words , they remained optimistic without being able to express specific likelihoods for benefits and acknowledged the possibility that gt may not generate visual benefits . similar to other studies , patient hope for direct benefits greatly surpassed aspirational and collateral benefits as a motivator for trial participation , a finding indicative of a therapeutic misconception . nevertheless , lazarus - like responses have occurred in phase i trials , generating debate on the appropriateness of discussing direct benefits . in oncology trials , critics raise concerns about the use of surrogate measures ( e.g. these may obscure the research - treatment distinction in early - phase trials , further confounding communications about the safety focus of phase i trials . other clinical trial design decisions , such as the inclusion of children in a phase i lca trial to accommodate the limited therapeutic window of patients , further blurs the research - treatment distinction . the implications of such adaptations to standard phase i clinical trial designs for the therapeutic hopes of patients and clinical investigators require further investigation . therapeutic misestimation engages not only overestimation of potential benefits , but also underestimation risks of harm . as such , patient attention was diverted from risks of harm and directed toward gaining access to trials . trust allows patients to reinterpret uncertainties in phase i trials as opportunities for potential benefits rather than as risks of harm . communications of clinicians and advocacy organizations with patients must account for both therapeutic misconception and misestimation . the former demands a detailed explanation of the safety focus of early - stage clinical trials . however , the safety focus of phase i trials does not preclude the prospect of direct benefit . communicators should therefore highlight that , while choroideremia gt could theoretically result in direct visual benefit , clinical equipoise exists given the lack of empirical data . ensuring patient comprehension of the relevant risks of harm and uncertainties , qualifying incremental evidence in cases where medical benefits were observed in the lca trials ( i.e. , measures of long - term efficacy and clinically meaningful improvement in quality of life are still under development ) , and emphasizing that aspirational and collateral benefits are more likely than direct medical benefits , are key strategies in promoting informed hope among patients and informed consent for participation in gt trials . to address therapeutic misestimation , patient communications should be clear about the full spectrum of theoretically feasible visual outcomes from gt , in general , and in light of patient - specific therapeutic windows . communicators should clearly situate the current state of choroideremia gt within the context of clinical research stages and time frames . while grounded in realistic visual benefits and time frames , patient communicators should not downplay the hope raised by promising novel biotechnologies . with this understanding , clinicians and patient advocacy organizations should focus on the quality of their communications in terms of presenting a balanced account of risks , benefits , and the state of research advances , rather than attempting to manage patient expectations . with these strategies , communicators may counter the sensationalism historically associated with gt , promote informed consent , and honor patient hope while grounding communications in current clinical realities . communications of clinicians and advocacy organizations with patients must account for both therapeutic misconception and misestimation . the former demands a detailed explanation of the safety focus of early - stage clinical trials . however , the safety focus of phase i trials does not preclude the prospect of direct benefit . communicators should therefore highlight that , while choroideremia gt could theoretically result in direct visual benefit , clinical equipoise exists given the lack of empirical data . ensuring patient comprehension of the relevant risks of harm and uncertainties , qualifying incremental evidence in cases where medical benefits were observed in the lca trials ( i.e. , measures of long - term efficacy and clinically meaningful improvement in quality of life are still under development ) , and emphasizing that aspirational and collateral benefits are more likely than direct medical benefits , are key strategies in promoting informed hope among patients and informed consent for participation in gt trials . communicators should clearly situate the current state of choroideremia gt within the context of clinical research stages and time frames . while grounded in realistic visual benefits and time frames , patient communicators should not downplay the hope raised by promising novel biotechnologies . with this understanding , clinicians and patient advocacy organizations should focus on the quality of their communications in terms of presenting a balanced account of risks , benefits , and the state of research advances , rather than attempting to manage patient expectations . with these strategies , communicators may counter the sensationalism historically associated with gt , promote informed consent , and honor patient hope while grounding communications in current clinical realities .

primary health care ( phc ) in the islamic republic of iran has experienced three 12-year periods : the first twelve years , i.e. 1972 through 1983 , gaining experiences and period and development of the program , the second 12-year period , i.e. 1984 through1995 , quantitative and qualitative development of the health care network , and during the third one ( 1996 - 2008 ) , the country has witnessed efforts for effecting fundamental reforms in the health care networks which despite its gradual speed , is so important . generally , the importance of human resources management ( hrm ) to the success or failure of health system performance has , until recently , been generally overlooked . in recent years , it has been increasingly recognized that getting hr policy and management right has to be at the core of any sustainable solution to health system performance . today , there is a growing interest in the psychosocial work environment of health care staff since they are at high risk for burnout , role conflict and job dissatisfaction . from many accounts healthcare professionals professional burnout is generally described as prolonged stress that impairs one 's ability to perform his or her job in demanding situations ; because they are exposed to psychological , emotional , and also physical stress . the previous studies generally have been reviewed the job stressors , psychological - behavioral responses to the job stress , organizational support and practices , physical and psychological responses to work , patient relationships and other job content , and other external factors in the nurses . a few studies have been done about factors associated with the burnout and burnout dimensions in the physicians and health workers . very limited studies in the world and iran also have been done about job satisfaction , job stress , and affecting factors on the burnout and its consequences in the primary health staff . according to the recent studies , there are too many limitations and high pressures which indicated the high risk of job stress for the employees in the position of the community health care . furthermore , the frequency , intensity and the effect of job stress in the occurrence of various dimensions of job stress and depression have been studied and reviewed largely on the nurses and in a relatively lower rate on the physicians and health workers . the present study aimed to describe and interpret the experiences of the employees , such as family physician , midwives , and health workers , from their own working condition in the rural health centers three decades after their vast and valuable activities using qualitative approach . the present study conducted in a qualitative research approach and content analysis method through individual and group interviews with 26 employed primary health care providers ( including 7 family physicians , 7 midwives and 12 health workers ) in the rural health centers in isfahan from october 2008 through september 2010 . the inclusion criteria were having at least one year experience and providing full - time services in the health houses and health centers . in order to collect the data , the participants were invited to form the focus group meeting after obtaining permission from the deputy vice chancellor research and cooperation with health deputy of isfahan university of medical sciences and providing introduction letter to provincial health center . explanation and justification about the aims of study was clearly stated and written consent form was received from the participants . the in - depth and semi - structured interviews -suitable for the qualitative studies- conducted in individual and group interviews using open ended questions . analyzing the data was done using content analysis method . then , in order for inductive analysis , organizing data was done using open coding process , creating subcategories and categories and abstraction . in this study , each one of the interviews formed the study analysis unit . the interviews divided into semantic units and then the semantic units were compressed and summarized . the summarized semantic units had been more abstracted and were converted to the codes . at the time of compressing and coding the semantic units , temporary categories were discussed and revised by the researchers . at the end , the basic and infrastructure meaning i.e. the hidden content in the categories were regulated in the form of six themes and these themes ultimately were put into the overall and central theme of the burnout which indicated the main theme and substantial data . trustworthiness of the data was analyzed through revising by the participants and reviewing by the non - participants . credibility of the findings and data quality coverage was evaluated by the categories through showing quotations method from the written text and also searching the coincidence and agreement between other researchers , experts , and the participants . verifiability and auditing the data was done through description of full details of analysis process and obtaining the results so that the readers could have a clear understanding of done analysis method , its strengths , and limitations . conformability or authentication had been strengthened by offering rich and strong results along with appropriate quotations and peer review debriefing by some of the professors and other people who were experienced in qualitative researches . participants profile based on profession , age , sex , work experience and marital status in the first step of the content analysis from analysis of the data obtained from individual interviews and focus group , 991 codes , 55 subcategory , and 21 categories were obtained . in the second step the mentioned themes indicated a main and more important theme called burnout , which are as the following : six central themes associated with the burnout frequent ( and often unexpected ) changes in type of the services and instability in definition of the target population and recipients of the services following the new programs and instructions which every day are sent from the higher centers and authorities with no clear purpose , cause instability , turbulence , and tiredness in the health care providers : in my opinion , changes should be purposeful . it means , in every system , we naturally need changes because the system needs improvement . our processes should be improved too , but these changes are not purposeful[d2 c28 ] ; the number of offices have increased dramatically with too many changes which are not really helpful for people and health . some of them expressed dissatisfaction from lack of awareness from their future obligations and tasks , being unimportant in the system and not being the consultant party to determine the type of the reforms and its procedure as an executor and also a beneficiary person : that means some unnecessary tasks . we really have no idea about what we are supposed to do tomorrow[d2 c314 ] ; they never ask about our opinions in any tasks [ b11 c227 ] . the physicians expressed their dissatisfaction from the coverage population instability and their increase ; the midwives from the frequency of the covered villages and providing services to other villages with unfamiliar people ; and mid - wives and health workers dissatisfied from providing service to the high risk afghan population . the care providers complained about multiple , inappropriate , hindrance and non - beneficial regulations , and had an unpleasant feeling and experience toward them : they waste our useful time and this is the same for doctors and midwives too . we suggested several times , a doctor and a midwife should be incharge and assistants can have a time out , but no one paid attention and the system did nt accept [ d6 c57 ] . they also complained about rigid and inflexible regulations and laws , hindrance instructions and inconsistent laws to the community conditions : in the present situation , the physicians are evaluated based on the statistics and the recorded issues due to the little flexibility of the system . one of the most important factors for their dissatisfaction was increasing their working hours regardless of the actual needs of the people , because all those hours were in the afternoon which was non - beneficial and boring:our work time is too long . we are too exhausted to get home at 5:00 [ b4 c24 ] . unbalanced workload and manpower caused stress and pressure on the care providers due to high population coverage and lack of sufficient manpower : there is too much work and it is too diverse too [ b4 c266 ] ; every year and even everyday , they increase the responsibilities of the health workers [ b3 c28 ] . all of them complained about high number of the clients and providing service to the high population in a limited time : i think each of us should visit at least about 50 or 60 patients , but i think it is not practically possible [ d7 c2 ] . all of them , particularly the health workers , expressed dissatisfaction from paperwork and spending so much time to register the services . they believed so much registration of the cases was one of the time consuming and useless factors in addition to reduction of the care time , failure in the services quality , waiting of the customers and making frequent mistakes : these days our job is just a kind of bureaucracy [ b8 c107 ] . some of them were dissatisfied from repeated and parallel services in the form of new services : another problem is that we have a series of parallel tasks . i say that i had already filled out the health case of this woman why should i do that in a form of professional health care again [ d2 c312 ] . job stress and the current psychological pressures in the work were experienced by all of them and highly affected them : we need our psychological health too . sense of frustration in the duties and unfinished services was one of the most common issues of the provided services , so that all of them expressed dissatisfaction and severe pressure from lack of enough time due to plurality and diversity of the tasks : how much energy does a health worker have ? [ b9 c189 ] ; there are a lot of expectations from family doctors[d7 c24 ] . we do nt have enough time to do all the tasks furthermore , interference of the heterogeneous tasks and also conflict between roles and tasks had caused disorder and confusion in implementing the tasks and also created many problems for them which consequently caused severe work pressure and dissatisfaction in them : because of too much work and different tasks that needs to be done and because of too many patients that we have , we have no time to educate people as we should do [ d6 c5 ] . the participants expressed this feeling due to impossibility of the scientific promotion , lack of suitable conditions to update scientific information , lack of opportunity for acquiring new skills , and lack of opportunity for independent decision - making : according to the project of family doctor , there would be no opportunities for doctors scientific improvement the health workers were worried about lack of the opportunity to promote scientific level and acquiring scientific capabilities and physicians discomfort from lack of enough time to study and access to update scientific resources and references : during the recent years , i really had no time to study at nights , therefore , i had to use my previous knowledge [ d2 c337 ] ; it 's true that one person is doing too many tasks , but maybe he did nt realize some of them completely most of the time in visiting a patient , i should be really careful to diagnose the sickness [ d1 c21 ] . the majority of the care providers expressed dissatisfaction from information resources , improper evaluation methods , underestimation and quantitative attitude in the system , number of the monitoring and observers , unfair judgment of the observers , and ultimately improper evaluation of the feedback . this event , in many of them was manifested as low self - feeling , defenseless , and depressed which were appeared as negative worthlessness feeling , insignificance , and unimportance : the health workers are under too much pressure[b9-c174 ] ; in my opinion , in this system , doctors have no good social level[d5 c31 ] ; in some cases , they really scorned us[b11 the defenseless feeling had been created due to negative experience of the authorities hearken and lack of right to protest : whenever we complain , they say you should nt have chosen this major they were also dissatisfied from focusing on the written evaluation and reports without regard to the quality of the provided work : they they would never accept our performance , even if we had already done that , unless we record our work.[d7 the second analysis and comparison of the mentioned six themes indicated that those themes showed a more inner and more abstract feeling in the participants toward the working condition which was the very burnout . so that the themes of instability , pressure , threat , frustration , deprivation and worthlessness along with emotional exhaustion and depersonalization in all the care providers , and individual failure of the health workers , lack of motivation in the work and extreme fatigue all indicated the inner feeling and event , general and more important called burnout : we are really frustrated with no motivation in our job [ b8 c103 ] . the feeling of individual failure was evident in the health workers and they had experienced all the dimensions of the burnout : there is no internal satisfaction either [ b11 c71 ] . frequent ( and often unexpected ) changes in type of the services and instability in definition of the target population and recipients of the services following the new programs and instructions which every day are sent from the higher centers and authorities with no clear purpose , cause instability , turbulence , and tiredness in the health care providers : in my opinion , changes should be purposeful . it means , in every system , we naturally need changes because the system needs improvement . our processes should be improved too , but these changes are not purposeful[d2 c28 ] ; the number of offices have increased dramatically with too many changes which are not really helpful for people and health . some of them expressed dissatisfaction from lack of awareness from their future obligations and tasks , being unimportant in the system and not being the consultant party to determine the type of the reforms and its procedure as an executor and also a beneficiary person : that means some unnecessary tasks . we really have no idea about what we are supposed to do tomorrow[d2 c314 ] ; they never ask about our opinions in any tasks [ b11 c227 ] . the physicians expressed their dissatisfaction from the coverage population instability and their increase ; the midwives from the frequency of the covered villages and providing services to other villages with unfamiliar people ; and mid - wives and health workers dissatisfied from providing service to the high risk afghan population . the care providers complained about multiple , inappropriate , hindrance and non - beneficial regulations , and had an unpleasant feeling and experience toward them : they waste our useful time and this is the same for doctors and midwives too . we suggested several times , a doctor and a midwife should be incharge and assistants can have a time out , but no one paid attention and the system did nt accept [ d6 c57 ] . they also complained about rigid and inflexible regulations and laws , hindrance instructions and inconsistent laws to the community conditions : in the present situation , the physicians are evaluated based on the statistics and the recorded issues due to the little flexibility of the system . one of the most important factors for their dissatisfaction was increasing their working hours regardless of the actual needs of the people , because all those hours were in the afternoon which was non - beneficial and boring:our work time is too long . we are too exhausted to get home at 5:00 [ b4 c24 ] . unbalanced workload and manpower caused stress and pressure on the care providers due to high population coverage and lack of sufficient manpower : there is too much work and it is too diverse too [ b4 c266 ] ; every year and even everyday , they increase the responsibilities of the health workers [ b3 c28 ] . all of them complained about high number of the clients and providing service to the high population in a limited time : i think each of us should visit at least about 50 or 60 patients , but i think it is not practically possible [ d7 c2 ] . all of them , particularly the health workers , expressed dissatisfaction from paperwork and spending so much time to register the services . they believed so much registration of the cases was one of the time consuming and useless factors in addition to reduction of the care time , failure in the services quality , waiting of the customers and making frequent mistakes : these days our job is just a kind of bureaucracy [ b8 c107 ] . some of them were dissatisfied from repeated and parallel services in the form of new services : another problem is that we have a series of parallel tasks . i say that i had already filled out the health case of this woman why should i do that in a form of professional health care again [ d2 c312 ] . job stress and the current psychological pressures in the work were experienced by all of them and highly affected them : we need our psychological health too . sense of frustration in the duties and unfinished services was one of the most common issues of the provided services , so that all of them expressed dissatisfaction and severe pressure from lack of enough time due to plurality and diversity of the tasks : how much energy does a health worker have ? [ b9 c189 ] ; there are a lot of expectations from family doctors[d7 c24 ] . we do nt have enough time to do all the tasks [ d7 c33 ] . furthermore , interference of the heterogeneous tasks and also conflict between roles and tasks had caused disorder and confusion in implementing the tasks and also created many problems for them which consequently caused severe work pressure and dissatisfaction in them : because of too much work and different tasks that needs to be done and because of too many patients that we have , we have no time to educate people as we should do [ d6 c5 ] . the participants expressed this feeling due to impossibility of the scientific promotion , lack of suitable conditions to update scientific information , lack of opportunity for acquiring new skills , and lack of opportunity for independent decision - making : according to the project of family doctor , there would be no opportunities for doctors scientific improvement the health workers were worried about lack of the opportunity to promote scientific level and acquiring scientific capabilities and physicians discomfort from lack of enough time to study and access to update scientific resources and references : during the recent years , i really had no time to study at nights , therefore , i had to use my previous knowledge [ d2 c337 ] ; it 's true that one person is doing too many tasks , but maybe he did nt realize some of them completely [ b1 c90 ] ; most of the time in visiting a patient , i should be really careful to diagnose the sickness [ d1 c21 ] . the majority of the care providers expressed dissatisfaction from information resources , improper evaluation methods , underestimation and quantitative attitude in the system , number of the monitoring and observers , unfair judgment of the observers , and ultimately improper evaluation of the feedback . low self - feeling , defenseless , and depressed which were appeared as negative worthlessness feeling , insignificance , and unimportance : the health workers are under too much pressure[b9-c174 ] ; in my opinion , in this system , doctors have no good social level[d5 c31 ] ; in some cases , they really scorned us[b11 the defenseless feeling had been created due to negative experience of the authorities hearken and lack of right to protest : whenever we complain , they say you should nt have chosen this major they were also dissatisfied from focusing on the written evaluation and reports without regard to the quality of the provided work : they they would never accept our performance , even if we had already done that , unless we record our work.[d7 the second analysis and comparison of the mentioned six themes indicated that those themes showed a more inner and more abstract feeling in the participants toward the working condition which was the very burnout . so that the themes of instability , pressure , threat , frustration , deprivation and worthlessness along with emotional exhaustion and depersonalization in all the care providers , and individual failure of the health workers , lack of motivation in the work and extreme fatigue all indicated the inner feeling and event , general and more important called burnout : we are really frustrated with no motivation in our job [ b8 c103 ] . sometimes i wish there were no visitors when i get to work [ b2 c284 ] . the feeling of individual failure was evident in the health workers and they had experienced all the dimensions of the burnout : there is no internal satisfaction either [ b11 c71 ] . frequent ( and often unexpected ) changes in type of the services and instability in definition of the target population and recipients of the services following the new programs and instructions which every day are sent from the higher centers and authorities with no clear purpose , cause instability , turbulence , and tiredness in the health care providers : in my opinion , changes should be purposeful . it means , in every system , we naturally need changes because the system needs improvement . our processes should be improved too , but these changes are not purposeful[d2 c28 ] ; the number of offices have increased dramatically with too many changes which are not really helpful for people and health . some of them expressed dissatisfaction from lack of awareness from their future obligations and tasks , being unimportant in the system and not being the consultant party to determine the type of the reforms and its procedure as an executor and also a beneficiary person : that means some unnecessary tasks . we really have no idea about what we are supposed to do tomorrow[d2 c314 ] ; they never ask about our opinions in any tasks [ b11 c227 ] . the physicians expressed their dissatisfaction from the coverage population instability and their increase ; the midwives from the frequency of the covered villages and providing services to other villages with unfamiliar people ; and mid - wives and health workers dissatisfied from providing service to the high risk afghan population . the care providers complained about multiple , inappropriate , hindrance and non - beneficial regulations , and had an unpleasant feeling and experience toward them : they waste our useful time and this is the same for doctors and midwives too . we suggested several times , a doctor and a midwife should be incharge and assistants can have a time out , but no one paid attention and the system did nt accept [ d6 c57 ] . they also complained about rigid and inflexible regulations and laws , hindrance instructions and inconsistent laws to the community conditions : in the present situation , the physicians are evaluated based on the statistics and the recorded issues due to the little flexibility of the system . one of the most important factors for their dissatisfaction was increasing their working hours regardless of the actual needs of the people , because all those hours were in the afternoon which was non - beneficial and boring:our work time is too long . we are too exhausted to get home at 5:00 [ b4 c24 ] . unbalanced workload and manpower caused stress and pressure on the care providers due to high population coverage and lack of sufficient manpower : there is too much work and it is too diverse too [ b4 c266 ] ; every year and even everyday , they increase the responsibilities of the health workers [ b3 c28 ] . all of them complained about high number of the clients and providing service to the high population in a limited time : i think each of us should visit at least about 50 or 60 patients , but i think it is not practically possible all of them , particularly the health workers , expressed dissatisfaction from paperwork and spending so much time to register the services . they believed so much registration of the cases was one of the time consuming and useless factors in addition to reduction of the care time , failure in the services quality , waiting of the customers and making frequent mistakes : these days our job is just a kind of bureaucracy [ b8 c107 ] . some of them were dissatisfied from repeated and parallel services in the form of new services : another problem is that we have a series of parallel tasks . i say that i had already filled out the health case of this woman why should i do that in a form of professional health care again [ d2 c312 ] . job stress and the current psychological pressures in the work were experienced by all of them and highly affected them : we need our psychological health too . sense of frustration in the duties and unfinished services was one of the most common issues of the provided services , so that all of them expressed dissatisfaction and severe pressure from lack of enough time due to plurality and diversity of the tasks : how much energy does a health worker have ? [ b9 c189 ] ; there are a lot of expectations from family doctors[d7 c24 ] . we do nt have enough time to do all the tasks [ d7 c33 ] . furthermore , interference of the heterogeneous tasks and also conflict between roles and tasks had caused disorder and confusion in implementing the tasks and also created many problems for them which consequently caused severe work pressure and dissatisfaction in them : because of too much work and different tasks that needs to be done and because of too many patients that we have , we have no time to educate people as we should do [ d6 c5 ] . the participants expressed this feeling due to impossibility of the scientific promotion , lack of suitable conditions to update scientific information , lack of opportunity for acquiring new skills , and lack of opportunity for independent decision - making : according to the project of family doctor , there would be no opportunities for doctors scientific improvement the health workers were worried about lack of the opportunity to promote scientific level and acquiring scientific capabilities and physicians discomfort from lack of enough time to study and access to update scientific resources and references : during the recent years , i really had no time to study at nights , therefore , i had to use my previous knowledge [ d2 c337 ] ; it 's true that one person is doing too many tasks , but maybe he did nt realize some of them completely [ b1 c90 ] ; most of the time in visiting a patient , i should be really careful to diagnose the sickness [ d1 c21 ] . the majority of the care providers expressed dissatisfaction from information resources , improper evaluation methods , underestimation and quantitative attitude in the system , number of the monitoring and observers , unfair judgment of the observers , and ultimately improper evaluation of the feedback . this event , in many of them was manifested as low self - feeling , defenseless , and depressed which were appeared as negative worthlessness feeling , insignificance , and unimportance : the health workers are under too much pressure[b9-c174 ] ; in my opinion , in this system , doctors have no good social level[d5 c31 ] ; in some cases , they really scorned us[b11 the defenseless feeling had been created due to negative experience of the authorities hearken and lack of right to protest : whenever we complain , they say you should nt have chosen this major they were also dissatisfied from focusing on the written evaluation and reports without regard to the quality of the provided work : they they would never accept our performance , even if we had already done that , unless we record our work.[d7 the second analysis and comparison of the mentioned six themes indicated that those themes showed a more inner and more abstract feeling in the participants toward the working condition which was the very burnout . so that the themes of instability , pressure , threat , frustration , deprivation and worthlessness along with emotional exhaustion and depersonalization in all the care providers , and individual failure of the health workers , lack of motivation in the work and extreme fatigue all indicated the inner feeling and event , general and more important called burnout : we are really frustrated with no motivation in our job [ b8 c103 ] . sometimes i wish there were no visitors when i get to work [ b2 c284 ] . the feeling of individual failure was evident in the health workers and they had experienced all the dimensions of the burnout : there is no internal satisfaction either [ b11 c71 ] . the results of this study indicated that understanding and feeling of the health care providers in the primary rural health centers were as the following : feeling instability and frequent changes with no purpose in the organization ; involved in laws and regulations ; pressure and stress due to unbalanced workload and manpower ; frustrated in performing the tasks ; deprivation of the professional development ; and sense of identity threat and low self - understanding , which these themes can represent the indices of a theme i.e. burnout . reliable sources consider burnout as a syndrome which includes exhausted , fatigue , and overworking which appear as negative attitude and feeling toward people whom work with , toward their own professional roles , and also emotional exhaustion . it also is a syndrome which includes emotional exhaustion , such as fatigue and severe emotional pressure , depersonalization , such as lack of kindness and tenderness toward others , and reduction of the adequacy feeling and personal success e.g. negative assessment from his / her potential abilities for success in the work . this syndrome may be seen more among the professions which directly deal with people than other jobs . the significant note in the study results were job stress and pressure due to frequent changes which was resulted from undesirable nature of the changes structure and not involving the care providers in decision - making about its quality and quantity had caused increase in workload , inconsistencies , and contradictions in the roles and ambiguity and amaze for the care providers and eventually predisposing the incidence of different dimensions of burnout in them . although changing is considered necessary because of the importance of dynamic health care delivery system , however , frequent changes in the services causes instability , turbulence , and turmoil in the services and leads to pressure , dissatisfaction , and finally incompetent and burnout in the care providers . in the present study , the care providers described the frequent changes and instability in the system as a useless and purposeless case . in their opinion , they did not consider the changes motion as monotonous and believed it was fast at the beginning and then gradually slow and boring and they also believed that this was the reason of previous left unfinished services and also imposing repetitive and parallel services to the system in the form of new services , besides they expressed dissatisfaction from lack of participation in decision making about the changes . glasberg et al also mentioned that one of the major sources of burnout was the frequent changes due to budget reduction and instability in the work . all the care providers were dissatisfied from notification of the rules and inflexible and inconsistent instructions with the community condition . they also complained about their useless presence in the afternoon hours and anachronistic increase of working hours . in addition , they expressed dissatisfaction from being unauthorized to plan and regulate inappropriate services to the community under its circumstances and consequently neglect the health of some of the target population groups such as men . according to the study findings of glasberg et al , one of the factors associated with emotional exhaustion in the service providers was lack of enough time for providing the required care and inability to meet the needs of others , besides , one of the factors associated with depersonalization was reduction of the desire to provide optimal services and inadequate support of the partners in addition to the above mentioned cases . according to the majority of the care providers , due to lack of balance and coordination between workload and human power and also the increase of expectation of the system from them , high workload , and increasing the frequency and variety of tasks , the services left unfinished and were not given with an optimal quality with itself caused the disability in conducting the tasks and pressure and stress in them . furthermore , increase the documentations and paperwork caused reduction of the care time , reduction in the services quality and making frequent mistakes and severe anxiety in them . the study of ahmadinia et al indicated that high workload , lack of environmental hygiene observation and commuting problem in the women health workers , insufficient salary and benefits , insufficient life facilities , housing problem and lack of vehicle in the men health workers were the affecting factors on job dissatisfaction . moreover , the health workers were unhap - py from the reaction of the observers and highranking officials . furthermore , in the study of arab , rural population size and coverage satellite villages and provide basic facilities mentioned as the affecting factors on job dissatisfaction of the health workers . the study of weymouth in australia on the employed nurses in the remote distances showed that low information , high stress , inadequate resources , and unrealistic expectations of society and managers led to high workload and the feeling of lack of being understood and support by the managers . the study of grunfeld et al in canada also indicated that increasing the workload had been the main source of job stress in nurses and had caused concern in them due to negative effects resulted from high workload and reduction of the services quality and loss their spirit . the study of gotz in germany showed that general practitioners were dissatisfied from the incoming level , working hours , and mental conditions of the work . in addition to the above mentioned cases , high workload along with the social and demographic factors as well as smoking had been found the affecting factors on physical and mental health of the physicians and nurses . multiplicity of the tasks and difference in their nature caused interference in the tasks and sometimes even conflict in playing the roles and confusion in the service providers caused many problems for them . lu et al in their study showed the organizational factors , factors related to job and patient care , ambiguity and conflict in the role , job stress and organizational commitment as related factors to job satisfaction and also willingness to keep the job . the care providers were dissatisfied from the current evaluation process and recognized resource information of the evaluators and their assessment method as an inappropriate method and believed underestimation and quantitative attitude in the system have been ruled out and judgments were unfair about the quality of their services . lack of professional development opportunities means lack of scientific promotion and lack of updating the information and the care providers complained about lack of opportunity to acquire skills and lack of enough opportunity for independent decision making . while , in the study of buciuniene on reforming in the health care system in lithuania showed that physicians had the most satisfaction from their independence level and relation with the partners and also they had quality of management . however , despite physicians satisfaction from job independency , they were dissatisfied from social status and high workload . the study of kushnir et al showed that continuing medical education had negative correlation with job stress and positive correlation with job satisfaction among the family physicians and the family physicians that had necessary opportunity and possibility for keeping the professional information updated had lower stress and burnout . the present study indicated that after emotional exhaustion with high intensity and after depersonalization with relatively more limited extent were observed in all the care providers , such as family physicians , midwives and health workers , but the important thing is that after individual failure and inability in performing the tasks with high intensity was observed only in the health workers and in addition to the inner sense in the health workers , its outer understanding was done by the physicians and mid - wives and proved this issue . severinsson in his study on burnout in the australian nurses showed that the process of high demand from the nurses caused reduction in control level of the affairs and weakness and inability feeling and led to inability to perform the tasks and job stress and physical problems , such as blood pressure , in them . the feeling of disability in the nurses was revealed in depression , frustrated , and loneliness . suspension between suffering and desire related to the nurses experience was one of the events that led to emotional and mental problems in the nurses . analyzing and discussing the above mentioned findings from the perception of the care providers from their working condition indicated that burnout was their main and central theme from understanding and experience of their working conditions . health service providers in the health centers who are the messengers and life and health guards of the people , due to stressful and challenging working condition , are being suffered from deprivation of something for which they are responsible for the society , therefore , it is worthy that in order for full effectiveness , the policy makers and managers consider the organizational factors affecting on performance method of the staff . the managers should look for the early symptoms and signs of the burnout in order for development the willingness of the employees to keep working and increase their spirit and also consider the improvement of their health and working condition which is like a container for the gift of the health . the services providers need emotional support , besides they also have the right to be principally monitored regularly and their services carefully be controlled and guided and a fair judgment should be done about their services . they should be helped so that they can realize their own capabilities and some effective measures should be done in order to eliminate the defects and solve their working problems . it should be tried to minimize the environmental stressful conditions and regulate the system 's expectations with their capabilities so that stress and pressure of the work , inability , weakness , and depression be minimized in them and subsequently the job satisfaction be increased and thus , the quality of services and health of the coverage community be improved .

objectives : health care providers in the rural centers offer the primary health services in the form of proficiencies and professions to the most required target population in the health system . these services are provided in certain condition and population with a verity of limitations . this study aimed to describe and interpret the experiences of the employees from their own working condition in the rural health centers.methods:the present study conducted in a qualitative research approach and content analysis method through individual and group interviews with 26 employed primary health care providers ( including 7 family physicians , 7 midwives , and 12 health workers ) in the rural health centers in isfahan in 2009 . sampling was done using purposive sampling method . the data were analyzed using qualitative content analysis as constant comparative basis.results:during the content analysis process , six themes were obtained ; instability and frequent changes , involved in laws and regulations , pressure and stress due to unbalanced workload and manpower , helplessness in performing the tasks and duties , sense of identity threat and low self - concept , and deprivation of professional development . the mentioned themes indicate a main and more important theme called burnout.conclusions:health services providers in the rural health centers are working in stressful and challenging work conditions and are suffered from deprivation of something for which are responsible to the community .

INTRODUCTION METHODS RESULTS None 1. Instability and frequent changes 2. Involved in laws and regulations 3. Pressure and stress due to unbalanced workload and manpower 4. Helplessness and frustration in performing the tasks and duties 5. Deprivation of professional development 6. Sense of identity threat and low self-understanding The central theme: Burnout DISCUSSION CONCLUSION

1984 through1995 , quantitative and qualitative development of the health care network , and during the third one ( 1996 - 2008 ) , the country has witnessed efforts for effecting fundamental reforms in the health care networks which despite its gradual speed , is so important . according to the recent studies , there are too many limitations and high pressures which indicated the high risk of job stress for the employees in the position of the community health care . the present study aimed to describe and interpret the experiences of the employees , such as family physician , midwives , and health workers , from their own working condition in the rural health centers three decades after their vast and valuable activities using qualitative approach . the present study conducted in a qualitative research approach and content analysis method through individual and group interviews with 26 employed primary health care providers ( including 7 family physicians , 7 midwives and 12 health workers ) in the rural health centers in isfahan from october 2008 through september 2010 . analyzing the data was done using content analysis method . the hidden content in the categories were regulated in the form of six themes and these themes ultimately were put into the overall and central theme of the burnout which indicated the main theme and substantial data . verifiability and auditing the data was done through description of full details of analysis process and obtaining the results so that the readers could have a clear understanding of done analysis method , its strengths , and limitations . participants profile based on profession , age , sex , work experience and marital status in the first step of the content analysis from analysis of the data obtained from individual interviews and focus group , 991 codes , 55 subcategory , and 21 categories were obtained . in the second step the mentioned themes indicated a main and more important theme called burnout , which are as the following : six central themes associated with the burnout frequent ( and often unexpected ) changes in type of the services and instability in definition of the target population and recipients of the services following the new programs and instructions which every day are sent from the higher centers and authorities with no clear purpose , cause instability , turbulence , and tiredness in the health care providers : in my opinion , changes should be purposeful . the physicians expressed their dissatisfaction from the coverage population instability and their increase ; the midwives from the frequency of the covered villages and providing services to other villages with unfamiliar people ; and mid - wives and health workers dissatisfied from providing service to the high risk afghan population . they also complained about rigid and inflexible regulations and laws , hindrance instructions and inconsistent laws to the community conditions : in the present situation , the physicians are evaluated based on the statistics and the recorded issues due to the little flexibility of the system . unbalanced workload and manpower caused stress and pressure on the care providers due to high population coverage and lack of sufficient manpower : there is too much work and it is too diverse too [ b4 c266 ] ; every year and even everyday , they increase the responsibilities of the health workers [ b3 c28 ] . some of them were dissatisfied from repeated and parallel services in the form of new services : another problem is that we have a series of parallel tasks . i say that i had already filled out the health case of this woman why should i do that in a form of professional health care again [ d2 c312 ] . sense of frustration in the duties and unfinished services was one of the most common issues of the provided services , so that all of them expressed dissatisfaction and severe pressure from lack of enough time due to plurality and diversity of the tasks : how much energy does a health worker have ? we do nt have enough time to do all the tasks furthermore , interference of the heterogeneous tasks and also conflict between roles and tasks had caused disorder and confusion in implementing the tasks and also created many problems for them which consequently caused severe work pressure and dissatisfaction in them : because of too much work and different tasks that needs to be done and because of too many patients that we have , we have no time to educate people as we should do [ d6 c5 ] . the participants expressed this feeling due to impossibility of the scientific promotion , lack of suitable conditions to update scientific information , lack of opportunity for acquiring new skills , and lack of opportunity for independent decision - making : according to the project of family doctor , there would be no opportunities for doctors scientific improvement the health workers were worried about lack of the opportunity to promote scientific level and acquiring scientific capabilities and physicians discomfort from lack of enough time to study and access to update scientific resources and references : during the recent years , i really had no time to study at nights , therefore , i had to use my previous knowledge [ d2 c337 ] ; it 's true that one person is doing too many tasks , but maybe he did nt realize some of them completely most of the time in visiting a patient , i should be really careful to diagnose the sickness [ d1 c21 ] . this event , in many of them was manifested as low self - feeling , defenseless , and depressed which were appeared as negative worthlessness feeling , insignificance , and unimportance : the health workers are under too much pressure[b9-c174 ] ; in my opinion , in this system , doctors have no good social level[d5 c31 ] ; in some cases , they really scorned us[b11 the defenseless feeling had been created due to negative experience of the authorities hearken and lack of right to protest : whenever we complain , they say you should nt have chosen this major they were also dissatisfied from focusing on the written evaluation and reports without regard to the quality of the provided work : they they would never accept our performance , even if we had already done that , unless we record our work. [d7 the second analysis and comparison of the mentioned six themes indicated that those themes showed a more inner and more abstract feeling in the participants toward the working condition which was the very burnout . so that the themes of instability , pressure , threat , frustration , deprivation and worthlessness along with emotional exhaustion and depersonalization in all the care providers , and individual failure of the health workers , lack of motivation in the work and extreme fatigue all indicated the inner feeling and event , general and more important called burnout : we are really frustrated with no motivation in our job [ b8 c103 ] . frequent ( and often unexpected ) changes in type of the services and instability in definition of the target population and recipients of the services following the new programs and instructions which every day are sent from the higher centers and authorities with no clear purpose , cause instability , turbulence , and tiredness in the health care providers : in my opinion , changes should be purposeful . the physicians expressed their dissatisfaction from the coverage population instability and their increase ; the midwives from the frequency of the covered villages and providing services to other villages with unfamiliar people ; and mid - wives and health workers dissatisfied from providing service to the high risk afghan population . they also complained about rigid and inflexible regulations and laws , hindrance instructions and inconsistent laws to the community conditions : in the present situation , the physicians are evaluated based on the statistics and the recorded issues due to the little flexibility of the system . unbalanced workload and manpower caused stress and pressure on the care providers due to high population coverage and lack of sufficient manpower : there is too much work and it is too diverse too [ b4 c266 ] ; every year and even everyday , they increase the responsibilities of the health workers [ b3 c28 ] . some of them were dissatisfied from repeated and parallel services in the form of new services : another problem is that we have a series of parallel tasks . i say that i had already filled out the health case of this woman why should i do that in a form of professional health care again [ d2 c312 ] . sense of frustration in the duties and unfinished services was one of the most common issues of the provided services , so that all of them expressed dissatisfaction and severe pressure from lack of enough time due to plurality and diversity of the tasks : how much energy does a health worker have ? furthermore , interference of the heterogeneous tasks and also conflict between roles and tasks had caused disorder and confusion in implementing the tasks and also created many problems for them which consequently caused severe work pressure and dissatisfaction in them : because of too much work and different tasks that needs to be done and because of too many patients that we have , we have no time to educate people as we should do [ d6 c5 ] . the participants expressed this feeling due to impossibility of the scientific promotion , lack of suitable conditions to update scientific information , lack of opportunity for acquiring new skills , and lack of opportunity for independent decision - making : according to the project of family doctor , there would be no opportunities for doctors scientific improvement the health workers were worried about lack of the opportunity to promote scientific level and acquiring scientific capabilities and physicians discomfort from lack of enough time to study and access to update scientific resources and references : during the recent years , i really had no time to study at nights , therefore , i had to use my previous knowledge [ d2 c337 ] ; it 's true that one person is doing too many tasks , but maybe he did nt realize some of them completely [ b1 c90 ] ; most of the time in visiting a patient , i should be really careful to diagnose the sickness [ d1 c21 ] . low self - feeling , defenseless , and depressed which were appeared as negative worthlessness feeling , insignificance , and unimportance : the health workers are under too much pressure[b9-c174 ] ; in my opinion , in this system , doctors have no good social level[d5 c31 ] ; in some cases , they really scorned us[b11 the defenseless feeling had been created due to negative experience of the authorities hearken and lack of right to protest : whenever we complain , they say you should nt have chosen this major they were also dissatisfied from focusing on the written evaluation and reports without regard to the quality of the provided work : they they would never accept our performance , even if we had already done that , unless we record our work. [d7 the second analysis and comparison of the mentioned six themes indicated that those themes showed a more inner and more abstract feeling in the participants toward the working condition which was the very burnout . so that the themes of instability , pressure , threat , frustration , deprivation and worthlessness along with emotional exhaustion and depersonalization in all the care providers , and individual failure of the health workers , lack of motivation in the work and extreme fatigue all indicated the inner feeling and event , general and more important called burnout : we are really frustrated with no motivation in our job [ b8 c103 ] . frequent ( and often unexpected ) changes in type of the services and instability in definition of the target population and recipients of the services following the new programs and instructions which every day are sent from the higher centers and authorities with no clear purpose , cause instability , turbulence , and tiredness in the health care providers : in my opinion , changes should be purposeful . the physicians expressed their dissatisfaction from the coverage population instability and their increase ; the midwives from the frequency of the covered villages and providing services to other villages with unfamiliar people ; and mid - wives and health workers dissatisfied from providing service to the high risk afghan population . they also complained about rigid and inflexible regulations and laws , hindrance instructions and inconsistent laws to the community conditions : in the present situation , the physicians are evaluated based on the statistics and the recorded issues due to the little flexibility of the system . unbalanced workload and manpower caused stress and pressure on the care providers due to high population coverage and lack of sufficient manpower : there is too much work and it is too diverse too [ b4 c266 ] ; every year and even everyday , they increase the responsibilities of the health workers [ b3 c28 ] . all of them complained about high number of the clients and providing service to the high population in a limited time : i think each of us should visit at least about 50 or 60 patients , but i think it is not practically possible all of them , particularly the health workers , expressed dissatisfaction from paperwork and spending so much time to register the services . i say that i had already filled out the health case of this woman why should i do that in a form of professional health care again [ d2 c312 ] . sense of frustration in the duties and unfinished services was one of the most common issues of the provided services , so that all of them expressed dissatisfaction and severe pressure from lack of enough time due to plurality and diversity of the tasks : how much energy does a health worker have ? furthermore , interference of the heterogeneous tasks and also conflict between roles and tasks had caused disorder and confusion in implementing the tasks and also created many problems for them which consequently caused severe work pressure and dissatisfaction in them : because of too much work and different tasks that needs to be done and because of too many patients that we have , we have no time to educate people as we should do [ d6 c5 ] . the participants expressed this feeling due to impossibility of the scientific promotion , lack of suitable conditions to update scientific information , lack of opportunity for acquiring new skills , and lack of opportunity for independent decision - making : according to the project of family doctor , there would be no opportunities for doctors scientific improvement the health workers were worried about lack of the opportunity to promote scientific level and acquiring scientific capabilities and physicians discomfort from lack of enough time to study and access to update scientific resources and references : during the recent years , i really had no time to study at nights , therefore , i had to use my previous knowledge [ d2 c337 ] ; it 's true that one person is doing too many tasks , but maybe he did nt realize some of them completely [ b1 c90 ] ; most of the time in visiting a patient , i should be really careful to diagnose the sickness [ d1 c21 ] . this event , in many of them was manifested as low self - feeling , defenseless , and depressed which were appeared as negative worthlessness feeling , insignificance , and unimportance : the health workers are under too much pressure[b9-c174 ] ; in my opinion , in this system , doctors have no good social level[d5 c31 ] ; in some cases , they really scorned us[b11 the defenseless feeling had been created due to negative experience of the authorities hearken and lack of right to protest : whenever we complain , they say you should nt have chosen this major they were also dissatisfied from focusing on the written evaluation and reports without regard to the quality of the provided work : they they would never accept our performance , even if we had already done that , unless we record our work. [d7 the second analysis and comparison of the mentioned six themes indicated that those themes showed a more inner and more abstract feeling in the participants toward the working condition which was the very burnout . so that the themes of instability , pressure , threat , frustration , deprivation and worthlessness along with emotional exhaustion and depersonalization in all the care providers , and individual failure of the health workers , lack of motivation in the work and extreme fatigue all indicated the inner feeling and event , general and more important called burnout : we are really frustrated with no motivation in our job [ b8 c103 ] . the feeling of individual failure was evident in the health workers and they had experienced all the dimensions of the burnout : there is no internal satisfaction either [ b11 c71 ] . the results of this study indicated that understanding and feeling of the health care providers in the primary rural health centers were as the following : feeling instability and frequent changes with no purpose in the organization ; involved in laws and regulations ; pressure and stress due to unbalanced workload and manpower ; frustrated in performing the tasks ; deprivation of the professional development ; and sense of identity threat and low self - understanding , which these themes can represent the indices of a theme i.e. the significant note in the study results were job stress and pressure due to frequent changes which was resulted from undesirable nature of the changes structure and not involving the care providers in decision - making about its quality and quantity had caused increase in workload , inconsistencies , and contradictions in the roles and ambiguity and amaze for the care providers and eventually predisposing the incidence of different dimensions of burnout in them . although changing is considered necessary because of the importance of dynamic health care delivery system , however , frequent changes in the services causes instability , turbulence , and turmoil in the services and leads to pressure , dissatisfaction , and finally incompetent and burnout in the care providers . in their opinion , they did not consider the changes motion as monotonous and believed it was fast at the beginning and then gradually slow and boring and they also believed that this was the reason of previous left unfinished services and also imposing repetitive and parallel services to the system in the form of new services , besides they expressed dissatisfaction from lack of participation in decision making about the changes . glasberg et al also mentioned that one of the major sources of burnout was the frequent changes due to budget reduction and instability in the work . in addition , they expressed dissatisfaction from being unauthorized to plan and regulate inappropriate services to the community under its circumstances and consequently neglect the health of some of the target population groups such as men . according to the majority of the care providers , due to lack of balance and coordination between workload and human power and also the increase of expectation of the system from them , high workload , and increasing the frequency and variety of tasks , the services left unfinished and were not given with an optimal quality with itself caused the disability in conducting the tasks and pressure and stress in them . multiplicity of the tasks and difference in their nature caused interference in the tasks and sometimes even conflict in playing the roles and confusion in the service providers caused many problems for them . while , in the study of buciuniene on reforming in the health care system in lithuania showed that physicians had the most satisfaction from their independence level and relation with the partners and also they had quality of management . the present study indicated that after emotional exhaustion with high intensity and after depersonalization with relatively more limited extent were observed in all the care providers , such as family physicians , midwives and health workers , but the important thing is that after individual failure and inability in performing the tasks with high intensity was observed only in the health workers and in addition to the inner sense in the health workers , its outer understanding was done by the physicians and mid - wives and proved this issue . analyzing and discussing the above mentioned findings from the perception of the care providers from their working condition indicated that burnout was their main and central theme from understanding and experience of their working conditions . health service providers in the health centers who are the messengers and life and health guards of the people , due to stressful and challenging working condition , are being suffered from deprivation of something for which they are responsible for the society , therefore , it is worthy that in order for full effectiveness , the policy makers and managers consider the organizational factors affecting on performance method of the staff . the managers should look for the early symptoms and signs of the burnout in order for development the willingness of the employees to keep working and increase their spirit and also consider the improvement of their health and working condition which is like a container for the gift of the health .

binaries containing white dwarfs , neutron stars , and black holes in compact orbits are over - represented in globular clusters compared with their population in the galactic field . from this population come a host of exotic objects such as ultracompact cataclysmic variables , non - flickering x - ray and uv sources , low - mass x - ray binaries , and millisecond pulsars . these objects and their dark counterparts in the population of relativistic binaries are also likely to be observable sources of gravitational radiation for low - frequency gravitational wave detectors such as the proposed spaceborne interferometer lisa . a relativistic binary is a product of interplay between stellar evolution and the gravitational interaction of a tight binary . the population of tight binaries in globular clusters is a product of the dynamical evolution of an n - body gravitational system . thus , relativistic binaries result from a combination of several of the more interesting processes in astrophysics . in keeping with the focus of this review article on relativistic binaries in globular clusters , we shall only touch on the aspects of globular clusters , observations , binary evolution , and n - body dynamics as they relate to populations of this specific class of binaries in globular clusters . we begin by looking at the physical structure and general history of the galactic globular cluster system that leads to the concentration of evolved stars , stellar remnants , and binary systems in the cores of the clusters . current observations of the cores of globular clusters that have revealed numerous tracer populations of relativistic binaries are also discussed . we will look at how mass transfer from one star in the presence of a nearby companion can dramatically alter the evolution of both stars in the process of binary evolution . the enhanced production of relativistic binaries in globular clusters results from dynamical processes that drive binaries toward tighter orbits and that preferentially exchange more massive and degenerate objects into binary systems . numerical simulations of globular cluster evolution , which can be used to predict the rate at which relativistic binaries are formed , are discussed . finally , we conclude with a brief discussion of the prospects for observing these systems in gravitational radiation . readers interested in further studies of the structure and evolution of globular clusters are invited to look at binney and tremaine , spitzer , and volumes i and ii of padmanabhan s theoretical astrophysics [ 116 , 117 ] for a good introduction to the physical processes involved . the recent review articles of meylan and heggie and meylan also provide a comprehensive look at the internal dynamics of globular clusters . although our focus is solely on the galactic globular cluster system , the physics of globular cluster systems associated with other galaxies is well covered in the review article by harris as well as his lecture notes from the saas - fee course on star clusters . an observational perspective on the role of binaries in globular clusters is presented in an excellent review by bailyn , while a good introduction to the details of observing binary systems in general can be found in an introduction to close binary stars . although slightly out of date , the review of binaries in globular clusters by hut et al . is an excellent introduction to the interaction between globular cluster dynamics and binary evolution . an updated , short article on globular cluster binaries by mcmillan , pryor , and phinney is also of value . have written recent reviews of numerical simulations of binary populations in globular clusters . a recent article by pfahl et al . has an in - depth discussion of the details of the retention of neutron stars in globular clusters . globular clusters comprising 10 to 10 stars were formed early in the history of the milky way , and are scattered throughout the halo . the age of the clusters is about 13 gyr , with an age spread of less than 5 gyr . according to a frequently updated catalog of globular cluster properties maintained on the web by harris , the globular cluster system numbers roughly 150 clusters . although the list is fairly complete , two new globular clusters were recently discovered at very low galactic latitudes , and there is the prospect for a few more clusters to be hidden behind the bulge or out in the far reaches of the galaxy . the distribution of globular clusters in galacto - centric coordinates is shown in figure 1 . figure 1globular cluster distribution about the galaxy . positions are from harris and are plotted as black circles on top of the cobe firas 2.2 micron map of the galaxy using a mollweide projection . positions are from harris and are plotted as black circles on top of the cobe firas 2.2 micron map of the galaxy using a mollweide projection . figure taken from brian chaboyer s website . because the clusters are of great age , most of the stars above about 0.8m have already evolved off the main sequence . thus , a large number of red giants are readily visible in most pictures of globular clusters ( see figure 2 ) . when viewing the color - magnitude diagram ( cmd ) for a globular cluster , one can clearly see the red giant branch lifting up away from the main sequence . the horizontal branch of evolved stars is also seen in the cmd for m80 shown in figure 3 . figure 2hubble space telescope photograph of the dense globular cluster m80 ( ngc 6093 ) . the diagram on the right focuses on the turn - off of the main sequence and the red giant branch . hb indicates the horizontal branch , rgb is the red giant branch , sgb is the subgiant branch , and bss indicate the blue stragglers . blue stragglers will be discussed later in this review and the interested reader can consult for a discription of the other objects . the diagram on the right focuses on the turn - off of the main sequence and the red giant branch . hb indicates the horizontal branch , rgb is the red giant branch , sgb is the subgiant branch , and bss indicate the blue stragglers . blue stragglers will be discussed later in this review and the interested reader can consult for a discription of the other objects . that there is a clearly visible turn - off point in the cmd for globular clusters is evidence for the roughly coeval nature of the stars in the cluster . during the early stages of the evolution of a globular cluster , subsequent replenishment of the intercluster gas by stellar winds from evolved stars is removed during periodic passages of the cluster through the plane of the galaxy . the overall structure of a globular cluster can be described in terms of an n - body system . these systems , each containing between 10 and 10 stars , have central densities in the range 10 to 10m/pc , with an average of 10m/pc . the important characteristic radii of a globular cluster are the core radius rc , the half - light radius rh , and the tidal radius rt . the core radius is defined to be the radius at which the surface brightness has dropped to half the central value . the half - light radius is the radius that contains half of the light of the cluster and the tidal radius is defined as the radius beyond which the external gravitational field of the galaxy dominates the dynamics . the half - mass radius is a three - dimensional construct , while the half - light radius is two - dimensional . typical values of these radii are 1.5 pc , 10 pc , and 50 pc , respectively [ 19 , 117 ] . these are the crossing time tcross , the relaxation time trelax , and the evaporation time tevap . the crossing time is the typical time required for a star in the cluster to travel the characteristic size r of the cluster ( typically taken to be the half - mass radius ) . / v , where v is a typical velocity ( 10 km / s ) . the relaxation time is the typical time for gravitational interactions with other stars in the cluster to remove the history of a star s original velocity . this amounts to the time required for gravitational encounters to alter the star s velocity by an amount comparable to its original velocity . since the relaxation time is related to the number and strength of the gravitational encounters of a typical cluster star , it is related to the number of stars in the cluster and the average energy of the stars in the cluster . thus , it can be shown that the mean relaxation time for a cluster is [ 19 , 116 ] ( 1)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { t_{{\rm{relax } } } } \simeq \frac{{0.1n}}{{\ln n}}{t_{{\rm{cross}}}}. $ $ \end{document } for a globular cluster with n=10 , a characteristic size of rrh10 pc , and a typical velocity of v10 km / s , the crossing time and relaxation time are tcross10 yr and t10 yr . it should be noted , however , that figure 1.3 of spitzer gives a median trelax1 gyr and padmanabhan gives trelax10 yr . the evaporation time for a cluster is the time required for the cluster to dissolve through the gradual loss of stars that gain sufficient velocity through encounters to escape its gravitational potential . in the absence of stellar evolution and tidal interactions with the galaxy , the evaporation time can be estimated by assuming that a fraction of the stars in the cluster are evaporated every relaxation time . the value of can be determined by noting that the escape speed ve at a point x is related to the gravitational potential (x ) at that point by ve2=-2(x ) . consequently , the mean - square escape speed in a cluster with density (x ) is ( 2)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left\langle { v_{\rm{e}}^2 } \right\rangle = \frac{{\int { \rho ( x ) } v_{\rm{e}}^2{d^3}x}}{{\int { \rho ( x ) } { d^3}x } } = - 2\frac{{\int { \rho ( x ) } \phi ( x){d^3}x}}{m } = - \frac{{4w}}{m } , $ $ \end{document } where w is the total potential energy of the cluster and m is its total mass . if the system is virialized ( as we would expect after a relaxation time ) , then -w=2k = mv , where k is the total kinetic energy of the cluster , and ( 3)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left\langle { v_{\rm{e}}^2 } \right\rangle = 4\left\langle { { v^2 } } \right\rangle . $ $ \end{document } thus , stars with speeds above twice the rms speed will evaporate . assuming a maxwellian distribution of speeds , the fraction of stars with v>2vrms is =7.3810 . therefore , the evaporation time is ( 4)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { t_{{\rm{evap } } } } = \frac{{{t_{{\rm{relax}}}}}}{\gamma } = 136{t_{{\rm{relax}}}}. $ $ \end{document } stellar evolution annd tidal interactions tend to shorten the evaporation time . see gnedin and ostriker and references therein for a thorough discussion of these effects . for a globular cluster , tevap10 yr , which is comparable to the observed age of globular clusters . the characteristic time scales differ significantly from each other : tcrosstrelaxtevap . when discussing stellar interactions during a given epoch of globular cluster evolution , it is possible to describe the background structure of the globular cluster in terms of a static model . these models describe the structure of the cluster in terms of a distribution function f that can be thought of as providing a probability of finding a star at a particular location in phase - space . the static models are valid over time scales which are shorter than the relaxation time so that gravitational interactions do not have time to significantly alter the distribution function . the structure of the globular cluster is then determined by the collisionless boltzmann equation , ( 5)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ v \cdot \nabla f - \nabla \phi \cdot \frac{{\partial f}}{{\partial v } } = 0 , $ $ \end{document } where the gravitational potential is found from f with ( 6)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \nabla ^2}\phi = 4\pi \int { f\left ( { x , v , m } \right){d^3}v\;dm . } $ $ \end{document } the solutions to equation ( 5 ) are often described in terms of the relative energy per unit mass #x03c8;v/2 with the relative potential defined as -+0 . the constant 0 is chosen so that there are no stars with relative energy less than 0 ( i.e. f>0 for >0 and f=0 for <0 ) . a simple class of solutions to equation ( 5 ) , ( 7)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f(\varepsilon ) = f{\varepsilon ^{7/2 } } , $ $ \end{document } a convenient class of models which admit anisotropy and a distribution in angular momenta l are known as king - michie models . the king - michie distribution function is : ( 8)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ f(\varepsilon , l ) = { \rho _ 1}{(2\pi { \sigma ^2})^ { - 3/2}}\exp \left ( { \frac { { - { l^2}}}{{2r_{\rm{a}}^2{\sigma ^2 } } } } \right)\left [ { { e^{\varepsilon /{\sigma ^2 } } } - 1 } \right],\;\varepsilon > 0 , $ $ \end{document } with f=0 for 0 and 1 a constant . the velocity dispersion is determined by and the anisotropy radius ra is defined so that the velocity distribution changes from nearly isotropic at the center to nearly radial at ra . an overview of the evolution of globular clusters can be found in hut et al . , we summarize here the aspects of globular cluster evolution that are relevant to the formation and concentration of relativistic binaries . the formation of globular clusters is not well understood and the details of the initial mass function ( imf ) are an ongoing field of star cluster studies . although the imf is expected to be flatter at the low - mass end ( see for a discussion of the local imf ) , many theorists assume an imf that follows a standard salpeter form : ( 9)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ dn \propto { m^ { - \alpha } } dm . $ $ \end{document } once the stars form out of the initial molecular cloud , the system will undergo violent relaxation as the protocluster first begins to collapse . the effect of this is that the stars are distributed widely in position and velocity , with a distribution that is independent of the stellar mass . these stars will then lose their kinetic energy to the less massive stars through stellar encounters , leading towards equipartition of energy . through virialization , this tends to concentrate the more massive stars in the center of the cluster . the process of mass segregation for stars of mass mi occurs on a timescale given by titrelaxm/mi . the higher concentration of stars in the center of the cluster increases the probability of an encounter , which , in turn , decreases the relaxation time . thus , the relaxation time given in equation ( 1 ) is an average over the whole cluster . the local relaxation time of the cluster is given in meylan and heggie and can be described by : ( 10)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { t_{\rm{r } } } = \frac{{0.065{{\left\langle { { v^2 } } \right\rangle } ^{3/2}}}}{{\rho \left\langle m \right\rangle { g^2}\ln \lambda } } , $ $ \end{document } where is the local mass density , v is the mass - weighted mean square velocity of the stars , m is the mean stellar mass , and 0.4n ( although a choice of 0.1n may be more appropriate in the presence of a mass spectrum ) . note that in the central regions of the cluster , the value of tr is much lower than the average relaxation time . this means that in the core of the cluster , where the more massive stars have concentrated , there are more encounters between these stars . the concentration of massive stars in the core of the cluster will occur within a few relaxation times , ttrelax10 yr . consequently , these stars will have evolved into carbon - oxygen ( co ) and oxygen - neon ( one ) white dwarfs , neutron stars , and black holes . after a few more relaxation times , the average mass of a star in the globular cluster will be around 0.5m and these degenerate objects will once again be the more massive objects in the cluster , despite having lost most of their mass during their evolution . thus , the population in the core of the cluster will be enhanced in degenerate objects . any binaries in the cluster that have a gravitational binding energy significantly greater than the average kinetic energy of a cluster star will act effectively as single objects with masses equal to their total mass . these objects , too , will segregate to the central regions of the globular cluster . the core would undergo what is known as core collapse within a few tens of relaxation times unless these binaries release some of their binding energy to the cluster . in core collapse , the central density increases to infinity as the core radius shrinks to zero . an example of core collapse can be seen in the comparison of two cluster evolution simulations shown in figure 4 . note the core collapse when the inner radius containing 1% of the total mass dramatically shrinks after t15trelax . since these evolution syntheses are single - mass , plummer models without binary interactions , the actual time of core collapse is not representative of a real globular cluster . figure 4lagrange radii indicating the evolution of a plummer model globular cluster for an n - body simulation and a monte carlo simulation . the radii correspond to radii containing 0.35 , 1 , 3.5 , 5 , 7 , 10 , 14 , 20 , 30 , 40 , 50 , 60 , 70 , and 80% of the total mass . lagrange radii indicating the evolution of a plummer model globular cluster for an n - body simulation and a monte carlo simulation . the radii correspond to radii containing 0.35 , 1 , 3.5 , 5 , 7 , 10 , 14 , 20 , 30 , 40 , 50 , 60 , 70 , and 80% of the total mass . the static description of the structure of globular clusters using king - michie or plummer models provides a framework for describing the environment of relativistic binaries and their pro - genitors in globular clusters . the short - term interactions between stars and degenerate objects can be analyzed in the presence of this background . over longer time scales ( comparable to trelax ) , the dynamical evolution of the distribution function as well as population changes due to stellar evolution can alter the overall structure of the globular cluster . we will discuss the dynamical evolution and its impact on relativistic binaries in section 5 . before moving on to the dynamical models and population syntheses of relativistic binaries , we will first look at the observational evidence for these objects in globular clusters . observations of relativistic binaries in globular clusters are hampered by the fact that most of the binaries have segregated to the cores of the clusters where crowding is a problem . furthermore , most of these systems are only visible during mass transfer or accretion stages of the evolution . one tracer of the processes that may lead to relativistic binaries is the population of blue stragglers . these stars appear on the main sequence to the left of the turn - off in the cmd of a globular cluster ( see figure 3 ) . they are hot and massive enough that they should have already evolved off the main sequence . blue stragglers are thought to arise from stellar coalescences either through the gradual coalescence of the components of binaries in the cluster or through direct collisions . these objects are some of the most visible and populous evidence of dynamical interactions in globular clusters that lead to the formation of ultracompact binary systems . ( see hut and bailyn for good reviews of the implications of blue stragglers on the dynamics of globular clusters . ) white dwarfs are generally too cool to have their binary properties readily measured . for example , the binary properties of double degenerate cataclysmic variables of the am cvn type are determined from observing the hot spot where the accreting matter from the donor dwarf collides with the accretion disk around the accretor ( see warner and references therein ) . with an expected mv10 , these objects would be virtually invisible at globular cluster distances . searches for cataclysmic variables generally focus on low - luminosity x - ray sources [ 87 , 64 , 159 ] and on ultraviolet - excess stars [ 62 , 93 , 103 ] , but these systems are usually a white dwarf accreting from a low mass star . the class of non - flickerers which have been detected recently [ 26 , 156 ] have been explained as he white dwarfs in binaries containing dark co white dwarfs [ 41 , 65 ] . pulsars , although easily seen in radio , are difficult to detect when they occur in hard binaries , due to the doppler shift of the pulse intervals . thanks to an improved technique known as an acceleration search , which assumes a constant acceleration of the pulsar during the observation period , more short orbital period binary pulsars are being discovered [ 20 , 21 , 29 , 30 , 48 , 51 , 130 ] . for a good review and description of this technique , see lorimer . the progenitors of the ultracompact millisecond pulsars are thought to pass through a low - mass x - ray binary ( lmxb ) phase [ 37 , 64 , 131 , 134 ] . these systems are very bright and all of them in the globular cluster system are known . in addition , a very recent observation has shown that the lmxb in m15 is , in fact , two bright sources . with the exception of m15 [ 53 , 66 ] , theoretical predictions of black holes in globular clusters indicate that there may be intermediatemass black holes ( m10m ) in as many as 20 globular clusters , or that stellar mass binary black holes may be generated and subsequently ejected from most globular clusters . if the velocity dispersion in globular clusters follows the same correlation to black hole mass as in galactic bulges , then there may be black holes with masses in the range 110m in many globular clusters . they have been detected in globular clusters through identification of the white dwarf itself or through evidence of the accretion process . white dwarfs managed to avoid detection until recent observations with the hubble space telescope revealed photometric sequences in several globular clusters [ 27 , 26 , 119 , 135 , 137 , 136 , 156 ] . spectral identification of white dwarfs in globular clusters has begun both from the ground with the vlt and in space with the hubble space telescope [ 26 , 41 , 156 ] . with spectral identification , it will be possible to identify those white dwarfs in hard binaries through doppler shifts in the h line . this approach has promise for detecting a large number of the expected double white dwarf binaries in globular clusters . identifications of globular cluster cvs have been made through such outbursts in the cores of m5 , 47 tuc , and ngc 6624 . with the exception of v101 in m5 , original searches for dwarf novae performed with ground this is primarily due to the fact that crowding obscured potential dwarf novae up to several core radii outside the center of the cluster [ 145 , 147 ] . since binaries tend to settle into the core , it is not surprising that none were found significantly outside of the core . subsequent searches using the improved resolution of the hubble space telescope eventually revealed a few dwarf novae close to the cores of selected globular clusters [ 144 , 146 , 148 ] . a more productive approach has been to look for direct evidence of the accretion around the white dwarf . this can be in the form of excess uv emission and strong h emission [ 44 , 63 , 93 ] from the accretion disk . this technique has resulted in the discovery of candidate cvs in 47 tuc [ 44 , 93 ] , m92 , ngc 6397 [ 26 , 41 , 156 ] , and ngc 6712 . these low luminosity x - ray binaries are characterized by a luminosity lx<10 erg / s , which distinguishes them from the low - mass x - ray binaries with lx > 10 erg / s . initial explanations of these objects focused on accreting white dwarfs , and a significant fraction of them are probably cvs . early searches performed with rosat data ( which had a detection limit of 10 erg / s ) revealed roughly 30 sources in 19 globular clusters . a more recent census of the rosat low luminosity x - ray sources , published by verbunt , lists 26 such sources that are probably related to globular clusters . recent observations with the improved angular resolution of chandra have begun to uncover numerous low luminosity x - ray candidates for cvs [ 64 , 65 , 73 , 79 ] . an attempt to identify an ir counterpart to the lmxb x - ray burster in liller 1 with chandra also resulted in the serendipitous discovery of 3 low luminosity x - ray sources , which may be quiescent lmxbs . observations of ngc 6652 have also discovered 3 new low luminosity x - ray sources , two of which may be cvs . the most comprehensive survey of x - ray sources in a globular cluster is that of grindlay et al . , which presents results of high resolution chandra images of 47 tuc . in addition to numerous other x - ray sources , they report 13 candidate cvs . the state of the field at this time is one of rapid change as chandra results come in and optical counterparts are found for the new x - ray sources . a living catalog of cvs has been created by downes et al . and may be the best source for confirmed cvs in globular clusters . the x - ray luminosities of low - mass x - ray binaries are in the range lx1010 erg / s . the upper limit is close to the eddington limit for accretion onto a neutron star , so these systems must contain an accreting neutron star or black hole . all of the lmxbs in globular clusters contain an accreting neutron star as they also exhibit x - ray bursts , indicating thermonuclear flashes on the surface of the neutron star . compared with 100 such systems in the galaxy , there are 13 lmxbs known in globular clusters . the globular cluster system contains roughly 0.1% of the mass of the galaxy and roughly 10% of the lmxbs . because these systems are so bright in x - rays , the globular cluster population is completely known we expect no new lmxbs to be discovered in the globular cluster system ( unless more multiple sources are resolved from these 13 sources ) . three have orbital periods greater than a few hours , four ultracompact systems have measured orbital periods less than 1 hour , and six have undetermined orbital periods . a member of the ultracompact group , 4u 1820 - 30 ( x1820 - 303 ) in the globular cluster ngc 6624 , has an orbital period of 11 minutes . this is the shortest known orbital period of any binary and most certainly indicates a degenerate companion . the orbital period , x - ray luminosity , and host globular clusters for these systems are given in table 1 . table 1 low - mass x - ray binaries in globular clusters . host clusters and lmxb properties . lmxb namecluster l x p orb ref.(10erg / s)(hr)x0512 - 401ngc 18511.9<0.85[37 , 149]x1724 - 307terzan 24.3 [37 , 149]x1745 - 203ngc 64400.9 [37 , 149]x1745 - 248terzan 5 x1746 - 370ngc 64417.65.70[37 , 123 , 149]x1747 - 313terzan 63.412.36[37 , 123 , 149]x1820 - 303ngc 662440.60.19[37 , 123 , 149]x1832 - 330ngc 66522.20.73[37 , 123]x1850 - 087ngc 67120.80.33[37 , 123 , 149]x2127 + 119ngc 70783.517.10[37 , 123 , 149 ] low - mass x - ray binaries in globular clusters . the improved resolution of chandra allows for the possibility of identifying optical counter - parts to lmxbs . if an optical counterpart can be found , a number of additional properties and constraints for these objects can be determined through observations in other wavelengths . in particular , the orbital parameters and the nature of the secondary can be determined . so far , optical counterparts have been found for x0512 - 401 in ngc 1851 , x1745 - 203 in ngc 6440 , x1746 - 370 in ngc 6441 , x1830 - 303 in ngc 6624 , x1832 - 330 in ngc 6652 [ 37 , 73 ] , x1850 - 087 in ngc 6712 [ 28 , 12 , 115 ] , and x2127 + 199 in ngc 7078 . recent observations with chandra of 47 tuc and ngc 6397 have revealed quiescent lmxbs which also harbor neutron stars in binaries and are detected by the thermal emission from the neutron star . work is ongoing , and new developments can be expected in the next few years . the population of known millisecond pulsars ( msps ) seems to be one of the fastest growing populations of relativistic binaries in globular clusters . in the past few years , improved searching techniques have revealed 47 pulsars in globular clusters . the lion s share of these are in 47 tuc , with 20 nine of which were recently discovered with the parkes radio telescope . additional recent discoveries have been made in ngc 6544 [ 29 , 30 , 130 ] , ngc 6266 , ngc 6397 , and ngc 6752 [ 29 , 30 ] . an approach by fruchter and goss uses deep multifrequency imaging to estimate the population of pulsars in globular clusters . in this approach , the expected number of pulsars beaming toward the earth , npuls , is determined by the total radio luminosity observed when the radio beam width is comparable in diameter to the core of the cluster . if the minimum pulsar luminosity is lmin and the total luminosity observed is ltot , then , with simple assumptions on the neutron star luminosity function , ( 11)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { n_{{\rm{puls } } } } = \frac{{{l_{{\rm{tot}}}}}}{{{l_{\min } } \ln ( { l_{{\rm{tot}}}}/{l_{\min } } } } . $ $ \end{document } in their observations of 7 globular clusters , fruchter and goss have recovered previously known pulsars in ngc 6440 , ngc 6539 , ngc 6624 , and 47 tuc . their estimates based on equation ( 11 ) give evidence of a population of between 60 and 200 previously unknown pulsars in terzan 5 , and about 15 each in liller 1 and ngc 6544 . chandra imaging of 47 tuc has also recovered those 15 msps with precise radio positions . one of these has now been identified with an optical counterpart that is almost certainly a he white dwarf . the x - ray spectral and temporal characteristics of these suggest that over 50 of the 108 x - ray sources identified in 47 tuc are millisecond pulsars . to obtain a full understanding of the nature of millisecond pulsars , it is necessary to develop full timing solutions so that the orbits can be determined . work has begun on the msps in 47 tuc , and freire et al . have obtained timing solutions for 15 of the known pulsars , including 8 binary systems . the properties of all the globular cluster msps with orbital periods less than 1 day are given in table 2 , which is a subset of table 5 in lorimer . table 2 short orbital period binary millisecond pulsars in globular pulsar p spin cluster p orb e m 2 ref.(ms)(days ) m j0023 - 7203j2.10147 tuc0.12 < 0.00020.02[20 , 48]j0024 - 7204o2.64347 tuc0.14 < 0.0040.02[20 , 48]j0024 - 72r3.4847 tuc0.066 > 0.00.03j0024 - 7203u4.34347 tuc0.430.0002 [20 , 48]j0024 - 72w2.35247 tuc0.11 > 0.00.15b1718 - 191004.037ngc 63420.26 > 0.00.1b1744 - 24a11.563terzan 50.08 > 0.00.1[101 , 114]j1807 - 243.059ngc 65440.071 > 0.00.009[29 , 130]j1910 - 593.266ngc 67520.865 > 0.00.19j1910 + 00043.619ngc 67600.14 > 0.00.02b2127 + 11c30.529 m 150.340.680.9 short orbital period binary millisecond pulsars in globular with the ongoing parkes multi - beam surveys of globular clusters and the use of the acceleration technique [ 98 , 108 ] , the population of known msps will be expected to grow dramatically in the next few years . this will help improve the understanding of these objects and their progenitors as well as the dynamics of globular clusters . although there have been hints of possible black hole binaries in extragalactic globular cluster systems [ 7 , 38 ] , there are no known black hole binaries in the galactic globular cluster system . all of the globular cluster lmxbs exhibit the x - ray variability that is indicative of nuclear burning on the surface of a neutron star . possible evidence of black holes in globular clusters can be found by extrapolating the velocity dispersion - black hole mass relation for galaxies with bulges to globular clusters . using velocity dispersion data for 22 galactic globular clusters , zheng finds possible black hole masses in the range of 1 1000m. although this is pure speculation , it does provide some indirect suggestion of possible black hole binaries in globular clusters . relativistic binaries are binary systems with at least one degenerate or collapsed object and an orbital period such that they will be brought into contact within a hubble time . ( note that this definition also includes binaries which are already in contact . ) outside of dense stellar clusters , most relativistic binary systems arise from primordial binary systems whose evolution drives them to tight , ultracompact orbits . the dynamical processes in globular clusters can drive wide binary systems toward short orbital periods and can also insert degenerate or collapsed stars into relativistic orbits with other stars . before addressing specific evolutionary scenarios , we will present the generic features of binary evolution that lead to the formation of relativistic binaries . the evolution of a binary system of two main - sequence stars can significantly affect the evolution of both component stars if the orbital separation is sufficiently small . if the orbital period is less than about 10 days , tidal interactions will have circularized the orbit during the pre- and early main - sequence phase . [ 60 , 167 , 168 ] both stars start in the main sequence with the mass of the primary mp , and the mass of the secondary ms , defined such that mpms . the binary system is described by the orbital separation a , and the mass ratio of the components qms / mp . the gravitational potential of the binary system is described by the roche model where each star dominates the gravitational potential inside regions called roche lobes . the two roche lobes meet at the inner lagrange point along the line joining the two stars . if either star fills its roche lobe , matter will stream from the roche lobe filling star through the inner lagrange point to the other star in a process known as roche lobe overflow ( rlof ) . this mass transfer affects both the evolution of the components of the binary as well as the binary properties such as orbital period and eccentricity . figure 5cross section of equipotential surfaces in the orbital plane of a binary with q=0.4 . the values of the potential surfaces are 5.0 , 3.9075 , 3.8 , 3.559 , 3.2 , 3.0 , and 2.8 . the values of the potential surfaces are 5.0 , 3.9075 , 3.8 , 3.559 , 3.2 , 3.0 , and 2.8 . roche lobe overflow can be triggered by the evolution of the binary properties or by evolution of the component stars . on the one hand , the orbital separation of the binary can change so that the roche lobe can shrink to within the surface of one of the stars . on the other hand , stellar evolution may eventually cause one of the stars to expand to fill its roche lobe . when both stars in the binary are main - sequence stars , the latter process is more common . since the more massive star will evolve first , it will be the first to expand and fill its roche lobe . at this stage , the mass exchange can be conservative ( no mass is lost from the binary ) or non - conservative ( mass is lost ) . depending on the details of the mass exchange and the evolutionary stage of the mass - losing star there are several outcomes that will lead to the formation of a relativistic binary . the primary star can lose its envelope , revealing its degenerate core as either a helium , carbon - oxygen , or oxygenneon white dwarf ; it can explode as a supernova , leaving behind a neutron star or a black hole ; or it can simply lose mass to the secondary so that they change roles . barring disruption of the binary , its evolution will then continue . in most outcomes , the secondary is now the more massive of the two stars and it may evolve off the main sequence to fill its roche lobe . the secondary can then initiate mass transfer or mass loss with the result that the secondary also can become a white dwarf , neutron star , or black hole . the relativistic binaries that result from this process fall into a number of observable categories . a wd - ms or wd - wd binary may eventually become a cataclysmic variable once the white dwarf begins to accrete material from its companion . if the companion is a main - sequence star , rlof can be triggered by the evolution of the companion . if the companion is another white dwarf , then rlof is triggered by the gradual shrinking of the orbit through the emission of gravitational radiation . if the total mass of the wd - wd binary is above the chandrasekhar mass , the system may be a progenitor to a type i supernova . the orbit of a ns - ms or ns - wd binary will shrink due to the emission of gravitational radiation . at the onset of rlof , the binary will become either a low - mass x - ray binary ( if the donor star is a wd or ms with m2m ) , or a high - mass x - ray binary ( if the donor is a more massive main - sequence star ) . these objects may further evolve to become millisecond pulsars if the ns is spun up during the x - ray binary phase [ 34 , 134 ] . a ns - ns binary will remain virtually invisible unless one of the neutron stars is observable as a pulsar . a bh - ms or bh - wd binary may also become a low- or high - mass x - ray binary . if the neutron star is observable as a pulsar , a bh - ns binary will appear as a binary pulsar . bh - bh binaries will be invisible unless they accrete matter from the interstellar medium . a comprehensive table of close binary types that can be observed in electromagnetic radiation can be found in hilditch . the type of binary that emerges depends upon the orbital separation and the masses of the component stars . during the evolution of a 10m star , the radius will slowly increase by a factor of about two as the star progresses from zero age main sequence to terminal age main sequence . the radius will then increase by about another factor of 50 as the star transitions to the red giant phase , and an additional factor of 10 during the transition to the red supergiant phase . these last two increases in size occur very quickly compared with the slow increase during the main - sequence evolution . depending upon the orbital separation , the onset of rlof can occur any time during the evolution of the star . mass transfer can be divided into three cases related to the timing of the onset of rlof . case a : if the orbital separation is small enough ( usually a few days ) , the star can fill its roche lobe during its slow expansion through the main - sequence phase while still burning hydrogen in its core.case b : if the orbital period is less than about 100 days , but longer than a few days , the star will fill its roche lobe during the rapid expansion to a red giant with a helium core . if the helium core ignites during this phase and the transfer is interrupted , the mass transfer is case bb.case c : if the orbital period is above 100 days , the star can evolve to the red supergiant phase before it fills its roche lobe . in this case case a : if the orbital separation is small enough ( usually a few days ) , the star can fill its roche lobe during its slow expansion through the main - sequence phase while still burning hydrogen in its core . case b : if the orbital period is less than about 100 days , but longer than a few days , the star will fill its roche lobe during the rapid expansion to a red giant with a helium core . if the helium core ignites during this phase and the transfer is interrupted , the mass transfer is case bb . case c : if the orbital period is above 100 days , the star can evolve to the red supergiant phase before it fills its roche lobe . in this case the typical evolution of the radius for a low metallicity star is shown in figure 6 . case a mass transfer occurs during the slow growth , case b during the first rapid expansion , and case c during the final expansion phase . the nature of the remnant depends upon the state of the primary during the onset of rlof and the orbital properties of the resultant binary depend upon the details of the mass transfer . although there are still many unanswered theoretical questions about the nature of the mass transfer phase , the basic properties of the evolution of a binary due to mass transfer can easily be described . the rate at which a star can adjust to changes in its mass is governed by three time scales . the dynamical time scale results from the adiabatic response of the star to restore hydrostatic equilibrium , and can be approximated by the free fall time across the radius of the star ( 12)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { t_{{\rm{dyn } } } } \simeq { \left ( { \frac{{2{r^3}}}{{gm } } } \right)^{1/2 } } \sim 40{\left [ { { { \left ( { \frac{r}{{{r _ \odot } } } } \right)}^3}\frac{{{m _ \odot } } } { m } } \right]^{1/2}}\min , $ $ \end{document } where m and r are the mass and radius of the star . the thermal equilibrium of the star is restored over a longer period given by the thermal time scale ( 13)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { t_{{\rm{th } } } } \simeq \frac{{g{m^2}}}{{rl } } \sim 3 \times { 10 ^ 7}{\left ( { \frac{m}{{{m _ \odot } } } } \right)^2}\frac{{{r _ \odot } } } { r}\frac{{{l _ \odot } } } { l}{\rm{yr , } } $ $ \end{document } where l is the luminosity of the star . finally , the main - sequence lifetime of the star itself provides a third time scale , which is also known as the nuclear time scale : ( 14)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { t_{{\rm{nuc } } } } \sim 7 \times { 10 ^ 9}\frac{m}{{{m _ \odot } } } \frac{{{l _ \odot } } } { l}{\rm{yr}}{\rm{. } } $ $ \end{document } the rate of mass transfer / loss from the roche lobe filling star is governed by how the star s radius changes in response to changes in its mass . hjellming and webbink describe these changes and the response of the roche lobe to mass changes in the binary using the radius - mass exponents , dlnr / dlnm , for each of the three processes described in equations ( 12 , 13 , 14 ) and defining ( 15)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \zeta _ { \rm{l } } } = ( 1 + q)\frac{{d\ln { r_{\rm{l}}}}}{{d\ln q } } $ $ \end{document } for the roche lobe radius - mass exponent . if l>dyn , the star can not adjust to the roche lobe , then the mass transfer takes place on a dynamical time scale and is limited only by the rate at which material can stream through the inner lagrange point . if dyn>l>th , then the mass transfer rate is governed by the slow expansion of the star as it relaxes toward thermal equilibrium , and it occurs on a thermal time scale . if both dyn and th are greater than l , then the mass loss is driven either by stellar evolution processes or by the gradual shrinkage of the orbit due to the emission of gravitational radiation . the time scale for both of these processes is comparable to the nuclear time scale . a good analysis of mass transfer in cataclysmic variables can be found in king et al . . conservative mass transfer occurs when there is no mass loss from the system . during conservative mass transfer consider a system with total mass m = m1+m2 and semi - major axis a. the total orbital angular momentum ( 16)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ j = { \left [ { \frac{{gm_1 ^ 2m_2 ^ 2a}}{m } } \right]^{1/2 } } $ $ \end{document } is a constant , and we can write a(m1m2 ) . using kepler s third law and denoting the initial values by a subscript i , we find : ( 17)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{p}{{{p_i } } } = { \left [ { \frac{{{m_{1i}}{m_{2i}}}}{{{m_1}{m_2 } } } } \right]^3}. $ $ \end{document } differentiating equation ( 17 ) and noting that conservative mass transfer requires 1=-2 gives : ( 18)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{\dot p}}{p } = \frac{{3{{\dot m}_1}({m_1 } - { m_2})}}{{{m_1}{m_2}}}. $ $ \end{document } note that if the more massive star loses mass , then the orbital period decreases and the orbit shrinks . usually , the initial phase of rlof takes place as the more massive star evolves . as a consequence , the orbit of the binary will shrink , driving the binary to a more compact orbit . in non - conservative mass transfer , both mass and angular momentum can be removed from the system . there are two basic non - conservative processes which are important to the formation of relativistic binaries the common - envelope process and the supernova explosion of one component of the binary . the result of the first process is often a short - period , circularized binary containing a white dwarf . although the most common outcome of the second process is the disruption of the binary , occasionally this process will result in an eccentric binary containing a neutron star . common envelope scenarios result when one component of the binary expands so rapidly that the mass transfer is unstable and the companion becomes engulfed by the donor star . the energy required to eject the envelope comes from the orbital energy of the binary and thus the orbit shrinks . the efficiency of this process determines the final orbital period after the common envelope phase . this is described by the efficiency parameter ( 19)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \alpha _ { { \rm{ce } } } } = \frac{{\delta { e_{{\rm{bind}}}}}}{{\delta { e_{{\rm{orb } } } } } } , $ $ \end{document } where ebind is the binding energy of the mass stripped from the envelope and eorb is the change in the orbital energy of the binary . the result of the process is the exposed degenerate core of the donor star in a tight , circular orbit with the companion . this process can result in a double degenerate binary if the process is repeated twice or if the companion has already evolved to a white dwarf through some other process . a brief description of the process is outlined by webbink , and a discussion of the factors involved in determining ce is presented in sandquist et al . . the effect on a binary of mass loss due to a supernova can be quite drastic . following padmanabhan , this process is outlined using the example of a binary in a circular orbit with radius a. let v be the velocity of one component of the binary relative to the other component . the initial energy of the binary is given by ( 20)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e = \frac{1}{2}\left ( { \frac{{{m_1}{m_2}}}{{{m_1 } + { m_2 } } } } \right){v^2 } - \frac{{g{m_1}{m_2}}}{a } = - \frac{{g{m_1}{m_2}}}{{2a}}. $ $ \end{document } following the supernova explosion of m1 , the expanding mass shell will quickly cross the orbit of m2 , decreasing the gravitational force acting on the secondary . the new energy of the binary is then ( 21)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e ' = \frac{1}{2}\frac{{{m_{{\rm{ns}}}}{m_2}}}{{{m_{{\rm{ns } } } } + { m_2}}}{v^2 } - \frac{{g{m_{{\rm{ns}}}}{m_2}}}{a } , $ $ \end{document } where mns is the mass of the remnant neutron star . we have assumed here that the passage of the mass shell by the secondary has negligible effect on its velocity ( a safe assumption , see pfahl et al . for a discussion ) , and that the primary has received no kick from the supernova ( not necessarily a safe assumption , but see davies and hansen for an application to globular cluster binaries ) . since we have assumed that the instantaneous velocities of both components have not been affected , we can replace them by v = g(m1+m2)/a , and so ( 22)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ e ' = \frac{{g{m_{{\rm{ns}}}}{m_2}}}{{2a}}\left ( { \frac{{{m_1 } + { m_2}}}{{{m_{{\rm{ns } } } } + { m_2 } } } - 2 } \right ) . $ $ \end{document } note that the final energy will be positive and the binary will be disrupted if mns<(1/2)(m1+m2 ) . this condition occurs when the mass ejected from the system is greater than half of the initial total mass : ( 23)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta m > \frac{1}{2}\left ( { { m_1 } + { m_2 } } \right ) , $ $ \end{document } where m = m1mns . if the binary is not disrupted , the new orbit becomes eccentric and expands to a new semi - major axis given by ( 24)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ a ' = a\left ( { \frac{{{m_1 } + { m_2 } - \delta m}}{{{m_1 } + { m_2 } - 2\delta m } } } \right ) , $ $ \end{document } and orbital period ( 25)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ p ' = p{\left ( { \frac{{a'}}{a } } \right)^{3/2}}{\left ( { \frac{{2a ' - a}}{{a ' } } } \right)^{1/2}}. $ $ \end{document } we have seen that conservative mass transfer can result in a tighter binary if the more massive star is the donor . non - conservative mass transfer can also drive the components of a binary together during a common envelope phase when mass and angular momentum are lost from the system . direct mass loss through a supernova explosion can also alter the properties of a binary , but this process generally drives the system toward larger orbital separation and can disrupt the binary entirely . with this exception , the important result of all of these processes is the generation of tight binaries with at least one degenerate object . they occur whenever the orbital separation of a progenitor binary is sufficiently small to allow for mass transfer or common envelope evolution . population distributions for relativistic binaries are derived from an initial mass function , a distribution in mass ratios , and a distribution in binary separations . these initial distributions are then fed into models for binary evolution in order to determine rates of production of relativistic binaries . the evolution of the binary is often determined by the application of some simple operational formulae such as those described by tout et al . . for example , hils , bender , and webbink estimated a population of close white dwarf binaries in the disk of the galaxy using a salpeter mass function , a mass ratio distribution strongly peaked at 1 , and a separation distribution that was flat in ln(a ) . other estimates of relativistic binaries differ mostly by using different distributions [ 14 , 86 , 113 , 112 ] . when the above evolutionary scenarios are played out in the environment of a globular cluster , additional mechanisms arise that enhance the production of relativistic binaries . new binary systems can be formed by dynamical interaction among three or more single stars or through tidal capture , while the period distribution and binary components of existing binary systems can be altered by interactions with other stars in the cluster . we will discuss here the broad features of these interactions and how they affect the evolution of binary systems toward relativistic binaries . the formation of binaries during the dynamical evolution of globular clusters can occur either through tidal capture or through n - body interactions . tidal capture occurs when an encounter between two stars is close enough that significant tides are raised on each . if the energy absorbed in these oscillations is great enough to leave the two stars with negative total energy , then the system will form a binary . this process was originally thought to be the dominant channel through which binaries were formed in globular clusters [ 19 , 43 ] . it is now thought to be quite rare , as detailed calculations have shown that the final result is more likely to be coalescence of the two stars [ 11 , 83 , 134 ] . although n - body interactions are less likely to occur than tidally significant two - body interactions , they are now thought to be the dominant channel for the formation of binaries during the evolution of a globular cluster . this process , however , is not likely to produce more than a few binaries during the lifetime of a cluster [ 19 , 117 ] . the existence of a population of primordial binaries provides a much more efficient channel for the transformation of the initial distribution in component masses and orbital periods towards higher mass components and shorter orbital periods . this process follows from the interaction of primordial binaries with single stars and other binaries . three results of the interaction are possible : complete disruption of the binary , an exchange of energy between the binary and the field star , or a replacement of one of the binary components by the field star . when a binary interacts with either a field star or with another binary , the energy of the interaction is shared among all stars in the interaction . the result is that the lowest mass object in the interaction will receive the largest velocity and be more likely to escape the interaction . in general , these interactions are quite complex , and must be studied numerically . a typical exchange interaction between a binary and a field star is shown in figure 7 . the binary comes in from the right ( red - white ) , while the field star ( green ) enters from the left . after a complicated interaction , the white star is ejected and the newly formed red - green binary is in a more tightly bound orbit . figure taken from mcmillan . the binary comes in from the right ( red - white ) , while the field star ( green ) enters from the left . after a complicated interaction , the white star is ejected and the newly formed red - green binary is in a more tightly bound orbit . figure taken from mcmillan . if the initial binding energy of the binary is large , the result of these interactions is to shrink the orbit of the new binary as the gravitational energy of the binary is used to bring the field star up to the speeds of the binary components . however , if the binding energy is low , the field star contributes energy to the components of the binary , thereby widening the orbit . heggie s law , which can be summarized as hard binaries get harder and soft binaries get softer . for roughly equal mass stars , a binary is considered hard if its binding energy is greater than the average kinetic energy of a field star in the cluster and soft if its binding energy is less . for unequal mass encounters , hills has shown that the ratio of the orbital speeds of the binary components to the speed of the impactor is a better indicator of whether the binding energy will increase or decrease . the average kinetic energy of a field star in the cluster is sometimes related to an effective temperature of the cluster [ 70 , 105 , 127 ] so that mv=3kt . numerical studies of the outcome of hard binary interactions indicate that the binding energy of the binary will increase by about 20% with each encounter [ 85 , 127 ] . since the encounter rate is proportional to the semi - major axis ( or 1/e ) and the energy increase per encounter is proportional to e , the rate of hardening per relaxation time is independent of the energy and is ebindi-0.6kt / trelax . a common feature of numerical studies of hard binary interactions is the preferential exchange of high - mass stars and stellar remnants with the least massive member of the binary . thus , the dynamical interactions in a globular cluster drive the initial orbital period distribution toward shorter periods by hardening the short period binaries while disrupting the softer binaries . through exchange interactions , the mass distribution of the binary components is also driven toward higher mass stars , which further enhances the number of mass - transferring systems that can evolve to become relativistic binaries . because stellar remnants can also be exchanged into hard binaries , globular cluster evolution opens up a new channel for the formation of relativistic binaries by introducing evolved components into binary systems that have not yet undergone a mass transfer phase . a particularly promising channel involves the exchange of a neutron star into a binary with a main - sequence star . the binary then undergoes case b or case c mass transfer with a common envelope phase , resulting in a ns - wd binary . similar interactions can occur to produce wd - wd binaries if a massive co or one white dwarf is exchanged into a hard binary . black hole binaries can also form as a result of exchange interactions , but the process is different because black hole progenitors will evolve so quickly in relation to the relaxation time of most globular clusters [ 96 , 150 ] . one scenario that generates black hole binaries in globular clusters is described by portegies zwart and mcmillan . stellar mass black holes of mass m10m will be born early in the life of a globular cluster and , through mass segregation , they will quickly sink to the core . once in the core , these black holes will be so much more massive than the field stars that they will effectively form their own cluster and interact solely with themselves . single black holes will form binaries with other black holes through three - body encounters ; any black holes which are in binaries with other stars will team up with another black hole through exchange encounters . this population of black holes and black hole binaries will then evolve separately from the rest of the cluster as no other stars will be massive enough to affect its dynamics . we have seen how the dynamics of globular clusters can enhance the population of progenitors to relativistic binaries , making the standard channels of mass - transfer more likely to occur . in addition , globular cluster dynamics can open up new channels for the formation of relativistic binaries by inserting evolved , stellar remnants such as neutron stars or white dwarfs into binary systems and by shrinking the orbits of binary systems to enhance the likelihood of mass exchange . finally , binary - single star encounters can simply create relativistic binaries by inserting two evolved objects into a binary and then shrinking the orbit to ultracompact periods . we next discuss the probable rates for the formation of such systems and the dynamical simulations that are used to synthesize globular cluster populations of relativistic binaries . simulations of the populations of relativistic binaries in globular clusters rely on the interplay between the evolution of individual stars in the progenitor systems and the evolution of globular clusters . the evolution of stars in the progenitor systems has been discussed in the previous section and we now turn to techniques for simulating the evolution of globular clusters . the evolution of a globular cluster is dominated by the gravitational interaction between the component stars in the cluster . the overall structure of the cluster as well as the dynamics of most of the stars in the cluster are determined by simple n - body gravitational dynamics . however , the evolutionary time scales of stellar evolution are comparable to the relaxation time and core collapse time of the cluster . consequently , stellar evolution affects the masses of the component stars of the cluster , which affects the dynamical state of the cluster . thus , the dynamical evolution of the cluster is coupled to the evolutionary state of the stars . also , as we have seen in the previous section , stellar evolution governs the state of the binary evolution and binaries provide a means of support against core collapse . thus , the details of binary evolution as coupled with stellar evolution must also be incorporated into any realistic model of the dynamical evolution of globular clusters . to close the loop , the dynamical evolution of the globular cluster affects the distribution and population of the binary systems in the cluster . in our case , we are interested in the end products of binary evolution , which are tied both to stellar evolution and to the dynamical evolution of the globular cluster . to synthesize the population of relativistic binaries , we need to look at the dynamical evolution of the globular cluster as well as the evolution of the binaries in the cluster . general approaches to this problem involve solving the n - body problem for the component stars in the cluster and introducing binary and stellar evolution when appropriate to modify the n - body evolution . there are two fundamental approaches to tackling this problem direct integration of the equations of motion for all n bodies in the system and large - n techniques , such as fokker - planck approximations coupled with monte carlo treatments of binaries ( see heggie et al . for a comparison of these techniques ) . in the next two subsections we conclude this section with a discussion of the recent relativistic binary population syntheses generated by dynamical simulations . the n - body approach to modeling globular cluster dynamics involves direct calculations of the gravitational interactions between all n bodies in the simulation . in principle the positions of the n objects in the cluster are determined by direct integration of the 3n equations of motion : ( 26)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { { \rm{\ddot r}}_i } = - \sum\limits_{j \ne i } { \frac{{g{m_j}\left ( { { { \rm{r}}_i } - { { \rm{r}}_j } } \right)}}{{{{\left| { { { \rm{r}}_j } - { { \rm{r}}_i } } \right|}^3}}}. } $ $ \end{document } when the positions indicate that objects are sufficiently close to each other , then the interaction is modeled to determine the outcome . in order to achieve realistic simulations with tidal interactions , possible mass transfer , and a mass spectrum of bodies , detailed stellar evolution and stellar collision models must be included and calculated . the kira integrator is part of the starlab environment which also includes stellar evolution codes and other modules for doing n - body simulations . the nbodyx codes have been developed and improved by aarseth since the early 1960 s . he has two excellent summmaries of the general properties and development of the nbodyx codes [ 4 , 3 ] . a good summmary of general n - body applications can also be found at the nemo website . most large n - body calculations are done with a special purpose computer called the grape ( gravity pipe ) invented by makino . the most recent incarnation of the grape is the grape 6 , which has a theoretical peak speed of 100 tflops . the grape calculates the accelerations and jerks for the interaction between each star in the cluster . the main advantage of n - body simulations is the small number of simplifying assumptions which must be made concerning the dynamical interactions within the cluster . therefore , the details for those specific interactions can be calculated during the simulation . within the limits of the numerical errors that accumulate during the calculation obviously , one of the main computational difficulties is simply the cpu cost necessary to integrate the equations of motion for n bodies . the other computational difficulty of the direct n - body method is the wide range of precision required [ 82 , 72 ] . consider the range of distances , from the size of neutron stars ( 10 km ) to the size of the globular cluster ( 50 pc 10 km ) , spanning 14 orders of magnitude . if the intent of the calculations is to determine the frequency of interactions with neutron stars , we have to know the relative position of every star to within 1 part in 10 . the range of time scales is worse yet . considering that the time for a close passage of two neutron stars is on the order of milliseconds and that the age of a globular cluster is 10 yr 10 ms these computational requirements coupled with hardware limitations mean that the number of bodies which can be included in a reasonable simulation is no more than 10 . this is about an order of magnitude less than the number of stars in a typical globular cluster . although one has great confidence in the results of an n - body simulation , these simulations are generally for systems that are smaller than globular clusters . consequently , applications of n - body simulations to globular cluster dynamics involve scaling lower n simulations up to the globular cluster regime . thus , one scales the results of an n - body simulation based upon the assumption of a dominant process . however , one can never be certain that the extrapolation is smooth and that there are no critical points in the scaling with n. one can also scale other quantities in the model , so that the quantity of interest is correctly scaled . an understanding of the nature of the scaling is crucial to understanding the applicability of n - body simulations to globular cluster dynamics ( see baumgardt for an example ) . . the computational limitations of n - body simulations can be sidestepped by describing the system in terms of distribution functions fm(x , v , t ) with the number of stars of mass m at time t in the range ( x , x+dx ) and ( v , v+dv ) given by dn = fmdxdv . this description requires that either the phase - space element dxdv be small enough to be infinitesimal yet large enough to be statistically meaningful , or that fm be interpreted as the probability distribution for finding a star of mass m at a location in phase space . the gravitational interaction is provided by a smoothed gravitational potential , which is determined by ( 27)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \nabla ^2}\phi = 4\pi \sum\limits_i { \left [ { { m_i}\int { { f_{{m_i}}}\left ( { { \rm{x , v,}}t } \right){d^3}v } } \right ] . } $ $ \end{document } the effect of gravitational interactions is modeled by a collision term [f ] ( see [ 19 , 117 ] for specific descriptions of ) . the dynamics of the globular cluster are then governed by the fokker - planck equation : ( 28)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{\partial f}}{{\partial t } } + { \rm{v } } \cdot \nabla f - \nabla \phi \cdot \frac{{\partial f}}{{\partial { \rm{v } } } } = \gamma \left [ f \right ] . $ $ \end{document } in the fokker - planck approach , the mass spectrum of stars is binned , with a separate fm for each bin . increasing the resolution of the mass spectrum requires increasing the number of distribution functions and thus increasing the complexity of the problem . methods for numerically solving the fokker - planck equation use either an orbit - averaged form of equation ( 28 ) , or a monte carlo approach [ 50 , 55 , 56 , 89 ] . the two time scales involved in the evolution of fm are tcross ( which governs changes in position ) and trelax ( which governs changes in energy ) . the orbit - averaged form of equation ( 28 ) derives from the realization that changes in position are essentially periodic with orbital period ttcrosstrelax . thus , one can average over the rapid changes in position and retain the slow changes in the phase space coordinates that occur over relaxation times . when one does this , the fokker - planck equation is reduced to an equation involving three phase - space variables and time . the orbit - averaged solutions of the fokker - planck equation can not easily handle the effect of binaries and the binary interactions that occur during the evolution of a globular cluster . the advantages of the orbit - averaged approach are that one can generalize it to handle anisotropy in velocity , thus allowing study of the effects of the galactic gravitational field and tidal stripping . monte carlo simulations do not actually solve the fokker - planck equation , but rather rely on a representative sample of n test stars which are followed numerically with random velocity perturbations applied as are appropriate to the diffusion coefficients in . the advantage of the monte carlo approach is that one can handle binary interactions and stellar evolution directly ( and then extrapolate the results of the n test stars to the n actual stars in the cluster ) . however , like fokker - planck , the monte carlo method currently requires the cluster to be spherically symmetric , so gravitational effects of the galaxy must be treated in an ad hoc way . there have been several recent works addressing binary populations in globular clusters [ 16 , 18 , 33 , 31 , 32 , 34 , 109 , 121 , 124 , 127 , 132 , 134 , 143 , 151 , 155 ] . although the motivations have been varied , it is often possible to extract information about the resulting populations of relativistic binaries . despite the differing models and population synthesis techniques , the predicted populations are in rough agreement . here , we summarize the different techniques and their predictions for relativistic binaries in globular clusters . although n - body simulations have the potential to provide the most detailed population syntheses of relativistic binaries in globular clusters , there are very few actual populations described in the literature . most of the current work that treats binaries in a consistent and detailed way is limited to open clusters [ 128 , 80 , 95 ] and is focused on a particular outcome of the binary population , such as blue stragglers in the case of hurley et al . , or brown dwarfs in the case of kroupa et al . . focus on photometric observations of open clusters , but promise a more detailed look at the binary population in a future spectroscopic paper . in their comparison of n - body and fokker - planck simulations of the evolution of globular clusters , takahashi and portegies zwart followed the evolution of n=1 k , 16 k , and 32 k systems with initial mass functions given by equation ( 9 ) and initial density profiles set up from king models . although they allowed for realistic stellar binary evolution in their comparisons , their focus was on the structural evolution of globular clusters . it is possible to generate a population distribution for black hole binaries in globular clusters using the n - body simulations of portegies zwart and mcmillan that were intended to describe the population of black hole binaries that were ejected from globular clusters . their scenario for black hole binary ejection describes a population of massive stars that evolves into black holes . as the black holes are significantly more massive than the other stars , they effectively form a separate sub - system , which interacts solely with itself . the black holes form binaries and then harden through binary - single black hole interactions that occasionally eject either the binary , the single black hole , or both . the results of their simulations roughly confirm a theoretical argument based on the recoil velocity that a binary receives during an interaction . noting that each encounter increases the binding energy by about 20% and that roughly 1/3 of this energy goes into binary recoil , the minimum binding energy eb min of an ejected black hole binary is ( 29)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { e_{{\rm{b min } } } } \sim 36{w_0}\frac{{{m_{{\rm{bh}}}}}}{{\left\langle m \right\rangle } } kt , $ $ \end{document } where m is the average mass of a globular cluster star and w0=m|0|/kt is the dimensionless central potential . after most binaries are ejected , m0.4m. after a few gigayears , nearly all of the black holes were ejected . at the end of this phase of black hole binary ejection , there is a 50% chance that a binary remains in the cluster with no other black hole to eject it . thus , there should be a stellar mass black hole binary remaining in about half of the galactic globular clusters . the maximum binding energy of the remaining black hole binary is eb min and is also given by equation ( 29 ) . we can then approximate the distribution in energies of the remaining black hole binaries as being flat in log(eb ) . dynamical monte carlo simulations can be used to study the evolution of binary populations within evolving globular cluster models . rasio et al . have used a monte carlo approach ( described in joshi et al . [ 88 , 89 ] ) to study the formation and evolution of ns wd binaries , which may be progenitors of the large population of millisecond pulsars being discovered in globular clusters ( see section 3.3 ) . in addition to producing the appropriate population of binary millisecond pulsars to match observations , the simulations also indicate the existence of a population of ns - wd binaries ( see figure 8) . figure 8results of the monte carlo simulation of ns - wd binary generation and evolution in 47 tuc . the circles and error bars are the 10 binary pulsars in 47 tuc with well measured orbits . systems in b have not yet evolved through gravitational radiation to begin rlof from the wd to the ns . results of the monte carlo simulation of ns - wd binary generation and evolution in 47 tuc . the circles and error bars are the 10 binary pulsars in 47 tuc with well measured orbits . systems in b have not yet evolved through gravitational radiation to begin rlof from the wd to the ns . the tail end of the systems in group b of figure 8 represents the ns - wd binaries that are in very short period orbits and are undergoing a slow inspiral due to gravitational radiation . these few binaries can be used to infer an order of magnitude estimate on the population of such objects in the galactic globular cluster system . if we consider that there are two binaries with orbital period less than 2000 s out of 10m in 47 tuc , and assume that this rate is consistent throughout the globular cluster system as a whole , we find a total of 60 such binaries . although this estimate is quite crude , it compares favorably with estimates arrived at through the encounter rate population syntheses , which are discussed in section 5.3.3 . there is also great promise for the hybrid gas / monte carlo method being developed by spurzem and giersz . their recent simulation of the evolution of a cluster of 300,000 equal point - mass stars and 30,000 binaries yields a wealth of detail about the position and energy distribution of binaries in the cluster . one expects that the inclusion of stellar evolution and a mass spectrum would produce similar detail concerning relativistic binaries . perhaps because of the paucity of observations of double white dwarf binaries ( there is only one candidate he - co binary ) , there have been few population syntheses of wd - wd binaries in globular clusters . sigurdsson and phinney use monte carlo simulations of binary encounters to infer populations using a static background cluster described by an isotropic king - michie model . their results are focused toward predicting the observable end products of binary evolution such as millisecond pulsars , cataclysmic variables , and blue stragglers . therefore , there are no clear descriptions of relativistic binary populations provided . davies and collaborators also use the technique of calculating encounter rates ( based on calculations of cross - sections for various binary interactions and number densities of stars using king - michie static models ) to determine the production of end products of binary evolution [ 33 , 31 ] . although they also do not provide a clear description of a population of relativistic binaries , their results allow the estimation of such a population . using the encounter rates of davies and collaborators [ 33 , 31 ] , one can follow the evolution of binaries injected into the core of a cluster . a fraction of these binaries will evolve into compact binaries which will then be brought into contact through the emission of gravitational radiation . by following the evolution of these binaries from their emergence from common envelope to contact , we can construct a population and period distribution for present day globular clusters . for a globular cluster with dimensionless central potential w0=12 the binaries were chosen from a salpeter imf with exponent =2.35 , and the common envelope evolution used an efficiency parameter ce=0.4 . one run was terminated after 15 gyr and the population of relativistic binaries which had been brought into contact through gravitational radiation emission was noted . the second run was allowed to continue until all binaries were either in merged or contact systems . there are four classes of relativistic binaries that are brought into contact by gravitational radiation : high mass white dwarf - white dwarf binaries wda2 with total mass above the chandrasekhar mass ; low mass white dwarf - white dwarf binaries wdb2 with total mass below the chandrasekhar mass ; neutron star - white dwarf binaries nw ; and neutron star - neutron star binaries ns . the number of systems brought into contact at the end of each run is given in table 3 . t evol wda2wdb2nwns15 gyr1101015707418 number of relativistic binaries brought into contact through binary interactions . in the second run , the relativistic binaries had all been brought into contact . in similar runs , this occurs after another 15 gyr . an estimate of the present - day period distribution can be made by assuming a constant merger rate over the second 15 gyr . consider the total number of binaries that will merge to be described by n(t ) . thus , the merger rate is =-dn / dt . assuming that the mergers are driven solely by gravitational radiation we define n(p ) to be the number of binaries with period less than p , and thus ( 30)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \eta = - \frac{{dn}}{{dt } } = - \frac{{dn}}{{dp}}\frac{{dp}}{{dt } } , $ $ \end{document } so ( 31)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{dn}}{{dp } } = \frac { { - \eta } } { { dp / dt}}. $ $ \end{document } the merger rate is given by the number of mergers of each binary type per 1000 primordial binaries per 15 gyr . if the orbits have been circularized ( which is quite likely if the binaries have been formed through a common envelope ) , the evolution of the period due to gravitational radiation losses is given by ( 32)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{dp}}{{dt } } = - { k_0}{p^ { - 5/3 } } , $ $ \end{document } where k0 is given by ( 33)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { k_0 } = \frac{{96}}{5}{\left ( { 2\pi } \right)^{8/3}}\frac{{{g^{5/3}}}}{{{c^5}}}{{\mathcal m}^{5/3 } } , $ $ \end{document } with the chirp mass mm1m2(m1+m2 ) . following this reasoning and using the numbers in table 3 , we can determine the present day population of relativistic binaries per 1000 primordial binaries . to find the population for a typical cluster , we need to determine the primordial binary fraction for globular clusters . estimates of the binary fraction in globular clusters range from 13% up to about 40% based on observations of either eclipsing binaries [ 5 , 165 , 166 ] or luminosity functions [ 138 , 139 ] . assuming a binary fraction of 30% , we can determine the number of relativistic binaries with short orbital period ( porb < pmax ) for a typical cluster with 10m and the galactic globular cluster system with 10m by simply integrating the period distribution from contact pc up to pmax ( 34)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n = \int_{{p_{\rm{c}}}}^{{p_{\max } } } { \frac{\eta } { { { k_0}}}{p^{5/3}}dp . } $ $ \end{document } the value of pc can be determined by using the roche lobe radius of eggleton , ( 35)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { r_{\rm{l } } } = \frac{{0.49{q^{2/3}}}}{{0.6{q^{2/3 } } + \ln \left ( { 1 + { q^{1/3 } } } \right)}}a , $ $ \end{document } and stellar radii as determined by lynden - bell and odwyer . the expected populations for an individual cluster and the galactic cluster system are shown in table 4 using neutron star masses of 1.4m , white dwarf masses of 0.6m and 0.3m , and pmax=2000 s. table 4encounter rate estimates of the population of relativistic binaries in a typical globular cluster and the galactic globular cluster system.objectwda2nwnscluster5.64.00.5system176.5125.216 encounter rate estimates of the population of relativistic binaries in a typical globular cluster and the galactic globular cluster system . although we have assumed the orbits of these binaries will be circularized , there is the possible exception of ns binaries , which may have a thermal distribution of eccentricities if they have been formed through exchange interactions rather than through a common envelope . in this case , an integration over both period and eccentricity , using the formulae of pierro and pinto , would be required . the small number of observed relativistic binaries can be used to infer the population of dark progenitor systems . for example , the low - mass x - ray binary systems are bright enough that we see essentially all of those that are in the galactic globular cluster system . if we assume that the ultracompact ones originate from detached wd - ns systems , then we can estimate the number of progenitor systems by looking at the time spent by the system in both phases . also , let ndet be the number of detached wd - ns systems that will evolve to become lmxbs , and tdet be the time spent during the inspiral due to the emission of gravitational radiation until the companion white dwarf fills its roche lobe . if the process is stationary , we must have ( 36)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{{n_{\rm{x}}}}}{{{t_{\rm{x } } } } } = \frac{{{n_{\det } } } } { { { t_{\det } } } } . $ $ \end{document } the time spent in the inspiral phase can be found from integrating equation ( 32 ) to get ( 37)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { t_{\det } } = \frac{3}{{8{k_0}}}\left ( { p_0^{8/3 } - p_{\rm{c}}^{8/3 } } \right ) $ $ \end{document } where p0 is the period at which the progenitor emerges from the common envelope and pc is the period at which rlof from the white dwarf to the neutron star begins . thus , the number of detached progenitors can be estimated from ( 38)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { n_{\det } } = \frac{{{n_{\rm{x}}}}}{{{t_{\rm{x}}}}}\frac{3}{{8{k_0}}}\left ( { p_0^{8/3 } - p_{\rm{c}}^{8/3 } } \right ) . $ $ \end{document } there are four known ultracompact lmxbs with orbital periods small enough to require a degenerate white dwarf companion to the neutron star . thus , 4nx10 . the lifetime tx is rather uncertain , depending upon the nature of the mass transfer and the timing when the mass transfer would cease . a standard treatment of mass transfer driven by gravitational radiation alone gives an upper bound of tx10 yr , but other effects such as tidal heating or irradiation may shorten this to tx10 yr [ 8 , 134 ] . the value of p0 depends critically upon the evolution of the neutron star - main - sequence binary , and is very uncertain . both k0 and pc depend upon the masses of the wd secondary and the ns primary . for a rough estimate , we take the mass of the secondary to be a typical he wd of mass 0.4m and the mass of the primary to be 1.4m. rather than estimate the typical period of emergence from the common envelope , we arbitrarily choose p0=2000 s. we can be certain that all progenitors have emerged from the common envelope by the time the orbital period is this low . the value of pc can be determined by using equation ( 35 ) and the radius of the white dwarf as determined by lynden - bell and odwyer . adopting the optimistic values of nx=10 and tx=10 yr , and evaluating equation ( 37 ) gives tdet10 yr . thus , we find ndet110 , which is within an order of magnitude of the numbers found through dynamical simulations ( section 5.3.2 ) and encounter rate estimations ( section 5.3.3 ) . continuing in the spirit of small number statistics , we note that there is one known radio pulsar in a globular cluster ns - ns binary ( b2127 + 11c ) and about 50 known radio pulsars in the globular cluster system as a whole ( although this number may continue to grow ) . we may estimate that ns - ns binaries make up roughly 1/50 of the total number of neutron stars in the globular cluster system . a lower limit on the number of neutron stars comes from estimates of the total number of active radio pulsars in clusters , giving \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$${n_{{\rm{n}}{{\rm{s}}^2 } } } \sim { 10 ^ 5}$$\end{document } . thus , we can estimate the total number of ns - ns binaries to be 2000 . not all of these will be in compact orbits , but we can again estimate the number of systems in compact orbits by assuming that the systems gradually decay through gravitational radiation and thus ( 39)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{{n_{{\rm{compact}}}}}}{{{n_{{\rm{n}}{{\rm{s}}^2 } } } } } = \frac{{{t_{{\rm{compact}}}}}}{{{t_{{\rm{coalesce } } } } } } $ $ \end{document } where ncompact is the number of systems in compact orbits ( porb<2000 s ) , tcompact is the time spent as a compact system , and tcoalesce is the typical time for a globular cluster ns - ns binary to coalesce due to gravitational radiation inspiral . adopting the coalescence time of b2127 + 11c as typical , tcoalesce=210 yr , and integrating equation ( 37 ) for two 1.4m neutron stars , we find ncompact25 . again a very exciting prospect for the observation of relativistic binaries in globular clusters lies in the fact that they will be sources of gravitational radiation . there is a phase in the evolution of most relativistic binaries during which the orbital period is slowly shrinking due to the emission of gravitational radiation . if the binary is in a circularized orbit , the gravitational radiation will be peaked strongly in the second harmonic of the orbital period , so fgw=2forb . although the strength of the gravitational radiation varies with the orientation of the binary , an angle - averaged estimate of the signal strength is ( 40)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { h_0 } = 1.5 \times { 10^ { - 21}}{\left ( { \frac{{{f_{{\rm{gw}}}}}}{{{{10}^ { - 3}}{\rm{hz } } } } } \right)^{2/3}}\left ( { \frac{{1{\rm { kpc}}}}{r } } \right){\left ( { \frac{{\mathcal m}}{{{m _ \odot } } } } \right)^{5/3}}. $ $ \end{document } at a typical globular cluster distance of r10 kpc and typical chirp mass of m0.5m , a relativistic wd - wd or wd - ns binary with porb=400 s will have a gravitational wave amplitude of 10 . this value is within the range of the proposed space - based gravitational wave observatory lisa . many globular clusters lie off the plane of the galaxy and are relatively isolated systems with known positions . the angular resolution of lisa improves with signal strength . by focusing the search for gravitational radiation using known positions of suspected sources , it is possible to increase the signal - to - noise ratio for the detected signal . thus , the angular resolution of lisa for globular cluster sources can be on the order of the angular size of the globular cluster itself at fgw>1 mhz . consequently , the orbital period distribution of a globular cluster s population of relativistic binaries can be determined through observations in gravitational radiation . we will discuss the prospects for observing each class of relativistic binaries covered in this review . white dwarf - white dwarf binaries that are formed from a common envelope phase will be briefly visible while the recently revealed hot core of the secondary cools . these objects are most likely the non - flickerers of cool et al . and edmonds et al . . wd - wd binaries formed through exchange interactions may very well harbor white dwarfs which are too cool to be observed . in either case , hardening through dynamical interactions will become less likely as the orbit shrinks and the effective cross section of the binary becomes too small . these objects will then be effectively invisible in electromagnetic radiation until they are brought into contact and rlof can begin . during this invisible phase , the orbital period is ground down through the emission of gravitational radiation until the orbital period is a few hundred seconds . with a frequency of 1 to 10 mhz , gravitational radiation from such white dwarf - neutron star binaries that are expected to be progenitors of the millisecond pulsars must pass through a phase of gravitational radiation after the degenerate core of the donor star emerges from the common envelope phase and before the spin - up phase begins with the onset of mass transfer from the wd to the neutron star . the orbital period at the onset of rlof will be on the order of 1 to 2 minutes and the gravitational wave signal will be received at lisa with a signal - to - noise of 50100 at a frequency of around 20 mhz for a globular cluster binary . ( section 5.3.4 ) to 125 from encounter rates ( table 4 ) . binaries with significant eccentricity will have a spectrum of harmonics of the orbital frequency , with the relative strength of the nth harmonic for eccentricity e given by ( 41)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ g\left ( { n , e } \right ) = \frac{{{n^4}}}{{32}}\left\ { { { { \left [ { { j_{n - 2}}\left ( { ne } \right ) - { j_{n - 1}}\left ( { ne } \right ) + \frac{2}{n}{j_n}\left ( { ne } \right ) + { j_{n + 1}}\left ( { ne } \right ) - { j_{n + 2}}\left ( { ne } \right ) } \right]}^2 } + \left ( { 1 - { e^2 } } \right){{\left [ { { j_{n - 2}}\left ( { ne } \right ) - 2{j_n}\left ( { ne } \right ) + { j_{n + 2}}\left ( { ne } \right ) } \right]}^2 } + \frac{4}{{3{n^2}}}{{\left [ { { j_n}\left ( { ne } \right ) } \right]}^2 } } \right\ } , $ $ \end{document } where jn is the bessel function . the higher harmonics of sufficiently eccentric binaries ( e>0.7 ) can be detected by lisa even though the fundamental orbital frequency is well below its sensitivity band of 1100 mhz . although the globular cluster population of ns - ns binaries is expected to be quite small ( 10 ) , they may have high eccentricities . the binary pulsar b2127 + 11cis an example of a ns - ns binary in a globular cluster . in terms of the unknown angle of inclination i if the globular cluster systems of other galaxies follow similar evolution as the milky way population , these binaries may be potential sources for ligo as gravitational radiation grinds them down to coalescence . with their high eccentricities and large chirp mass , black hole binaries will also be good potential sources for gravitational radiation from the galactic globular cluster system [ 17 , 15 ] . the relatively close proximity of the galactic globular cluster system and the separations between individual globular clusters allows for the identification of gravitational radiation sources with their individual host clusters . although the expected angular resolution of lisa is not small enough to allow for the identification of individual sources , knowledge of the positions of the clusters will allow for focused searches of the relativistic binary populations of the majority of the galactic globular clusters . armed with a knowledge of the orbital periods of any detected binaries , concentrated searches in electromagnetic radiation can be successful in identifying relativistic binaries that may have otherwise been missed . the populations of these objects are the result of an interplay between the gravitational dynamics of large n - body systems , the dynamics of mass transfer , the details of stellar evolution , and the effect of the gravitational field of the galaxy . the gravitational dynamics of globular clusters can enhance the population of short period binaries of main - sequence stars as well as inject compact objects such as white dwarfs and neutron stars into stellar binary systems . once they are in such systems , the details of stellar evolution and mass transfer in close binary systems govern the likely end products of the dynamical interaction between the two stars . furthermore , most models of the evolution of the core of a globular cluster rely on the gradual hardening and ejection of binary systems to delay the onset of core collapse . the hardening of binaries in the core of globular clusters will produce relativistic binaries , but it will also eventually eject these systems as they gain larger and larger recoil velocities in each subsequent encounter . the threshold for ejection from a globular cluster depends both upon the gravitational potential of the cluster itself and the gravitational potential of its environment generated by the milky way . as the globular cluster orbits the milky way , its local environment changes . consequently , if other dynamical processes ( such as gravothermal oscillations ) do not dominate , the globular cluster s population of relativistic binaries may also reflect the past orbital history of the globular cluster . over the last decade , observational techniques and technology have improved to the extent that significant discoveries are being made regularly . at this point , the bottleneck in observations of binary millisecond pulsars , low - mass x - ray binaries , and cataclysmic variables is time , not technology . as these observational techniques are brought to bear on more clusters , more discoveries are bound to be made . in the next decade , the possibility of using gravitational wave astronomy to detect relativistic binaries brings the exciting possibility of identifying the populations of electromagnetically invisible objects such as detached wd and ns binaries and black hole binaries in globular clusters . these observations can only help to improve the understanding of the complex and interesting evolution of these objects and their host globular clusters .

the galactic population of globular clusters are old , dense star systems , with a typical cluster containing 104 106 stars . as an old population of stars , globular clusters contain many collapsed and degenerate objects . as a dense population of stars , globular clusters are the scene of many interesting close dynamical interactions between stars . these dynamical interactions can alter the evolution of individual stars and can produce tight binary systems containing one or two compact objects . in this review , we discuss the theoretical models of globular cluster evolution and binary evolution , techniques for simulating this evolution which lead to relativistic binaries , and current and possible future observational evidence for this population . globular cluster evolution will focus on the properties that boost the production of hard binary systems and on the tidal interactions of the galaxy with the cluster , which tend to alter the structure of the globular cluster with time . the interaction of the components of hard binary systems alters the evolution of both bodies and can lead to exotic objects . direct n - body integrations and fokker - planck simulations of the evolution of globular clusters that incorporate tidal interactions and lead to predictions of relativistic binary populations are also discussed . we discuss the current observational evidence for cataclysmic variables , millisecond pulsars , and low - mass x - ray binaries as well as possible future detection of relativistic binaries with gravitational radiation .

Introduction Globular Clusters Observations Relativistic Binaries Dynamical Evolution Prospects of Gravitational Radiation Summary

from this population come a host of exotic objects such as ultracompact cataclysmic variables , non - flickering x - ray and uv sources , low - mass x - ray binaries , and millisecond pulsars . these objects and their dark counterparts in the population of relativistic binaries are also likely to be observable sources of gravitational radiation for low - frequency gravitational wave detectors such as the proposed spaceborne interferometer lisa . the population of tight binaries in globular clusters is a product of the dynamical evolution of an n - body gravitational system . in keeping with the focus of this review article on relativistic binaries in globular clusters , we shall only touch on the aspects of globular clusters , observations , binary evolution , and n - body dynamics as they relate to populations of this specific class of binaries in globular clusters . we begin by looking at the physical structure and general history of the galactic globular cluster system that leads to the concentration of evolved stars , stellar remnants , and binary systems in the cores of the clusters . current observations of the cores of globular clusters that have revealed numerous tracer populations of relativistic binaries are also discussed . we will look at how mass transfer from one star in the presence of a nearby companion can dramatically alter the evolution of both stars in the process of binary evolution . the enhanced production of relativistic binaries in globular clusters results from dynamical processes that drive binaries toward tighter orbits and that preferentially exchange more massive and degenerate objects into binary systems . numerical simulations of globular cluster evolution , which can be used to predict the rate at which relativistic binaries are formed , are discussed . readers interested in further studies of the structure and evolution of globular clusters are invited to look at binney and tremaine , spitzer , and volumes i and ii of padmanabhan s theoretical astrophysics [ 116 , 117 ] for a good introduction to the physical processes involved . although our focus is solely on the galactic globular cluster system , the physics of globular cluster systems associated with other galaxies is well covered in the review article by harris as well as his lecture notes from the saas - fee course on star clusters . during the early stages of the evolution of a globular cluster , subsequent replenishment of the intercluster gas by stellar winds from evolved stars is removed during periodic passages of the cluster through the plane of the galaxy . since the relaxation time is related to the number and strength of the gravitational encounters of a typical cluster star , it is related to the number of stars in the cluster and the average energy of the stars in the cluster . in the absence of stellar evolution and tidal interactions with the galaxy , the evaporation time can be estimated by assuming that a fraction of the stars in the cluster are evaporated every relaxation time . for a globular cluster , tevap10 yr , which is comparable to the observed age of globular clusters . when discussing stellar interactions during a given epoch of globular cluster evolution , it is possible to describe the background structure of the globular cluster in terms of a static model . an overview of the evolution of globular clusters can be found in hut et al . , we summarize here the aspects of globular cluster evolution that are relevant to the formation and concentration of relativistic binaries . after a few more relaxation times , the average mass of a star in the globular cluster will be around 0.5m and these degenerate objects will once again be the more massive objects in the cluster , despite having lost most of their mass during their evolution . the static description of the structure of globular clusters using king - michie or plummer models provides a framework for describing the environment of relativistic binaries and their pro - genitors in globular clusters . over longer time scales ( comparable to trelax ) , the dynamical evolution of the distribution function as well as population changes due to stellar evolution can alter the overall structure of the globular cluster . before moving on to the dynamical models and population syntheses of relativistic binaries , we will first look at the observational evidence for these objects in globular clusters . one tracer of the processes that may lead to relativistic binaries is the population of blue stragglers . these objects are some of the most visible and populous evidence of dynamical interactions in globular clusters that lead to the formation of ultracompact binary systems . searches for cataclysmic variables generally focus on low - luminosity x - ray sources [ 87 , 64 , 159 ] and on ultraviolet - excess stars [ 62 , 93 , 103 ] , but these systems are usually a white dwarf accreting from a low mass star . the progenitors of the ultracompact millisecond pulsars are thought to pass through a low - mass x - ray binary ( lmxb ) phase [ 37 , 64 , 131 , 134 ] . these low luminosity x - ray binaries are characterized by a luminosity lx<10 erg / s , which distinguishes them from the low - mass x - ray binaries with lx > 10 erg / s . the x - ray luminosities of low - mass x - ray binaries are in the range lx1010 erg / s . all of the lmxbs in globular clusters contain an accreting neutron star as they also exhibit x - ray bursts , indicating thermonuclear flashes on the surface of the neutron star . table 1 low - mass x - ray binaries in globular clusters . (10erg / s)(hr)x0512 - 401ngc 18511.9<0.85[37 , 149]x1724 - 307terzan 24.3 [37 , 149]x1745 - 203ngc 64400.9 [37 , 149]x1745 - 248terzan 5 x1746 - 370ngc 64417.65.70[37 , 123 , 149]x1747 - 313terzan 63.412.36[37 , 123 , 149]x1820 - 303ngc 662440.60.19[37 , 123 , 149]x1832 - 330ngc 66522.20.73[37 , 123]x1850 - 087ngc 67120.80.33[37 , 123 , 149]x2127 + 119ngc 70783.517.10[37 , 123 , 149 ] low - mass x - ray binaries in globular clusters . the population of known millisecond pulsars ( msps ) seems to be one of the fastest growing populations of relativistic binaries in globular clusters . (ms)(days ) m j0023 - 7203j2.10147 tuc0.12 < 0.00020.02[20 , 48]j0024 - 7204o2.64347 tuc0.14 < 0.0040.02[20 , 48]j0024 - 72r3.4847 tuc0.066 > 0.00.03j0024 - 7203u4.34347 tuc0.430.0002 [20 , 48]j0024 - 72w2.35247 tuc0.11 > 0.00.15b1718 - 191004.037ngc 63420.26 > 0.00.1b1744 - 24a11.563terzan 50.08 > 0.00.1[101 , 114]j1807 - 243.059ngc 65440.071 > 0.00.009[29 , 130]j1910 - 593.266ngc 67520.865 > 0.00.19j1910 + 00043.619ngc 67600.14 > 0.00.02b2127 + 11c30.529 m 150.340.680.9 short orbital period binary millisecond pulsars in globular with the ongoing parkes multi - beam surveys of globular clusters and the use of the acceleration technique [ 98 , 108 ] , the population of known msps will be expected to grow dramatically in the next few years . all of the globular cluster lmxbs exhibit the x - ray variability that is indicative of nuclear burning on the surface of a neutron star . before addressing specific evolutionary scenarios , we will present the generic features of binary evolution that lead to the formation of relativistic binaries . this mass transfer affects both the evolution of the components of the binary as well as the binary properties such as orbital period and eccentricity . at the onset of rlof , the binary will become either a low - mass x - ray binary ( if the donor star is a wd or ms with m2m ) , or a high - mass x - ray binary ( if the donor is a more massive main - sequence star ) . when the above evolutionary scenarios are played out in the environment of a globular cluster , additional mechanisms arise that enhance the production of relativistic binaries . we will discuss here the broad features of these interactions and how they affect the evolution of binary systems toward relativistic binaries . although n - body interactions are less likely to occur than tidally significant two - body interactions , they are now thought to be the dominant channel for the formation of binaries during the evolution of a globular cluster . a common feature of numerical studies of hard binary interactions is the preferential exchange of high - mass stars and stellar remnants with the least massive member of the binary . because stellar remnants can also be exchanged into hard binaries , globular cluster evolution opens up a new channel for the formation of relativistic binaries by introducing evolved components into binary systems that have not yet undergone a mass transfer phase . we have seen how the dynamics of globular clusters can enhance the population of progenitors to relativistic binaries , making the standard channels of mass - transfer more likely to occur . in addition , globular cluster dynamics can open up new channels for the formation of relativistic binaries by inserting evolved , stellar remnants such as neutron stars or white dwarfs into binary systems and by shrinking the orbits of binary systems to enhance the likelihood of mass exchange . we next discuss the probable rates for the formation of such systems and the dynamical simulations that are used to synthesize globular cluster populations of relativistic binaries . simulations of the populations of relativistic binaries in globular clusters rely on the interplay between the evolution of individual stars in the progenitor systems and the evolution of globular clusters . the evolution of stars in the progenitor systems has been discussed in the previous section and we now turn to techniques for simulating the evolution of globular clusters . the overall structure of the cluster as well as the dynamics of most of the stars in the cluster are determined by simple n - body gravitational dynamics . to close the loop , the dynamical evolution of the globular cluster affects the distribution and population of the binary systems in the cluster . in our case , we are interested in the end products of binary evolution , which are tied both to stellar evolution and to the dynamical evolution of the globular cluster . to synthesize the population of relativistic binaries , we need to look at the dynamical evolution of the globular cluster as well as the evolution of the binaries in the cluster . the dynamics of the globular cluster are then governed by the fokker - planck equation : ( 28)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{\partial f}}{{\partial t } } + { \rm{v } } \cdot \nabla f - \nabla \phi \cdot \frac{{\partial f}}{{\partial { \rm{v } } } } = \gamma \left [ f \right ] . $ $ \end{document } in the fokker - planck approach , the mass spectrum of stars is binned , with a separate fm for each bin . the orbit - averaged solutions of the fokker - planck equation can not easily handle the effect of binaries and the binary interactions that occur during the evolution of a globular cluster . however , like fokker - planck , the monte carlo method currently requires the cluster to be spherically symmetric , so gravitational effects of the galaxy must be treated in an ad hoc way . although n - body simulations have the potential to provide the most detailed population syntheses of relativistic binaries in globular clusters , there are very few actual populations described in the literature . in their comparison of n - body and fokker - planck simulations of the evolution of globular clusters , takahashi and portegies zwart followed the evolution of n=1 k , 16 k , and 32 k systems with initial mass functions given by equation ( 9 ) and initial density profiles set up from king models . although they allowed for realistic stellar binary evolution in their comparisons , their focus was on the structural evolution of globular clusters . it is possible to generate a population distribution for black hole binaries in globular clusters using the n - body simulations of portegies zwart and mcmillan that were intended to describe the population of black hole binaries that were ejected from globular clusters . [ 88 , 89 ] ) to study the formation and evolution of ns wd binaries , which may be progenitors of the large population of millisecond pulsars being discovered in globular clusters ( see section 3.3 ) . if we consider that there are two binaries with orbital period less than 2000 s out of 10m in 47 tuc , and assume that this rate is consistent throughout the globular cluster system as a whole , we find a total of 60 such binaries . their recent simulation of the evolution of a cluster of 300,000 equal point - mass stars and 30,000 binaries yields a wealth of detail about the position and energy distribution of binaries in the cluster . their results are focused toward predicting the observable end products of binary evolution such as millisecond pulsars , cataclysmic variables , and blue stragglers . davies and collaborators also use the technique of calculating encounter rates ( based on calculations of cross - sections for various binary interactions and number densities of stars using king - michie static models ) to determine the production of end products of binary evolution [ 33 , 31 ] . if the orbits have been circularized ( which is quite likely if the binaries have been formed through a common envelope ) , the evolution of the period due to gravitational radiation losses is given by ( 32)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \frac{{dp}}{{dt } } = - { k_0}{p^ { - 5/3 } } , $ $ \end{document } where k0 is given by ( 33)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { k_0 } = \frac{{96}}{5}{\left ( { 2\pi } \right)^{8/3}}\frac{{{g^{5/3}}}}{{{c^5}}}{{\mathcal m}^{5/3 } } , $ $ \end{document } with the chirp mass mm1m2(m1+m2 ) . assuming a binary fraction of 30% , we can determine the number of relativistic binaries with short orbital period ( porb < pmax ) for a typical cluster with 10m and the galactic globular cluster system with 10m by simply integrating the period distribution from contact pc up to pmax ( 34)\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ n = \int_{{p_{\rm{c}}}}^{{p_{\max } } } { \frac{\eta } { { { k_0}}}{p^{5/3}}dp . } the expected populations for an individual cluster and the galactic cluster system are shown in table 4 using neutron star masses of 1.4m , white dwarf masses of 0.6m and 0.3m , and pmax=2000 s. table 4encounter rate estimates of the population of relativistic binaries in a typical globular cluster and the galactic globular cluster system.objectwda2nwnscluster5.64.00.5system176.5125.216 encounter rate estimates of the population of relativistic binaries in a typical globular cluster and the galactic globular cluster system . for example , the low - mass x - ray binary systems are bright enough that we see essentially all of those that are in the galactic globular cluster system . although the expected angular resolution of lisa is not small enough to allow for the identification of individual sources , knowledge of the positions of the clusters will allow for focused searches of the relativistic binary populations of the majority of the galactic globular clusters . the populations of these objects are the result of an interplay between the gravitational dynamics of large n - body systems , the dynamics of mass transfer , the details of stellar evolution , and the effect of the gravitational field of the galaxy . the gravitational dynamics of globular clusters can enhance the population of short period binaries of main - sequence stars as well as inject compact objects such as white dwarfs and neutron stars into stellar binary systems . furthermore , most models of the evolution of the core of a globular cluster rely on the gradual hardening and ejection of binary systems to delay the onset of core collapse . consequently , if other dynamical processes ( such as gravothermal oscillations ) do not dominate , the globular cluster s population of relativistic binaries may also reflect the past orbital history of the globular cluster . at this point , the bottleneck in observations of binary millisecond pulsars , low - mass x - ray binaries , and cataclysmic variables is time , not technology .

however , mgp has other properties that may relate to its biological role . it is synthesized in a wide variety of tissues during embryonic life [ 15 ] . mgp is synthesized in embryonic kidney tubules and mgp protein and vitronectin colocalize at focal sites . mgp is highly expressed in cancers of the ovary , testes , kidney , prostate , and glioblastomas but its function in neoplastic cells is unknown [ 1215 ] . mgp is a migration - promoting protein for glioblastoma , suggesting that it can promote the cancer spreading . vitronectin binding suggested a mechanism to anchor mgp in the extracellular matrix where it may bind to bmps and/or calcium to prevent calcification . mgp has at least 2 functional domains , a vitronectin extracellular matrix binding domain in the c - terminal and a calcification inhibitory domain in the n - terminal half of the protein . mutations in human mgp that prevent synthesis of functional mgp suffer from keutel syndrome , with excessive calcification of cartilage and pulmonary artery stenosis [ 5 , 18 , 19 ] . excessive calcification occurs if mgp is absent , but the mechanism of calcification inhibition is unknown . a postulated mechanism of calcification inhibition is regulation of bone morphogenetic protein-2 ( bmp-2 ) , a cytokine / morphogen in the transforming growth factor beta superfamily [ 10 , 2023 ] . a role in transport of calcium - phosphate complexes has also been suggested , because mgp binds calcium phosphate crystals and forms a serum complex with fetuin - a and calcium phosphate [ 24 , 25 ] . calcification inhibition by mgp is connected to the gla residues contained in the n - terminal half of mgp which are involved in binding of bmp-2 , hydroxyapatite , and fetuin - a calcium phosphate complexes [ 22 , 2426 ] . the gla domain includes the vitamin k - dependent , gamma - carboxyglutamic acids ( gla ) , and all 3 phosphoserines [ 2 , 27 , 28 ] . the functional role of gla and the gla domain is supported by calcification defects that occur after treatment with warfarin , an inhibitor of gla formation [ 29 , 30 ] . the present studies show mgp binds fibronectin and augments cell adhesion and spreading on fibronectin . mgp localizes with fibronectin in many embryonic tissues , possibly augmenting cell matrix interaction in development . one aspect of mgp function is mediated by binding fibronectin and increasing cell interactions with fibronectin . matrix gla protein was purified as described from bovine bone , with the final step being reversed phase purification on a c18 column . after purification on hplc , mgp is a relatively insoluble protein with an approximate water solubility of 10 g / ml ; purified mgp was kept at 20c in a solution of approximately 40% acetonitrile and 0.06% trifluoroacetic acid in water . a typical purification peak contained 0.2 to 0.4 mg / ml . the final concentration of mgp was achieved by dilution into aqueous buffer at 10 g / ml or lower . concentration of mgp was estimated by absorbance at 280 nm , using an extinction coefficient of 1.0 mg / ml = 1.0 absorbance units . rabbit antibodies to bovine mgp and the purified igg fraction were produced as described using pierce immunopure columns [ 6 , 31 ] . the mgp peptide corresponding to amino acids 6177 in the secreted human protein nh2-eryamvygynaaynryf - cooh was synthesized by the molecular resource center of the university of tennessee , hsc . bovine serum albumin , human plasma fibronectin , and its proteolytic fragments , including the iii1-c polypeptide ( anastellin ) , guinea pig liver tissue transglutaminase ( ttg ) , human fibrinogen , gelatin , pooled normal rabbit serum , were purchased from sigma . the reducible crosslinking chemical dithiobis ( succinimidyl proprionate ) ( dsp ) and the immunopure igg purification kit were purchased from pierce . protein binding was assessed by the ability of filter immobilized target proteins to bind to iodinated mgp in an overlay buffer . the methods for assessing mgp binding by nitrocellulose transferred proteins from sds polyacrylamide gels ( sds - page ) and electroblotting were described previously [ 6 , 3133 ] . briefly , proteins were run in an sds - page and then transferred to shleicher and schuell bas - nc reinforced nitrocellulose membranes by electroblotting on a hoefer electroblot apparatus . in some experiments proteins were transferred to membranes by dot - blotting on a bio - rad bio - dot apparatus as described in . the membranes are incubated in 1% bovine serum albumin ( bsa ) , 10 mm phosphate , 140 mm nacl , and ph 7.4 ( bsa - pbs ) overnight at 4c . the filter overlay is performed in bsa - pbs with 0.1% tween 20 buffers containing radiolabeled mgp for 4 hours at ambient temperature and then washed in incubation buffer . the membrane is exposed to kodak biomax ms film in a kodak biomax transcreen - he intensifying screen and developed on an automatic film developer . fibronectin ( fn ) , fibrinogen ( fg ) , and vitronectin ( vn ) were mixed with tissue transglutaminase ( ttg ) and iodinated mgp , respectively , in buffer containing 0.05 m tris , 2.5 mm calcium , and 1 mm dithiothreitol ( tcd buffer ) at 37 degrees . final concentrations of 0.18 mg / ml fn , 0.18 mg / ml fg , or 0.18 mg / ml vn were incubated with 0.0125 mg / ml ttg and trace amounts of i - mgp ( 2.5 ng / ml ) containing 30,000 dpm . to determine the effect of antibodies to mgp , 31 g / ml rabbit anti - mgp igg was included during incubation ( see figure 2(c ) ) . after incubation , samples were processed immediately for sds page in sample buffer containing 2% sds and 2% 2-mercaptoethanol treated at 100 degrees for 2 min . the electrophoretic gels were processed by wrapping in plastic film and exposure to kodak biomax ms film with he enhancing screen for the appropriate time at 80c . after exposure 0.5 mg / ml fibronectin iii1-c polypeptide , 0.5 mg / ml fn , 0.5 mg / ml vn , or 0.5 mg / ml ovalbumin were incubated with trace amounts of -i - mgp ( 2.5 ng / ml , 30,000 dpm ) at ambient temperature for 2 hours in 5 mm phosphate , 75 mm nacl , 2.5 mm edta , and 0.05 mg / ml gelatin ph 7.4 . after 2 hours , either 1 mg / ml dsp or an equal volume of dmso for controls the reaction was quenched for 15 min at ambient temperature with tris ph 8 to react with free dsp the frozen at 20 degrees for sds gel electrophoresis or gel filtration chromatography . for sds - page , samples are diluted with 2% sds sample buffer ph 6.8 with or without 2% 2-mercaptoethanol and boiled for 2 min . for gel filtration chromatography , samples are diluted with 4 m guanidine hcl , 0.1 m tris , 0.1% tween 20 , and 0.5 mg / ml gelatin ph 8.0 . some samples were reduced with 2% 2-mercaptoethanol for 10 min at ambient temperature to break dsp crosslinks before dilution with buffer . gel filtration chromatography was performed on an ap ( pharmacia - lkb ) sephacryl hr s200 packed in a bio - rad column ( 1.6 19 cm ) equilibrated with 4 m guanidine , 0.1 m tris , and 0.1% tween 20 , ph 8.0 . constant volume fractions ( 30 drops / fraction ) were collected and counted on a packard auto - gamma counter to determine the elution position of radioactive mgp . controls were mgp and ovalbumin incubated with dsp , mgp alone , proteins incubated without dsp crosslinking , and dsp treated samples reduced with mercaptoethanol to reduce and break crosslinks . to determine whether mgp affected cell attachment to fibronectin , hela cells were used as an immortal cancer derived cell line that is popular as a model of cell behaviors such as attachment , spreading , and integrin action [ 3437 ] . initial studies demonstrated they had a dose - dependent cell attachment response to increasing concentrations of the matrix proteins . short term attachment studies within 1 hour after cell release from feeder plates were performed . hela cells are grown in liebovitz l-15 medium supplemented with 10% fetal bovine serum ( fbs ) with gentamicin and amphotericin b added at the recommended concentrations . 8095% confluent cells are harvested by release with 5 mm edta in calcium - free pbs ph 7.4 , washed with 10% fbs l-15 and resuspended to 250,000 cells / ml in serum - free l-15 . the cell suspension is used immediately for cell attachment assays below . in a typical cell attachment assay , 96-well plates are coated with the indicated concentrations of fibronectin in 50 mm sodium bicarbonate buffer ph 9.6 ( coat buffer ) . for control with no fibronectin the well is treated with coat buffer alone . after incubating overnight at 4c covered with parafilm to prevent evaporation , fibronectin solutions are removed and replaced with the indicated amount of mgp in coat buffer or coat buffer alone as control . after incubating at 37c in a humidified atmosphere for 3 hours , the second mgp or control solutions are removed and replaced with blocking solution made with 1% bsa in pbs ph 7.4 which has been heat inactivated at 65c for 30 min . after 1.5 hours at 37c in a humidified atmosphere , the wells are emptied and 25,000 hela cells added in 0.1 ml of serum - free l-15 medium using an 8-well multiwell pipet . vitronectin mediated cell attachment experiments were performed with the same procedure , except that vitronectin replaces fibronectin in the plate coating . the wells are immediately washed twice with serum - free l-15 medium and then fixed with 96% ethanol for 10 min and then stained with 0.1% crystal violet dye in distilled water for 30 min at ambient temperature . the dye is removed and the wells washed gently with 0.375 ml of distilled water 3 times . after wells have dried completely , 0.1 ml / well of 0.2% triton x100 is added and incubated at ambient temperature overnight covered with parafilm to prevent evaporation . the plates are read at 595 nm on a molecular devices spectramax 2500 multiwell plate reader . data is entered as replicates ( usually 6 but a minimum of 4 replicates for each data point ) into prism 2.0 for analysis , nonlinear regression plotting , and statistical analysis . in some experiments the first fibronectin coat is replaced by a first mgp coat and then followed by a second coat of fibronectin to ascertain the effect of switching the order of addition of fibronectin and mgp . in some experiments after an initial coat of fibronectin the remaining nonspecific protein binding sites on the plate are blocked with bsa - pbs before addition of mgp to see if mgp effects required nonspecific protein binding sites on wells . in other experiments the effect of rabbit anti - mgp antibody on mgp enhancement of fibronectin - cell binding was assessed by adding 31 g / ml ( 0.0312 a280/ml ) of anti - mgp igg in the second coat step containing mgp . hela cells are grown and harvested nonenzymatically as described above and , after washing once with l-15 supplemented with 10% fbs , cells are resuspended in serum - free l-15 medium to 15000 cells / ml . 9000 hela cells/0.6 ml are added to each chamber of lab - tek 4 chamber glass slides . lab - tek chamber slides are previously coated with 0.0 , 0.2 , or 0.4 g / ml of fibronectin in coat buffer overnight at 4c and then a second coat of 3 g / ml mgp for 3 hours at 37c and then blocked with heat inactivated bsa - pbs for 1.5 h at 37c as described above . cells attach for 2 hours and then are rinsed gently twice with serum - free l-15 medium . freshly made 1% paraformaldehyde in pbs is added to fix cells for 10 min at ambient temperature , washed pbs for 10 min , and then treated with 0.1% triton x-100 in pbs for 5 min to permeabilize cells . the bsa - pbs is then replaced with 1 : 40 dilution of phalloidin alexafluor488 in heat inactivated bsa - pbs for 20 min . after one wash with heat inactivated bsa - pbs the chambers are washed twice with pbs . the slides are placed in a 10 cm petri dish immersed in pbs and then imaged using 10x or 40x water immersion objectives on a zeiss axiophot fluorescence microscope equipped with a digital camera . 1416 random microscopic field images taken on the 10x objective were analyzed , with a minimum of 100 cells analyzed for each condition . the procedures in nih image are as follows : an introduction and tutorial were used to determine cell areas . briefly , low power images of cells taken with the 10x objective were modified by autocontrast and grayscale conversion in photoshop . the images were opened in nih image , scaled to fit the window , and modified with the lutz tool to turn cells red then under analyze , analyze particle . after examination to manually remove clumped / adherent cells from analysis , the results are copied into excel as area / cell . the cell spreading analysis is similar to that used in a previous publication measuring cell attachment and spreading on titanium surfaces . paraffin embedded sections of formalin fixed day 19 embryos were obtained as described previously [ 4 , 6 ] . the sections were deparaffinized with xylene , rehydrated , and boiled for 2 min in 0.01 m sodium citrate , ph 6 for antigen retrieval as described previously . sections were blocked with buffer containing normal horse serum at 1.5% then incubated with rabbit anti - mgp at 20 g / ml or mouse antifibronectin monoclonal antibody ( clone ist-3 sigma ) at 1 : 5000 dilution . after an overnight 4c incubation and washing , biotinylated horse antiuniversal igg ( vector ) is added . images were collected on a zeiss axiophot microscope equipped with a pc and 6.3x and 40x objectives . as seen in figure 1(a ) , overlay of dot - blotted proteins with radioactive mgp demonstrates that mgp binds to fibronectin and fibronectin fragment iii1-c ( anastellin ) . mgp did not bind ovalbumin or other fragments of fibronectin with the exception of the 110 kilodalton fibronectin fragment that weakly binds to mgp . mgp binds to the sds - page band of fibronectin iii1-c ( figure 1(b ) ) . we previously reported that mgp binds to vitronectin and fibronectin but not laminin , type ii collagen , osteocalcin , tissue transglutaminase , chondroitin sulfate glycosaminoglycan , biglycan , beta - casein , ovalbumin , or bovine serum albumin . figure 1(c ) shows that mgp61 - 77 also binds to the first type iii fibronectin domain . ttg was incubated in mixtures of i - mgp with fibronectin , fibrinogen , or vitronectin , respectively . the products were analyzed by autoradiographs of samples run on sds - page to identify shifts in radioactive mgp ( figure 2(a ) ) . radioiodinated mgp was incorporated into higher fibronectin ( fn ) and fibrinogen ( fg ) bands . there was no mgp incorporated into higher molecular weight bands for mgp incubated with fn or fg the absence of ttg . transglutaminase shifted a small amount of mgp to higher molecular weight but not to the size or extent found with either fibrinogen or fibronectin . the band at the bottom of the figure 2(a ) is free radioiodinated mgp . figure 2(b ) shows the coomassie blue stained gel exposed to film in figure 2(a ) . ttg converted only a small fraction of the fibronectin ( fn ) to multimers as there is little change in lanes with and without ttg . ttg was more efficient at crosslinking fibrinogen ( fg ) to multimers which can be as additional bands when ttg is present but not in samples without ttg ( figure 2(b ) ) . note that the coomassie blue stained band in all lanes is bsa , a component of the buffer used to separate radioiodinated mgp from unincorporated iodine during iodination . mgp in the ttg crosslinked fibronectin lane comigrated at the stacking gel surface or the top of the running gel . the presence of anti - mgp polyclonal antibodies blocked the ability of tissue transglutaminase to incorporate mgp into a fibronectin multimer ( figure 2(c ) ) . increasing the concentration of ttg increased the amount of mgp shifted to higher molecular weight with fibronectin . transglutaminase could incorporate mgp into larger complexes of fibronectin but not with vitronectin , ovalbumin , or bovine serum albumin . chemical crosslinking could confirm that the mgp and iii1-c are in close proximity in solution . i - mgp and iii1-c , vitronectin , or ovalbumin was incubated under associative conditions then reacted with dsp , a disulfide containing homobifunctional crosslinking agent . the prediction is that interacting proteins would become covalently crosslinked in the presence of a crosslinking agent . if dsp is added to a mixture of fibronectin iii1-c and i - mgp , a higher molecular weight crosslinked species is observed on autoradiographs after sds - page ( figure 3(a ) , iiic , 2me , and + dsp lane ) . if samples are reduced with 2-mercaptoethanol ( 2me ) , the dsp crosslink is reduced and larger bands disappear ( figure 3(a ) iiic , + 2me , and + dsp lane ) . no additional higher molecular weight bands are present for iiic in the absence of dsp crosslinker ( iiic , 2me , and dsp lane ) . the lack of higher molecular weight species is due to disruption of protein interactions in 2% sds buffer heated to 100c . mgp and either vitronectin or fibronectin was crosslinked by dsp although not to the same extent as seen with iii1-c ( figure 3(a ) , vn and fn , 2me , and + dsp lanes ) . it is possible that the increased crosslinking of iii1-c and mgp results from aggregation of the bound complex that increases crosslinking efficiency . ovalbumin is a negative control for binding ; mgp and ovalbumin were not crosslinked by dsp . if the products of dsp crosslinked i - mgp and fibronectin iii1-c are denatured in 4 m guanidine containing buffer then analyzed by sephacryl s200 gel filtration , an elution profile with mgp shifted to a peak near the excluded volume , ve , is the result ( figure 3(b ) , filled circles ) . if the crosslinked sample is reduced with mercaptoethanol prior to column loading , the elution profile is similar to controls , for example , i - mgp alone ( figure 3(b ) , open circles ) . the i mgp peak normally elutes around fraction 37 with a minor peak at fraction 28 ( figure 3(b ) , open triangles ) . control reactions with i - mgp with dsp , i - mgp + iii1-c in the absence of dsp were identical in gel filtration elution profile with mgp alone or the reduced crosslink ( open diamonds or squares , resp . ) . a four - fold decreased amount of iii1-c in the mixture with labeled mgp and dsp resulted in a smaller s200 column ve peak ( data not shown ) . the data show that iii1-c and the crosslinking agent were necessary to produce the shift in the mgp peak and are consistent with a close interaction between fibronectin iii1-c and mgp . figure 4(a ) shows that cell attachment increases with increasing fibronectin until a maximum is reached at fibronectin coating concentrations between 0.8 and 3.2 g / ml ( solid line with closed circles ) . mgp enhanced cell attachment to fibronectin ( open circles with the dashed line , figure 4(a ) ) . the cell attachment curve for equal amounts of fibronectin is shifted to the left if mgp is added at 3 g / ml . the result is analogous to increasing the apparent affinity of cells for equivalent amounts of fibronectin ( figure 4(a ) ) . in addition , mgp did not increase cell attachment beyond the maximum observed for fibronectin alone . the lack of inherent cell attachment activity for mgp is in agreement with the absence of cell binding activity previously reported . in contrast to mgp augmenting cell attachment to fibronectin , mgp did not augment attachment of cells to vitronectin . there was a small inhibitory effect of mgp on cell binding to vitronectin , but in some experiments there was no significant effect . under the same conditions and cell line , mgp consistently augments cell attachment on fibronectin but not on vitronectin . antibody to mgp inhibited the ability of mgp to augment cell attachment ( figure 4(b ) ) . anti - mgp rabbit igg abolished the ability of mgp to enhance cell attachment to fibronectin ( mgp compared to mgp + antimgp , p .05 ) , but an equal amount of nonspecific rabbit igg did not . the presence of igg at the concentrations used did not affect cell attachment to fibronectin , because anti - mgp igg or control igg added in the absence of mgp was identical to the control fibronectin alone ( control , figure 4(b ) ) . increasing amounts of mgp dose dependently enhanced cell attachment to fibronectin ( figure 4(c ) ) . a dose response curve of increasing mgp ( 0.0 , 0.8 , 3.0 , and 6.0 g / ml ) reveals a change in apparent affinity with an increasing shift of the binding curve to the left indicating increased cell attachment at each concentration of fibronectin . again , maximum binding of cells to fibronectin was not affected ( figure 4(c ) ) . there was no inherent mgp cell attachment activity , because cells did not attach even at the highest amounts of mgp ( 6.0 g / ml ) if fibronectin was not present . cells attach and spread on fibronectin as integrins and other cell adhesive proteins form focal adhesions which connect to the actin cytoskeleton . to determine whether mgp affects cell spreading on fibronectin , the average area of cells attached to fibronectin alone or fibronectin plus mgp was determined by calculation of average cell area from over 100 cells as described in experimental procedures . mgp induced significantly greater cell spreading at each fibronectin concentration ( figure 5(a ) , fn versus fn + mgp , p .0001 indicated by asterisk ) . there were too few cells attached in the mgp alone wells to measure spreading ( mgp does not have cell adhesive activity on its own ) . the appearance of selected microscopic fields of cells adherent on 0.4 g / ml fibronectin coated surfaces is shown is in figures 5(b ) and 5(c ) . the appearance of selected microscopic fields of cells on the same concentration of fibronectin plus mgp is shown in figures 5(d ) and 5(e ) . it was previously shown that mgp in the rat embryonic kidney localizes in the developing ureter and the collecting tubules . fibronectin is widely distributed in the rat embryonic kidney , including in the ureter and collecting tubules ( figures 6(a ) , 6(b ) , and 6(c ) ) . localization of mgp in a serial section reveals that fibronectin is present in some of the same collecting tubules and ureter epithelium as mgp ( figures 6(d ) , 6(e ) , and 6(f ) ) . arrows in figures 6(b ) , 6(c ) , 6(e ) , and 6(f ) indicate similar structures in serial sections with both fibronectin and mgp . fibronectin has a wider distribution within the developing kidney , being present in other tubules with little or no mgp , in connective tissue areas , and in forming glomeruli ( figures 6(a)6(c ) ) . developing glomeruli has little or no mgp at this stage of development ( figure 6(d ) ) . this confirms the results from a previous publication that showed that mgp binds to fibronectin , vitronectin , and fibrinogen but not tissue transglutaminase , type ii collagen , fibromodulin , osteocalcin , osteonectin , heparin , decorin , casein , or ovalbumin . the current study shows that mgp binds to a region of fibronectin made up by its first type iii repeat called iii1-c or anastellin . mgp did not bind the 30 , 45 kilodalton fragments of fibronectin that do not contain a type iii repeat [ 40 , 41 ] . mgp showed some affinity for a 110 kilodalton fragment of fibronectin that is comprised of many type iii repeats [ 40 , 41 ] . a region of mgp comprised of amino acids 6177 was proposed as an extracellular matrix binding region . the current study shows that mgp6177 peptide binds to fibronectin and also to the fibronectin iii1-c fragment . for example , human mgp is originally 84 amino acids long but has 7 amino acids removed from the c - terminus , so that the mature protein is 77 amino acids long and ends with the peptide sequence identical to the mgp6177 peptide used for this study . while bovine mgp was used in this study , the results imply that the human mgp will bind to extracellular matrix proteins via a sequence at its carboxyl terminal . the proteolytic processing of the mgp c - terminal to remove amino acids 7884 may relate to activating a protein interaction site . the crosslinked multimerized fibronectin formed by incubation with ttg is analogous to the insoluble matrix fibronectin present in many tissues . the close association of mgp and fibronectin suggested that mgp may become incorporated into fibronectin crosslinked multimers . indeed , addition of mgp during crosslinking of fibronectin by ttg resulted in its incorporation into a larger multimer form of fibronectin . the i mgp remained with the fibronectin multimer after boiling in 2% sds that dissociates the mgp and fibronectin interaction . it is possible that transglutaminase crosslinks mgp to fibronectin , although further studies are necessary to prove crosslinking occurs and determine which amino acids of mgp and fibronectin are involved . the shift of smaller amounts of mgp to higher molecular weight by ttg suggested it may become crosslinked to itself or to ttg , but we were unable to directly establish that mgp is a substrate of ttg . to prove mgp was a substrate for ttg we attempted incorporation of radiolabeled putrescine as described . the association of mgp with crosslinked fibronectin confirms a close association of mgp with fibronectin . although mgp binds to vitronectin , ttg did not incorporate mgp into a high molecular weight component with vitronectin . the results suggest that mgp may become a component of crosslinked fibronectin and fibrinogen in vivo . this has been established by overlay assays and by dsp crosslinking of mgp to the fibronectin iii1-c polypeptide . overlay assays show that the c - terminal mgp 6177 amino acid peptide can bind to fibronectin and to the iii1-c polypeptide . a noncarboxylated form of mgp ( ucmgp or glu - mgp ) accumulates in calcified atherosclerotic plaque [ 22 , 30 ] . further studies will be necessary to determine whether glu - mgp and mgp share binding for fibronectin and the iii1-c domain . this is not simply an additive effect of providing more adhesive protein , because mgp by itself has no adhesive activity . the lack of inherent cell adhesion activity suggests that mgp acts by altering the ability of cells to bind fibronectin . mgp binding of fibronectin seems to be the key , because the enhanced cell attachment was observed in bsa - coated surfaces in which mgp - fibronectin binding was the only way that the enhancement could occur . mgp enhanced cell attachment to fibronectin but not to vitronectin , another extracellular matrix protein bound by mgp . these studies explored the mechanism of mgp binding to fibronectin and the effect of exogenous fibronectin and mgp on cell attachment and spreading . hela cells are a well characterized cell line that is a popular model for cell attachment . hela cells express the integrins for fibronectin and vitronectin binding [ 3437 ] . the goal was to characterize the effect of mgp for fibronectin binding and cell - fibronectin interactions in a short , 1 hour period when cellular synthesis and secretion of fibronectin , mgp , and other attachment proteins could be minimized . there has been a positive correlation of mgp expression with tumor progression and poor prognosis in glioma [ 15 , 16 , 45 ] , but a negative correlation of mgp expression with tumor progression and metastasis in renal and prostate carcinoma and decreased mgp has been found in colon carcinoma . future studies should determine whether mgp has the effect in multiple normal and neoplastic cell types . other studies could determine the effect of mgp for migration and invasion assays in hela compared to other cancer cell types , but longer term studies need to consider the ability of cells to synthesize fibronectin , mgp , integrins , or metalloproteinases that affect migration and invasion . the expression of mgp mrna is very high in the embryonic rat kidney [ 1 , 4 ] . we demonstrated that mgp protein is also synthesized during this time and that localization of mgp mrna was strongest in the developing ureter and collecting tubules on embryonic days 1719 . immunolocalization studies showed mgp localized in embryonic ureters and collecting tubules also contained abundant fibronectin . calcification inhibition and bmp-2 binding requires posttranslational carboxylation to produce gla in a vitamin k - dependent step [ 22 , 26 , 29 ] . the phosphorylation of 3 serine residues at the amino terminal end of mgp is necessary for its secretion to inhibit deposition of hydroxyapatite in the extracellular matrix [ 2 , 47 ] . the amino terminal 154 amino acids of mgp contains the phosphoserines and gla necessary for calcification inhibition , whereas a c - terminal peptide mgp6177 has been shown previously to bind vitronectin and in the current studies to bind fibronectin [ 6 , 26 ] . it is not clear whether the binding of fibronectin or modulating the cell response to fibronectin is related to the calcification inhibitory activity of mgp . it is more likely that this mgp property is related to migration - promoting activity that is demonstrated for glioblastoma cells . it is possible that mgp is a multifunctional protein that can act to modify cell - matrix interactions during embryonic life or in cancer cells and also as a calcification inhibitor in selected adult tissues . this could help explain why many tissues that contain mgp synthesizing cells do not become calcified in mgp knockout animals and in keutel syndrome , a human disease characterized by loss of mgp function . mgp binds via a 6177 amino acid sequence present at the physiologic c - terminus . mgp can become part of transglutaminase crosslinked multimers of fibronectin , suggesting it may be a component of fibronectin matrices . cells attach and spread better on fibronectin and mgp coated surfaces than to fibronectin alone , even though mgp itself has no cell attachment activity . in tissues , mgp localizes near fibronectin , suggesting that interactions between the proteins are possible in vivo . the ability of mgp to alter cell interactions with fibronectin is a potential reason for cancer cells and certain embryonic cells to overexpress mgp .

background . matrix gla protein ( mgp ) is a vitamin k - dependent , extracellular matrix protein . mgp is a calcification inhibitor of arteries and cartilage . however mgp is synthesized in many tissues and is especially enriched in embryonic tissues and in cancer cells . the presence of mgp in those instances does not correlate well with the calcification inhibitory role . this study explores a potential mechanism for mgp to bind to matrix proteins and alter cell matrix interactions . methods . to determine whether mgp influences cell behavior through interaction with fibronectin , we studied mgp binding to fibronectin , the effect of mgp on fibronectin mediated cell attachment and spreading and immunolocalized mgp and fibronectin . results . first , mgp binds to fibronectin . the binding site for mgp is in a specific fibronectin fragment , called iii1-c or anastellin . the binding site for fibronectin is in a mgp c - terminal peptide comprising amino acids 6177 . second , mgp enhances cell attachment and cell spreading on fibronectin . mgp alone does not promote cell adhesion . third , mgp is present in fibronectin - rich regions of tissue sections . conclusions . mgp binds to fibronectin . the presence of mgp increased cell - fibronectin interactions .

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

it is synthesized in a wide variety of tissues during embryonic life [ 15 ] . mgp is synthesized in embryonic kidney tubules and mgp protein and vitronectin colocalize at focal sites . mgp is a migration - promoting protein for glioblastoma , suggesting that it can promote the cancer spreading . vitronectin binding suggested a mechanism to anchor mgp in the extracellular matrix where it may bind to bmps and/or calcium to prevent calcification . mgp has at least 2 functional domains , a vitronectin extracellular matrix binding domain in the c - terminal and a calcification inhibitory domain in the n - terminal half of the protein . calcification inhibition by mgp is connected to the gla residues contained in the n - terminal half of mgp which are involved in binding of bmp-2 , hydroxyapatite , and fetuin - a calcium phosphate complexes [ 22 , 2426 ] . the gla domain includes the vitamin k - dependent , gamma - carboxyglutamic acids ( gla ) , and all 3 phosphoserines [ 2 , 27 , 28 ] . the present studies show mgp binds fibronectin and augments cell adhesion and spreading on fibronectin . mgp localizes with fibronectin in many embryonic tissues , possibly augmenting cell matrix interaction in development . one aspect of mgp function is mediated by binding fibronectin and increasing cell interactions with fibronectin . matrix gla protein was purified as described from bovine bone , with the final step being reversed phase purification on a c18 column . after purification on hplc , mgp is a relatively insoluble protein with an approximate water solubility of 10 g / ml ; purified mgp was kept at 20c in a solution of approximately 40% acetonitrile and 0.06% trifluoroacetic acid in water . the mgp peptide corresponding to amino acids 6177 in the secreted human protein nh2-eryamvygynaaynryf - cooh was synthesized by the molecular resource center of the university of tennessee , hsc . protein binding was assessed by the ability of filter immobilized target proteins to bind to iodinated mgp in an overlay buffer . the methods for assessing mgp binding by nitrocellulose transferred proteins from sds polyacrylamide gels ( sds - page ) and electroblotting were described previously [ 6 , 3133 ] . the membrane is exposed to kodak biomax ms film in a kodak biomax transcreen - he intensifying screen and developed on an automatic film developer . to determine the effect of antibodies to mgp , 31 g / ml rabbit anti - mgp igg was included during incubation ( see figure 2(c ) ) . constant volume fractions ( 30 drops / fraction ) were collected and counted on a packard auto - gamma counter to determine the elution position of radioactive mgp . controls were mgp and ovalbumin incubated with dsp , mgp alone , proteins incubated without dsp crosslinking , and dsp treated samples reduced with mercaptoethanol to reduce and break crosslinks . to determine whether mgp affected cell attachment to fibronectin , hela cells were used as an immortal cancer derived cell line that is popular as a model of cell behaviors such as attachment , spreading , and integrin action [ 3437 ] . initial studies demonstrated they had a dose - dependent cell attachment response to increasing concentrations of the matrix proteins . in a typical cell attachment assay , 96-well plates are coated with the indicated concentrations of fibronectin in 50 mm sodium bicarbonate buffer ph 9.6 ( coat buffer ) . for control with no fibronectin the well is treated with coat buffer alone . after incubating overnight at 4c covered with parafilm to prevent evaporation , fibronectin solutions are removed and replaced with the indicated amount of mgp in coat buffer or coat buffer alone as control . after incubating at 37c in a humidified atmosphere for 3 hours , the second mgp or control solutions are removed and replaced with blocking solution made with 1% bsa in pbs ph 7.4 which has been heat inactivated at 65c for 30 min . after 1.5 hours at 37c in a humidified atmosphere , the wells are emptied and 25,000 hela cells added in 0.1 ml of serum - free l-15 medium using an 8-well multiwell pipet . vitronectin mediated cell attachment experiments were performed with the same procedure , except that vitronectin replaces fibronectin in the plate coating . in some experiments the first fibronectin coat is replaced by a first mgp coat and then followed by a second coat of fibronectin to ascertain the effect of switching the order of addition of fibronectin and mgp . in some experiments after an initial coat of fibronectin the remaining nonspecific protein binding sites on the plate are blocked with bsa - pbs before addition of mgp to see if mgp effects required nonspecific protein binding sites on wells . in other experiments the effect of rabbit anti - mgp antibody on mgp enhancement of fibronectin - cell binding was assessed by adding 31 g / ml ( 0.0312 a280/ml ) of anti - mgp igg in the second coat step containing mgp . briefly , low power images of cells taken with the 10x objective were modified by autocontrast and grayscale conversion in photoshop . the cell spreading analysis is similar to that used in a previous publication measuring cell attachment and spreading on titanium surfaces . after an overnight 4c incubation and washing , biotinylated horse antiuniversal igg ( vector ) is added . as seen in figure 1(a ) , overlay of dot - blotted proteins with radioactive mgp demonstrates that mgp binds to fibronectin and fibronectin fragment iii1-c ( anastellin ) . mgp did not bind ovalbumin or other fragments of fibronectin with the exception of the 110 kilodalton fibronectin fragment that weakly binds to mgp . mgp binds to the sds - page band of fibronectin iii1-c ( figure 1(b ) ) . we previously reported that mgp binds to vitronectin and fibronectin but not laminin , type ii collagen , osteocalcin , tissue transglutaminase , chondroitin sulfate glycosaminoglycan , biglycan , beta - casein , ovalbumin , or bovine serum albumin . ttg was incubated in mixtures of i - mgp with fibronectin , fibrinogen , or vitronectin , respectively . transglutaminase shifted a small amount of mgp to higher molecular weight but not to the size or extent found with either fibrinogen or fibronectin . the band at the bottom of the figure 2(a ) is free radioiodinated mgp . the presence of anti - mgp polyclonal antibodies blocked the ability of tissue transglutaminase to incorporate mgp into a fibronectin multimer ( figure 2(c ) ) . increasing the concentration of ttg increased the amount of mgp shifted to higher molecular weight with fibronectin . the prediction is that interacting proteins would become covalently crosslinked in the presence of a crosslinking agent . mgp and either vitronectin or fibronectin was crosslinked by dsp although not to the same extent as seen with iii1-c ( figure 3(a ) , vn and fn , 2me , and + dsp lanes ) . ovalbumin is a negative control for binding ; mgp and ovalbumin were not crosslinked by dsp . if the products of dsp crosslinked i - mgp and fibronectin iii1-c are denatured in 4 m guanidine containing buffer then analyzed by sephacryl s200 gel filtration , an elution profile with mgp shifted to a peak near the excluded volume , ve , is the result ( figure 3(b ) , filled circles ) . if the crosslinked sample is reduced with mercaptoethanol prior to column loading , the elution profile is similar to controls , for example , i - mgp alone ( figure 3(b ) , open circles ) . control reactions with i - mgp with dsp , i - mgp + iii1-c in the absence of dsp were identical in gel filtration elution profile with mgp alone or the reduced crosslink ( open diamonds or squares , resp . ) a four - fold decreased amount of iii1-c in the mixture with labeled mgp and dsp resulted in a smaller s200 column ve peak ( data not shown ) . figure 4(a ) shows that cell attachment increases with increasing fibronectin until a maximum is reached at fibronectin coating concentrations between 0.8 and 3.2 g / ml ( solid line with closed circles ) . mgp enhanced cell attachment to fibronectin ( open circles with the dashed line , figure 4(a ) ) . the cell attachment curve for equal amounts of fibronectin is shifted to the left if mgp is added at 3 g / ml . in addition , mgp did not increase cell attachment beyond the maximum observed for fibronectin alone . the lack of inherent cell attachment activity for mgp is in agreement with the absence of cell binding activity previously reported . in contrast to mgp augmenting cell attachment to fibronectin , mgp did not augment attachment of cells to vitronectin . there was a small inhibitory effect of mgp on cell binding to vitronectin , but in some experiments there was no significant effect . under the same conditions and cell line , mgp consistently augments cell attachment on fibronectin but not on vitronectin . antibody to mgp inhibited the ability of mgp to augment cell attachment ( figure 4(b ) ) . anti - mgp rabbit igg abolished the ability of mgp to enhance cell attachment to fibronectin ( mgp compared to mgp + antimgp , p .05 ) , but an equal amount of nonspecific rabbit igg did not . the presence of igg at the concentrations used did not affect cell attachment to fibronectin , because anti - mgp igg or control igg added in the absence of mgp was identical to the control fibronectin alone ( control , figure 4(b ) ) . increasing amounts of mgp dose dependently enhanced cell attachment to fibronectin ( figure 4(c ) ) . a dose response curve of increasing mgp ( 0.0 , 0.8 , 3.0 , and 6.0 g / ml ) reveals a change in apparent affinity with an increasing shift of the binding curve to the left indicating increased cell attachment at each concentration of fibronectin . again , maximum binding of cells to fibronectin was not affected ( figure 4(c ) ) . there was no inherent mgp cell attachment activity , because cells did not attach even at the highest amounts of mgp ( 6.0 g / ml ) if fibronectin was not present . to determine whether mgp affects cell spreading on fibronectin , the average area of cells attached to fibronectin alone or fibronectin plus mgp was determined by calculation of average cell area from over 100 cells as described in experimental procedures . there were too few cells attached in the mgp alone wells to measure spreading ( mgp does not have cell adhesive activity on its own ) . the appearance of selected microscopic fields of cells on the same concentration of fibronectin plus mgp is shown in figures 5(d ) and 5(e ) . fibronectin is widely distributed in the rat embryonic kidney , including in the ureter and collecting tubules ( figures 6(a ) , 6(b ) , and 6(c ) ) . localization of mgp in a serial section reveals that fibronectin is present in some of the same collecting tubules and ureter epithelium as mgp ( figures 6(d ) , 6(e ) , and 6(f ) ) . fibronectin has a wider distribution within the developing kidney , being present in other tubules with little or no mgp , in connective tissue areas , and in forming glomeruli ( figures 6(a)6(c ) ) . this confirms the results from a previous publication that showed that mgp binds to fibronectin , vitronectin , and fibrinogen but not tissue transglutaminase , type ii collagen , fibromodulin , osteocalcin , osteonectin , heparin , decorin , casein , or ovalbumin . the current study shows that mgp binds to a region of fibronectin made up by its first type iii repeat called iii1-c or anastellin . a region of mgp comprised of amino acids 6177 was proposed as an extracellular matrix binding region . the current study shows that mgp6177 peptide binds to fibronectin and also to the fibronectin iii1-c fragment . for example , human mgp is originally 84 amino acids long but has 7 amino acids removed from the c - terminus , so that the mature protein is 77 amino acids long and ends with the peptide sequence identical to the mgp6177 peptide used for this study . while bovine mgp was used in this study , the results imply that the human mgp will bind to extracellular matrix proteins via a sequence at its carboxyl terminal . the proteolytic processing of the mgp c - terminal to remove amino acids 7884 may relate to activating a protein interaction site . the crosslinked multimerized fibronectin formed by incubation with ttg is analogous to the insoluble matrix fibronectin present in many tissues . the close association of mgp and fibronectin suggested that mgp may become incorporated into fibronectin crosslinked multimers . indeed , addition of mgp during crosslinking of fibronectin by ttg resulted in its incorporation into a larger multimer form of fibronectin . the i mgp remained with the fibronectin multimer after boiling in 2% sds that dissociates the mgp and fibronectin interaction . it is possible that transglutaminase crosslinks mgp to fibronectin , although further studies are necessary to prove crosslinking occurs and determine which amino acids of mgp and fibronectin are involved . the shift of smaller amounts of mgp to higher molecular weight by ttg suggested it may become crosslinked to itself or to ttg , but we were unable to directly establish that mgp is a substrate of ttg . the association of mgp with crosslinked fibronectin confirms a close association of mgp with fibronectin . although mgp binds to vitronectin , ttg did not incorporate mgp into a high molecular weight component with vitronectin . this has been established by overlay assays and by dsp crosslinking of mgp to the fibronectin iii1-c polypeptide . overlay assays show that the c - terminal mgp 6177 amino acid peptide can bind to fibronectin and to the iii1-c polypeptide . a noncarboxylated form of mgp ( ucmgp or glu - mgp ) accumulates in calcified atherosclerotic plaque [ 22 , 30 ] . further studies will be necessary to determine whether glu - mgp and mgp share binding for fibronectin and the iii1-c domain . the lack of inherent cell adhesion activity suggests that mgp acts by altering the ability of cells to bind fibronectin . mgp binding of fibronectin seems to be the key , because the enhanced cell attachment was observed in bsa - coated surfaces in which mgp - fibronectin binding was the only way that the enhancement could occur . mgp enhanced cell attachment to fibronectin but not to vitronectin , another extracellular matrix protein bound by mgp . these studies explored the mechanism of mgp binding to fibronectin and the effect of exogenous fibronectin and mgp on cell attachment and spreading . hela cells are a well characterized cell line that is a popular model for cell attachment . hela cells express the integrins for fibronectin and vitronectin binding [ 3437 ] . the goal was to characterize the effect of mgp for fibronectin binding and cell - fibronectin interactions in a short , 1 hour period when cellular synthesis and secretion of fibronectin , mgp , and other attachment proteins could be minimized . future studies should determine whether mgp has the effect in multiple normal and neoplastic cell types . other studies could determine the effect of mgp for migration and invasion assays in hela compared to other cancer cell types , but longer term studies need to consider the ability of cells to synthesize fibronectin , mgp , integrins , or metalloproteinases that affect migration and invasion . immunolocalization studies showed mgp localized in embryonic ureters and collecting tubules also contained abundant fibronectin . calcification inhibition and bmp-2 binding requires posttranslational carboxylation to produce gla in a vitamin k - dependent step [ 22 , 26 , 29 ] . the phosphorylation of 3 serine residues at the amino terminal end of mgp is necessary for its secretion to inhibit deposition of hydroxyapatite in the extracellular matrix [ 2 , 47 ] . the amino terminal 154 amino acids of mgp contains the phosphoserines and gla necessary for calcification inhibition , whereas a c - terminal peptide mgp6177 has been shown previously to bind vitronectin and in the current studies to bind fibronectin [ 6 , 26 ] . it is not clear whether the binding of fibronectin or modulating the cell response to fibronectin is related to the calcification inhibitory activity of mgp . it is possible that mgp is a multifunctional protein that can act to modify cell - matrix interactions during embryonic life or in cancer cells and also as a calcification inhibitor in selected adult tissues . this could help explain why many tissues that contain mgp synthesizing cells do not become calcified in mgp knockout animals and in keutel syndrome , a human disease characterized by loss of mgp function . mgp binds via a 6177 amino acid sequence present at the physiologic c - terminus . mgp can become part of transglutaminase crosslinked multimers of fibronectin , suggesting it may be a component of fibronectin matrices . cells attach and spread better on fibronectin and mgp coated surfaces than to fibronectin alone , even though mgp itself has no cell attachment activity . in tissues , mgp localizes near fibronectin , suggesting that interactions between the proteins are possible in vivo . the ability of mgp to alter cell interactions with fibronectin is a potential reason for cancer cells and certain embryonic cells to overexpress mgp .

noncarious cervical lesions ( nccls ) are becoming an increasingly important factor when considering the long - term health of the dentition . in fact , the occurrence of this condition is steadily increasing [ 14 ] . according to the present literature available , it is not possible to determine a unique etiological factor , but there is a concern that it is a multifactorial condition [ 58 ] . these lesions can affect tooth sensitivity , plaque retention , caries incidence , structural integrity , and pulp vitality , and they present unique challenges for successful restoration [ 59 ] . these challenges involve each step of the restoration process , including isolation , adhesion , insertion technique , and finishing and polishing . a successful diagnosis and treatment plan requires keen observation , a thorough patient history , and careful evaluation . this work aims to provide some knowledge of the nccls ' characteristics and etiologic covariables as well as improve assessment of prognosis by aiding in proper case selection for treatment and in the selection of appropriate treatment protocols . this could be reached with a complete patient anamnesis accompanied by a careful clinical examination . some studies suggest that treatment provided for nccls may not be based on the correct diagnosis [ 3 , 4 ] . it is important to diagnose the tooth wear process in children and adults as early as possible . diagnosing early forms of erosion is difficult , as erosion is accompanied by few signs and fewer , if any , symptoms . therefore , clinical appearance is the most important feature for dental professionals when diagnosing this condition . commonly , when the nccl is painless and does not affect esthetics , there is no complaint by the patient . sometimes , it is not completely painless , but the dentin is partially ( or completely ) covered by dental plaque , tartar , or gum . a simple removal ( or displacement ) of this coverage followed by the application of some stimulus ( like a delicate air blast ) can initiate a pain process . when pain is present , the location of the lesion becomes easier to detect . pain is one of the factors that will directly influence the decision for restorative therapy as well as the technique employed . as soon as the dental caries is eliminated as primary cause , the possible factors involved have to be identified . these noncarious processes may include abrasion , corrosion , and ( possibly ) abfraction , acting alone or in combination . there are factors associated directly with the genesis of nccl , such as occlusion , saliva , age , sex , diet , and parafunctional habits [ 11 , 12 ] . if teeth are worn on their occlusal surfaces , incisal surfaces , or both by friction from the food bolus , this wear is termed masticatory abrasion . masticatory abrasion can also occur on the facial and lingual aspects of teeth , as coarse food is forced against these surfaces by the tongue , lips , and cheeks during mastication . we should not underestimate the relevance of some current diet habits , which are considered but potentially destructive to the teeth ( granolas , nuts , all bran cereal , and acid juices ) . abrasion can also occur as a result of overzealous tooth brushing , improper use of dental floss and toothpicks , or detrimental oral habits . frequently , they appear as painless cavities with polished surfaces , but pain is not an uncommon occurrence . typically , when improper tooth brushing is one of the causes of the nccls , the enamel resists differently than the dentin which erodes following the path made by the toothbrush [ 39 ] . in dentistry , the term erosion is used to define the loss of dental hard tissues by chemical action not involving bacteria . erosion , as defined by the american society for testing and materials committee on standards , is the progressive loss of a material from a solid surface due to mechanical interaction between that surface and a fluid , a multi component fluid , impinging liquid or solid particles . corrosion is the more appropriate term and represents tooth surface loss caused by chemical or electrochemical action . there are both endogenous and exogenous sources of corrosion . in cases of endogenous sources of corrosion , such as bulimia or gastro esophageal reflux disease ( gerd ) , the enamel appears thin and translucent , enamel is lost on the posterior occlusal and anterior palatal surfaces , and depressions occur at the cervical areas of upper anterior teeth . cupped , or invaginated , areas develop where dentin has been exposed on the occlusal surfaces of posterior teeth because of wear . in the exogenous sources of corrosion , the aspect is similar , but the tissue loss location modifies following the areas related to the passage of the corrosive element . it has been reported that any food substance with a critical ph value of less than 5.5 can become a corrodent and demineralize teeth . this may occur as a result of consuming highly acidic foods and beverages such as citrus fruits , carbonated soft drinks , and sucking on sour candies . acidulated carbonated soft drinks have become a major component of many diets , particularly among adolescents and young children . it is evident that this condition does not exclusively affect the cervical areas , but , in association with other factors , it will act synergistically . abfraction is thought to take place when excessive cyclic , nonaxial tooth loading leads to cusp flexure and stress concentration in the vulnerable cervical region of teeth . such stress is then believed to directly or indirectly contribute to the loss of cervical tooth substance [ 5 , 7 , 8 , 1623 ] . although there is theoretical evidence in support of abfraction , predominantly from finite element analysis studies , caution is advised when interpreting results of these studies due to their limitations [ 9 , 2426 ] . frequently , more than two mechanisms may be involved in the etiology of tooth surface lesions , featuring a multifactorial phenomenon . when to these two mechanisms are added the effect of stress ( abfraction ) resulting from bruxism or occlusal interference , these lesions then become corrosive - abrasive abfractive in nature . the interplay of chemical , biological , and behavioral factors is crucial and helps to explain why some individuals exhibit more erosion than others [ 5 , 7 ] . therefore , awareness of a multifactorial etiology in noncarious cervical lesions may help the clinician to formulate an appropriate treatment plan for the patient . abrasion is the most cited etiological factor for development of nccl . in clinical surveys , 94% of respondent dentists classified the lesion as abrasion , and 66% rated tooth brushing as the most likely cause . cervical toothbrush abrasions are generally thought to be a consequence of toothbrush factors such as frequent or forceful tooth brushing , faulty or vigorous techniques , filament stiffness or design , dominant hand dexterity , or abrasive dentifrices . however , investigations can not conclusively establish one factor as the primary etiology because of conflicting results . therefore , an array of aspects related to toothbrush factors may operate in conjunction with dental erosion and occlusal loading . the clarification of patients , their orientation about brushing techniques , and the change of some of the above factors can bring tangible results and must be performed . another etiology that can be effectively modified is the chemical corrosion ( also called dental erosion ) and should be correctly diagnosed . when derived from eating disorders ( bulimia ) or and gerd , the treatment may require the participation of a physician . the extrinsic etiology is more easily treatable ; removing or altering the harmful habit , as in the abrasion etiology , provides consistent results . when the abfraction etiology is diagnosed , no consensus on treatment strategies exists . it is important that oral health professionals understand that abfraction is still a theoretical concept , as it is not proved . as a result of the reported associations between occlusal interferences and abfraction lesions , and between loading direction ( influenced by cusp inclines ) and unfavorable tensile stresses , occlusal adjustment has been advocated to prevent their initiation and progression and to minimize failure of cervical restorations . occlusal adjustments may involve altering cusp inclines , reducing heavy contacts , and removing premature contacts . in fact , inappropriate occlusal adjustments may increase the risk of certain conditions such as caries , occlusal tooth wear , and dentine hypersensitivity . the science of occlusion is complex , and the treatment requires understanding , care , and experience . although it is desirable to reduce lateral forces on teeth with stress - induced cervical lesions , extensive restorative procedures , such as the reestablishment of anterior guidance or orthodontic movement , require cost - and - benefit justification . occlusal adjustment should be undertaken only in cases where the interferences are well established and diagnosed . the professional must be enabled to do the adjustments and be aware that this procedure must be performed only when strictly indicated . the adjustment must be carried out in order to remove only the interferences , preserving the original points of centric occlusion . it is a conservative procedure , since it involves only the application of a composite resin , but it is important to carefully observe the possibility of excessive stress concentrated on this tooth . in fact , it is recommended that destructive , irreversible treatments aimed at treating so - called abfraction lesions , such as occlusal adjustment , must be avoided or implemented only in exceptional cases . occlusal splints , aimed at reducing the amount of nocturnal bruxism and nonaxial tooth forces , have been recommended to prevent the initiation and progression of abfraction lesions . however , it should be noted that the use of occlusal splints to reduce bruxism is still a controversial topic . although they provide a conservative treatment option for managing suspected abfraction lesions , according to some authors , there is no evidence base to support their use [ 9 , 24 ] . in the presence of evidence of the relevance of the abfraction mechanism in the development of lesions , the occlusal splint should be considered as a good treatment strategy due to its conservative nature . it should be noted that when restoring nccls , clinicians are not treating the etiology but are merely replacing what has been lost . others recommend early intervention [ 6 , 16 , 24 , 26 , 27 ] . there are no generally accepted , specific guidelines in the literature stating that all lesions should be restored . logic and good clinical judgment would suggest that they should be restored when clinical consequences ( e.g. , dentine hypersensitivity ) have developed or are likely to develop in the near future . cervical restorations may contribute to increased plaque accumulation potentially leading to caries and periodontal disease [ 11 , 24 , 25 ] . there are different reasons for the need for restorative treatment : the structural integrity of the tooth is threatened , the exposed dentin is hypersensitive , the defect is esthetically unacceptable to the patient , or pulp exposure is likely to occur . when the dentist is against nonsensitive shallow cavities that do not provide additional plaque retention the possible causes of the nccls should be identified and eliminated ( or treated ) . if the abfraction etiology is considered , the occlusion should be marked with red and blue articulating paper to check whether there has been any change , and photographic records from an occlusal view should be taken . if a progression of the nccls is diagnosed , changes in the therapy should be considered , providing restorative treatment if necessary [ 6 , 19 ] . once the restorative treatment is indicated , the dentist has to know the different causes and aspects of each situation and choose the best strategy to employ . unfortunately , although nccl restorations are a very common occurrence in clinics , they also represent one of the less durable types of restorations and have a high index of loss of retention , marginal excess , and secondary caries . despite these restorations being a continuing problem in restorative dentistry , failure of cervical adhesive restorations is often attributed to inadequate moisture control , adhesion to different opposite substrates ( enamel and dentin ) , differences in dentin composition , and also cusp movement during occlusion . in order to help adopt the best restorative strategy , problems with restoring nccls include difficulty in obtaining moisture control and gaining access to subgingival margins [ 10 , 2830 ] . rubber dam clamps , gingival retraction cord , and periodontal surgery are methods that can be used to retract and control the gingival tissues , and thus facilitate access and also control moisture . the exudation of gingival fluid is possibly one of the challenges to adhesion in cervical region , which is already impaired by other factors ( such as the absence of enamel in the gingival wall of the cavity and the characteristics of the dentin in nccls ) . intrinsic anatomical and morphological characteristics of the cervical region create limitations in the placement of the rubber dam and clamp . proper isolation , is very difficult , sometimes impossible , when lesions extend proximally or under the gingiva . sometimes part of the structure can not be isolated and the dam promotes restorative material accumulation . when adequate rubber dam isolation is not possible another isolation method has to be employed . another option is a proposed association of mylar matrix with wood wedges and a photocured gingival barrier . in any case , a proper isolation is the first step for the success in restoring nccls but , despite being the basis for the other subsequent steps , is probably the most underestimated one . even with advanced destruction , minimally invasive restorative intervention , such as sealing or covering with composite material it is evident from the recent literature that there is no place for metallic materials such as amalgam and gold in the modern day restoration of nccls . glass ionomer cements ( gics ) , resin - modified gics ( rmgics ) , a gic / rmgic liner base laminated with a resin composite , and resin composite in combination with a dentine bonding agent are all restorative options [ 24 , 3135 ] . some authors recommend that rmgic should be the first preference for restoration of nccls or , in aesthetically demanding cases , a gic / rmgic liner base with resin composite [ 32 , 33 ] . indeed gic presents several characteristics that make them a good choice : biocompatibility , adhesion to calcified substrates ( especially in cases of dentin sclerosis where traditional adhesion may underperform ) , and elastic modulus similar to the dentin . however , some other characteristics make its use infrequent : technical difficulties related to the material 's stickiness , poor esthetics , solubility particularly in acidic oral environments , and retention failure occurrences . some authors claim that under the action of parafunctional loadings , fracture - induced failure of cervical gic restorations occurs at the cervical margin . it is further shown that prior to fracture , the restorative material undergoes strain softening , which in turn introduces damage and weakens the materials involved . the softening of the material occurs in the cervical region of the restoration area which has been linked to the location of most of the clinical observed failures . the author does not indicate gic or rmgic frequently , but it is a good indication in deep nccls , where a laminate technique ( sandwich technique with composite resins ) can be used . the best materials for restoration of nccls are the composite resins . within this group of materials , some authors recommend that nccls suspected of being caused primarily by abfraction should be restored with a microfilled resin composite or a flowable resin that has a low modulus of elasticity , as it will thus flex with the tooth and not compromise retention [ 34 , 3638 ] . however , no definitive conclusion can be found in the literature addressing the difference between failures rates of resin composites of different stiffness used to restore nccls . nevertheless , in must situation , the authors recommend low modulus composites or associations of composites with different modulus . after the isolation another important , and commonly neglected , step should be performed : the prophylaxis of the cavity . due to their nature , nccls are lined with a contaminated layer that resists adhesion . the gingival proximity ( sometimes partially or totally covering the cavity ) makes this procedure a more complex step . in some cases , rotary prophylactic brushes can not be used in order to avoid mechanical aggression and bleeding . in nonsensitive cavities , the authors recommend rubbing the cavity and its periphery with a cotton pellet soaked with an anionic detergent , followed by rinsing with water , drying , and conventional total acid etching ( 37% phosphoric acid10 seconds on dentins and 20 seconds on enamel ) with the aim of removing the sticky layer . even when the roughening procedure is performed , the same sequence is recommended . in the presence of sensitivity , rubbing with detergent is still indicated but the phosphoric acid should be applied only on enamel . when a conventional gic is chosen , the previous conditioning with polyacrylic acid is indicated in order to provide a good surface wetting . if an rmgic is chosen , pretreatment of dentin with self - etch adhesive systems , before filling , seems to be a good alternative to the conventional dentin conditioner provided by the manufacturer . some recent studies demonstrate important histological differences between prepared dentin and the affected dentin from nccls . one work based on raman analysis showed that the distinct compositional and structural alterations in mineral and matrix components of nccls affected dentin . a heterogeneous hypermineralized layer , with characteristic features such as high phosphate / low carbonate content , high degree of crystallinity , and partially denatured collagen , was revealed in the affected dentin substrate of nccls [ 39 , 40 ] . in another study focusing on adhesion to sclerotic dentin , the authors observed that most dentinal tubules were obliterated by rod - like sclerotic casts and could not be dissolved by acid etching . both the hybrid zone and the resin tags were observed in sclerotic dentin after restoration . although resin tags were fewer , and in lack of communications , the length of resin tags and the thickness of the hybrid zone were almost similar to those of the sound dentin . they concluded that bonding to sclerotic dentin is different from bonding to sound dentin and may be compromised by fewer resin tags and communications . transmission electron microscopy revealed that in addition to occlusion of the tubules by mineral crystals , many parts of wedge - shaped cervical lesions contain a hyper mineralized surface that resists the etching action of both self - etching primers and phosphoric acid . examination of both sides of the failed bonds revealed a wide variation in fracture patterns that involved all of these structures . microtensile bond strengths to the occlusal , gingival , and deepest portions of these wedge - shaped lesions were significantly lower than similar areas artificially prepared in normal teeth . further studies are required to understand the role that these alterations play in response to acid etching and bonding to these clinically relevant substrates . further , some authors agree that restorations placed in teeth whose dentin / enamel had been prepared , or roughened , showed a statistically significant higher retention rate than those placed in teeth with unprepared dentin [ 10 , 43 ] . considering these studies and the author 's clinical experience , a mild roughening of the superficial dentin with a diamond point is indicated when restoring polished nonsensitive nccls . this procedure does not create additional sensitivity and aims to get a more reliable adhesion in this specific situation . if the cavity is deep and provides sufficient thickness , a sandwich technique may be performed , taking advantage of the gic 's good adhesion to calcium . it is important to note that adhesives with direct interaction with calcium have been recently developed and present a promising option in these cases . , it is logical to conclude , based on hydrodynamic theory , that the dentin tubules are not obliterated ; on the contrary , they are probably opened . thus , the etching should be gentle in order to provide a good substrate to adhesion without enhancing sensitivity . based on this , and considering the available adhesives , the self - conditioning ( se ) adhesives should be the first choice . although several articles doubt their efficiency in aspects such as bond strength and marginal discoloration , others demonstrate acceptable clinical performance [ 4549 ] . a previous acid etching of the surrounding enamel is indicated because , as known , the microretentions created by the se adhesives are not enough to give adhesive strength similar to that achieved by conventional acid etching . within this group , the self - etching primers ( two steps ) present better results than the self - etching adhesives ( one step ) [ 5052 ] . one must always remember that an active application of these adhesives should be employed , rubbing the surface with a soaked microbrush for 15-seconds , waiting other 15 second period to allow volatilization of solvents . this is important because the cervical wall of the cavity tends to retain excess of adhesive which leads to future discoloration and gap formation . despite the apparent easy access and insertion , nccl presents some particularities that should be emphasized . this may justify the high documented failure rate [ 30 , 33 , 5355 ] and the number of published articles about this theme [ 10 , 34 , 36 , 5667 ] . the first point that creates difficulties is that the cavity limits are not well defined , especially the proximal limits location . . every effort should be made to delimit the future restoration , because the excess removal and the finishing and polishing present other difficulties . the simple fact of working with cavities on opposite walls from dissimilar tissues like dentin and enamel already creates intrinsic problems . several restorative techniques have been proposed to minimize shrinkage due to polymerization and also to achieve better marginal adaptation in class v cavities . because bond strength to enamel is usually greater than to dentin , it was suggested that cavities could be restored in multiple layers , starting with incremental placement in the occlusal wall of the preparation . it has also been suggested that the contraction gap at the gingival margin caused by polymerization shrinkage could be prevented by the incremental placement of a composite material starting in the dentin portion of the preparation . regarding the possibility of bulk placement , it has been stated that this often results in open dentin margins , thus increasing microleakage . since enamel adhesion is stronger , more stable , and more predictable , the insertion of material should begin from the gingival wall , without surrounding enamel . avoiding concomitant insertion on opposite walls and leaving a free surface , the adhesion to the cervical wall can be achieved without antagonistic forces . whenever possible , the cavity should be restored with three , or at least two , increments . employing a careful technique is possible to achieve a restoration with minimum or no finishing and polishing procedures needed . considering esthetics , the color of the cervical area is easy to obtain , usually with a higher saturation and smaller translucency compared to the color of the other two thirds of the tooth . any excess or roughness plaque retention , gingival inflammation , and occurrence of caries lesions represent not only a failure of the restoration but also a creation of new problems to the patient . poorly performed finishing and polishing procedures can lead to damage to the soft and hard tissues . techniques with minimum need of finishing and polishing are ideal , but properly contoured restorations are seldom achieved without the need to remove excess material [ 10 , 6872 ] . when they are needed , a good option is the use of delicate diamond finishing points followed by application of a surface sealant or a liquid polisher [ 10 , 72 , 73 ] . as emphasized before , treatment of nccls is not easy , and sometimes , new procedures or different approaches are needed . semiannual appointments should be performed in order to observe the evolution of the lesions , the conditions of the restorations , and other concerns of the patient . also , the maintenance of the surface polish can be performed with a new surface sealant application . treating nccls necessarily involves these steps : problem identification , diagnosis , etiological factor removal , or treatment , and , if necessary , restoration . due to the multifactorial character , it is not a simple procedure . a successful diagnosis and treatment plan requires a thorough patient history and careful observations and evaluations .

at this time , restoration of noncarious cervical lesions ( nccls ) is a common occurrence in clinics nowadays . some reasons for this are the growth of the elderly population , a smaller rate of tooth loss , and possibly the increase of some etiologic factors . these factors include inadequate brushing techniques in gingival recession cases , corrosive food and drink consumption , and occlusal stress concentrating factors ( occlusal interferences , premature contacts , habits of bruxism , and clenching ) . unfortunately , class v restorations also represent one of the less durable types of restorations and have a high index of loss of retention , marginal excess , and secondary caries . some causes for these problems include difficulties in isolation , insertion , contouring , and finishing and polishing procedures . this work aims to help dentists in choosing the best treatment strategy , which necessarily involves steps of problem identification , diagnosis , etiological factor removal or treatment , and , if necessary , restoration . finally , appropriate restorative techniques are suggested for each situation .

1. Introduction 2. Identification of the Problem and Etiology 3. Removing (or Treating) the Causes 4. Conclusions

noncarious cervical lesions ( nccls ) are becoming an increasingly important factor when considering the long - term health of the dentition . according to the present literature available , it is not possible to determine a unique etiological factor , but there is a concern that it is a multifactorial condition [ 58 ] . these lesions can affect tooth sensitivity , plaque retention , caries incidence , structural integrity , and pulp vitality , and they present unique challenges for successful restoration [ 59 ] . these challenges involve each step of the restoration process , including isolation , adhesion , insertion technique , and finishing and polishing . a successful diagnosis and treatment plan requires keen observation , a thorough patient history , and careful evaluation . this work aims to provide some knowledge of the nccls ' characteristics and etiologic covariables as well as improve assessment of prognosis by aiding in proper case selection for treatment and in the selection of appropriate treatment protocols . some studies suggest that treatment provided for nccls may not be based on the correct diagnosis [ 3 , 4 ] . it is important to diagnose the tooth wear process in children and adults as early as possible . diagnosing early forms of erosion is difficult , as erosion is accompanied by few signs and fewer , if any , symptoms . a simple removal ( or displacement ) of this coverage followed by the application of some stimulus ( like a delicate air blast ) can initiate a pain process . when pain is present , the location of the lesion becomes easier to detect . pain is one of the factors that will directly influence the decision for restorative therapy as well as the technique employed . as soon as the dental caries is eliminated as primary cause , the possible factors involved have to be identified . these noncarious processes may include abrasion , corrosion , and ( possibly ) abfraction , acting alone or in combination . there are factors associated directly with the genesis of nccl , such as occlusion , saliva , age , sex , diet , and parafunctional habits [ 11 , 12 ] . masticatory abrasion can also occur on the facial and lingual aspects of teeth , as coarse food is forced against these surfaces by the tongue , lips , and cheeks during mastication . we should not underestimate the relevance of some current diet habits , which are considered but potentially destructive to the teeth ( granolas , nuts , all bran cereal , and acid juices ) . abrasion can also occur as a result of overzealous tooth brushing , improper use of dental floss and toothpicks , or detrimental oral habits . frequently , they appear as painless cavities with polished surfaces , but pain is not an uncommon occurrence . typically , when improper tooth brushing is one of the causes of the nccls , the enamel resists differently than the dentin which erodes following the path made by the toothbrush [ 39 ] . in dentistry , the term erosion is used to define the loss of dental hard tissues by chemical action not involving bacteria . erosion , as defined by the american society for testing and materials committee on standards , is the progressive loss of a material from a solid surface due to mechanical interaction between that surface and a fluid , a multi component fluid , impinging liquid or solid particles . corrosion is the more appropriate term and represents tooth surface loss caused by chemical or electrochemical action . in cases of endogenous sources of corrosion , such as bulimia or gastro esophageal reflux disease ( gerd ) , the enamel appears thin and translucent , enamel is lost on the posterior occlusal and anterior palatal surfaces , and depressions occur at the cervical areas of upper anterior teeth . cupped , or invaginated , areas develop where dentin has been exposed on the occlusal surfaces of posterior teeth because of wear . in the exogenous sources of corrosion , the aspect is similar , but the tissue loss location modifies following the areas related to the passage of the corrosive element . this may occur as a result of consuming highly acidic foods and beverages such as citrus fruits , carbonated soft drinks , and sucking on sour candies . acidulated carbonated soft drinks have become a major component of many diets , particularly among adolescents and young children . it is evident that this condition does not exclusively affect the cervical areas , but , in association with other factors , it will act synergistically . such stress is then believed to directly or indirectly contribute to the loss of cervical tooth substance [ 5 , 7 , 8 , 1623 ] . frequently , more than two mechanisms may be involved in the etiology of tooth surface lesions , featuring a multifactorial phenomenon . when to these two mechanisms are added the effect of stress ( abfraction ) resulting from bruxism or occlusal interference , these lesions then become corrosive - abrasive abfractive in nature . the interplay of chemical , biological , and behavioral factors is crucial and helps to explain why some individuals exhibit more erosion than others [ 5 , 7 ] . therefore , awareness of a multifactorial etiology in noncarious cervical lesions may help the clinician to formulate an appropriate treatment plan for the patient . abrasion is the most cited etiological factor for development of nccl . in clinical surveys , 94% of respondent dentists classified the lesion as abrasion , and 66% rated tooth brushing as the most likely cause . cervical toothbrush abrasions are generally thought to be a consequence of toothbrush factors such as frequent or forceful tooth brushing , faulty or vigorous techniques , filament stiffness or design , dominant hand dexterity , or abrasive dentifrices . however , investigations can not conclusively establish one factor as the primary etiology because of conflicting results . therefore , an array of aspects related to toothbrush factors may operate in conjunction with dental erosion and occlusal loading . the clarification of patients , their orientation about brushing techniques , and the change of some of the above factors can bring tangible results and must be performed . another etiology that can be effectively modified is the chemical corrosion ( also called dental erosion ) and should be correctly diagnosed . the extrinsic etiology is more easily treatable ; removing or altering the harmful habit , as in the abrasion etiology , provides consistent results . as a result of the reported associations between occlusal interferences and abfraction lesions , and between loading direction ( influenced by cusp inclines ) and unfavorable tensile stresses , occlusal adjustment has been advocated to prevent their initiation and progression and to minimize failure of cervical restorations . occlusal adjustments may involve altering cusp inclines , reducing heavy contacts , and removing premature contacts . in fact , inappropriate occlusal adjustments may increase the risk of certain conditions such as caries , occlusal tooth wear , and dentine hypersensitivity . the science of occlusion is complex , and the treatment requires understanding , care , and experience . although it is desirable to reduce lateral forces on teeth with stress - induced cervical lesions , extensive restorative procedures , such as the reestablishment of anterior guidance or orthodontic movement , require cost - and - benefit justification . occlusal adjustment should be undertaken only in cases where the interferences are well established and diagnosed . the adjustment must be carried out in order to remove only the interferences , preserving the original points of centric occlusion . it is a conservative procedure , since it involves only the application of a composite resin , but it is important to carefully observe the possibility of excessive stress concentrated on this tooth . in the presence of evidence of the relevance of the abfraction mechanism in the development of lesions , the occlusal splint should be considered as a good treatment strategy due to its conservative nature . it should be noted that when restoring nccls , clinicians are not treating the etiology but are merely replacing what has been lost . others recommend early intervention [ 6 , 16 , 24 , 26 , 27 ] . there are no generally accepted , specific guidelines in the literature stating that all lesions should be restored . logic and good clinical judgment would suggest that they should be restored when clinical consequences ( e.g. cervical restorations may contribute to increased plaque accumulation potentially leading to caries and periodontal disease [ 11 , 24 , 25 ] . there are different reasons for the need for restorative treatment : the structural integrity of the tooth is threatened , the exposed dentin is hypersensitive , the defect is esthetically unacceptable to the patient , or pulp exposure is likely to occur . when the dentist is against nonsensitive shallow cavities that do not provide additional plaque retention the possible causes of the nccls should be identified and eliminated ( or treated ) . if the abfraction etiology is considered , the occlusion should be marked with red and blue articulating paper to check whether there has been any change , and photographic records from an occlusal view should be taken . if a progression of the nccls is diagnosed , changes in the therapy should be considered , providing restorative treatment if necessary [ 6 , 19 ] . once the restorative treatment is indicated , the dentist has to know the different causes and aspects of each situation and choose the best strategy to employ . unfortunately , although nccl restorations are a very common occurrence in clinics , they also represent one of the less durable types of restorations and have a high index of loss of retention , marginal excess , and secondary caries . despite these restorations being a continuing problem in restorative dentistry , failure of cervical adhesive restorations is often attributed to inadequate moisture control , adhesion to different opposite substrates ( enamel and dentin ) , differences in dentin composition , and also cusp movement during occlusion . in order to help adopt the best restorative strategy , problems with restoring nccls include difficulty in obtaining moisture control and gaining access to subgingival margins [ 10 , 2830 ] . rubber dam clamps , gingival retraction cord , and periodontal surgery are methods that can be used to retract and control the gingival tissues , and thus facilitate access and also control moisture . the exudation of gingival fluid is possibly one of the challenges to adhesion in cervical region , which is already impaired by other factors ( such as the absence of enamel in the gingival wall of the cavity and the characteristics of the dentin in nccls ) . intrinsic anatomical and morphological characteristics of the cervical region create limitations in the placement of the rubber dam and clamp . proper isolation , is very difficult , sometimes impossible , when lesions extend proximally or under the gingiva . sometimes part of the structure can not be isolated and the dam promotes restorative material accumulation . another option is a proposed association of mylar matrix with wood wedges and a photocured gingival barrier . in any case , a proper isolation is the first step for the success in restoring nccls but , despite being the basis for the other subsequent steps , is probably the most underestimated one . even with advanced destruction , minimally invasive restorative intervention , such as sealing or covering with composite material it is evident from the recent literature that there is no place for metallic materials such as amalgam and gold in the modern day restoration of nccls . glass ionomer cements ( gics ) , resin - modified gics ( rmgics ) , a gic / rmgic liner base laminated with a resin composite , and resin composite in combination with a dentine bonding agent are all restorative options [ 24 , 3135 ] . some authors recommend that rmgic should be the first preference for restoration of nccls or , in aesthetically demanding cases , a gic / rmgic liner base with resin composite [ 32 , 33 ] . indeed gic presents several characteristics that make them a good choice : biocompatibility , adhesion to calcified substrates ( especially in cases of dentin sclerosis where traditional adhesion may underperform ) , and elastic modulus similar to the dentin . however , some other characteristics make its use infrequent : technical difficulties related to the material 's stickiness , poor esthetics , solubility particularly in acidic oral environments , and retention failure occurrences . it is further shown that prior to fracture , the restorative material undergoes strain softening , which in turn introduces damage and weakens the materials involved . the softening of the material occurs in the cervical region of the restoration area which has been linked to the location of most of the clinical observed failures . the author does not indicate gic or rmgic frequently , but it is a good indication in deep nccls , where a laminate technique ( sandwich technique with composite resins ) can be used . the best materials for restoration of nccls are the composite resins . however , no definitive conclusion can be found in the literature addressing the difference between failures rates of resin composites of different stiffness used to restore nccls . nevertheless , in must situation , the authors recommend low modulus composites or associations of composites with different modulus . after the isolation another important , and commonly neglected , step should be performed : the prophylaxis of the cavity . due to their nature , nccls are lined with a contaminated layer that resists adhesion . the gingival proximity ( sometimes partially or totally covering the cavity ) makes this procedure a more complex step . in some cases , rotary prophylactic brushes can not be used in order to avoid mechanical aggression and bleeding . in nonsensitive cavities , the authors recommend rubbing the cavity and its periphery with a cotton pellet soaked with an anionic detergent , followed by rinsing with water , drying , and conventional total acid etching ( 37% phosphoric acid10 seconds on dentins and 20 seconds on enamel ) with the aim of removing the sticky layer . in the presence of sensitivity , rubbing with detergent is still indicated but the phosphoric acid should be applied only on enamel . if an rmgic is chosen , pretreatment of dentin with self - etch adhesive systems , before filling , seems to be a good alternative to the conventional dentin conditioner provided by the manufacturer . one work based on raman analysis showed that the distinct compositional and structural alterations in mineral and matrix components of nccls affected dentin . a heterogeneous hypermineralized layer , with characteristic features such as high phosphate / low carbonate content , high degree of crystallinity , and partially denatured collagen , was revealed in the affected dentin substrate of nccls [ 39 , 40 ] . although resin tags were fewer , and in lack of communications , the length of resin tags and the thickness of the hybrid zone were almost similar to those of the sound dentin . transmission electron microscopy revealed that in addition to occlusion of the tubules by mineral crystals , many parts of wedge - shaped cervical lesions contain a hyper mineralized surface that resists the etching action of both self - etching primers and phosphoric acid . examination of both sides of the failed bonds revealed a wide variation in fracture patterns that involved all of these structures . microtensile bond strengths to the occlusal , gingival , and deepest portions of these wedge - shaped lesions were significantly lower than similar areas artificially prepared in normal teeth . further studies are required to understand the role that these alterations play in response to acid etching and bonding to these clinically relevant substrates . considering these studies and the author 's clinical experience , a mild roughening of the superficial dentin with a diamond point is indicated when restoring polished nonsensitive nccls . this procedure does not create additional sensitivity and aims to get a more reliable adhesion in this specific situation . if the cavity is deep and provides sufficient thickness , a sandwich technique may be performed , taking advantage of the gic 's good adhesion to calcium . it is important to note that adhesives with direct interaction with calcium have been recently developed and present a promising option in these cases . , it is logical to conclude , based on hydrodynamic theory , that the dentin tubules are not obliterated ; on the contrary , they are probably opened . thus , the etching should be gentle in order to provide a good substrate to adhesion without enhancing sensitivity . based on this , and considering the available adhesives , the self - conditioning ( se ) adhesives should be the first choice . a previous acid etching of the surrounding enamel is indicated because , as known , the microretentions created by the se adhesives are not enough to give adhesive strength similar to that achieved by conventional acid etching . within this group , the self - etching primers ( two steps ) present better results than the self - etching adhesives ( one step ) [ 5052 ] . this is important because the cervical wall of the cavity tends to retain excess of adhesive which leads to future discoloration and gap formation . despite the apparent easy access and insertion , nccl presents some particularities that should be emphasized . this may justify the high documented failure rate [ 30 , 33 , 5355 ] and the number of published articles about this theme [ 10 , 34 , 36 , 5667 ] . the first point that creates difficulties is that the cavity limits are not well defined , especially the proximal limits location . every effort should be made to delimit the future restoration , because the excess removal and the finishing and polishing present other difficulties . several restorative techniques have been proposed to minimize shrinkage due to polymerization and also to achieve better marginal adaptation in class v cavities . because bond strength to enamel is usually greater than to dentin , it was suggested that cavities could be restored in multiple layers , starting with incremental placement in the occlusal wall of the preparation . it has also been suggested that the contraction gap at the gingival margin caused by polymerization shrinkage could be prevented by the incremental placement of a composite material starting in the dentin portion of the preparation . since enamel adhesion is stronger , more stable , and more predictable , the insertion of material should begin from the gingival wall , without surrounding enamel . avoiding concomitant insertion on opposite walls and leaving a free surface , the adhesion to the cervical wall can be achieved without antagonistic forces . employing a careful technique is possible to achieve a restoration with minimum or no finishing and polishing procedures needed . considering esthetics , the color of the cervical area is easy to obtain , usually with a higher saturation and smaller translucency compared to the color of the other two thirds of the tooth . any excess or roughness plaque retention , gingival inflammation , and occurrence of caries lesions represent not only a failure of the restoration but also a creation of new problems to the patient . poorly performed finishing and polishing procedures can lead to damage to the soft and hard tissues . techniques with minimum need of finishing and polishing are ideal , but properly contoured restorations are seldom achieved without the need to remove excess material [ 10 , 6872 ] . when they are needed , a good option is the use of delicate diamond finishing points followed by application of a surface sealant or a liquid polisher [ 10 , 72 , 73 ] . as emphasized before , treatment of nccls is not easy , and sometimes , new procedures or different approaches are needed . semiannual appointments should be performed in order to observe the evolution of the lesions , the conditions of the restorations , and other concerns of the patient . also , the maintenance of the surface polish can be performed with a new surface sealant application . treating nccls necessarily involves these steps : problem identification , diagnosis , etiological factor removal , or treatment , and , if necessary , restoration . due to the multifactorial character , it is not a simple procedure . a successful diagnosis and treatment plan requires a thorough patient history and careful observations and evaluations .

over 90 percent of head and neck cancers are squamous cell carcinomas ( hnsccs ) that arise from the mucosal lining of the upper aerodigestive tract . hnscc is the fifth most common malignancy worldwide , with more than 500 000 new cases diagnosed each year . it is estimated that these tumors accounted for 45 700 new cases and 11 210 deaths in 2007 in the united states . patients often present with advanced stage disease and , despite combined therapy , the 5-year survival rate of approximately 50% has improved only marginally in recent years . tumors are typically staged by combining clinical and pathological parameters of the primary tumor and its metastases . there are no reliable markers of early detection and prognosis , and the overall genetic and molecular basis of hnscc remains ill - defined . the major risk factor is epithelial exposure to tobacco and alcohol but , more recently , human papillomavirus ( hpv ) , an etiological agent in cervical cancer , has been linked to hnscc , especially in the oropharynx [ 4 , 5 ] . hnsccs are frequently resistant to the growth inhibition mediated by transforming growth factor- ( tgf- ) . in the majority of cases , defects in the tgf- type ii receptor ( tr - ii ) have been shown to play an important role in this resistance . in a subset of tumors , however , the mechanism responsible is not yet fully understood [ 6 , 7 ] . the tgf- superfamily is a set of multifunctional cytokines that regulate numerous cellular functions including proliferation , differentiation , organ development , wound healing , and immunity . tgf- effects are mediated by a membrane - bound serine / threonine kinase receptor complex , consisting of type i and type ii receptors [ 8 , 9 ] and their downstream signal transducers , the smad proteins . tumor cells escape tgf--mediated growth regulation via the loss of one or more functional tgf- receptors and/or smad proteins . since these abnormalities can result in unregulated cell growth , various components of the tgf- signaling pathway are considered tumor suppressor genes . genetic alterations and alterations of epigenetic information are associated with malignant transformation and progression in most cancers . modification of dna methylation patterns and chromatin remodeling contribute to epigenetic alterations of gene expression . it has been suggested that methylation silencing is as important as loss of heterogeneity or mutations in cancer development and that each tumor appears to have a characteristic profile of methylated genes . mutations in tr - ii have been frequently found in colon and gastric cancers but are less frequent in hnsccs [ 15 , 16 ] and cancers of prostate and breast . mutations in tr - i are less frequent and have been reported in lymphoma and in ovarian [ 20 , 21 ] and pancreatic cancers . a germline mutation , int7g24a , associated with susceptibility to cancer , has been detected in carcinomas of the lung , kidney and bladder , and breast . one study found no somatic mutations in the tr - i gene in 30 primary hnsccs while another found them in 4 of 21 metastatic hnsccs . inactivating mutations of the smad2 gene have been detected in a small group of human colorectal , lung , hepatocellular , and cervical cancers . moreover , smad4/dpc4 is inactivated by somatic mutations in pancreatic , colonic , and pulmonary carcinomas . while methylation of the tr - ii promoter region has been reported in esophageal and nonsmall cell pulmonary carcinomas , aberrant methylation of tr - i has been reported both in gastric cancer cell lines and in primary gastric adenocarcinomas [ 32 , 33 ] . recently , we reported that smad4 expression is significantly reduced in hpv16-positive compared to hpv16-negative hnsccs . in the same study , we detected a significant reduction in the expression of tr - i in most of the hnsccs tested . in order to understand the molecular mechanisms underlying this decrease in tr - i expression in hnsccs , we investigated the possible presence of mutations and aberrant methylation of the tr - i gene promoter . fifty puerto rican patients who had undergone surgery for hnscc were included in this study . institutional review board approvals were obtained from both the university of puerto rico medical sciences campus and the moffitt cancer center . there were 42 males ( 84% ) and 8 females ( 16% ) ranging in age from 38 to 84 years with a mean of 61.5 years . clinicopathological data collected included stage , tumor site , degree of tumor differentiation , treatment method , date and site of tumor recurrence and date and cause of death . tissue sections , 4 m in thickness , were deparaffinized , rehydrated , incubated with 0.3% peroxide , washed in water and subjected to antigen retrieval . blocking serum sections were then incubated overnight , at 4c in a humidified atmosphere , with a primary anti tr - i antibody ( santa cruz biotechnology , santa cruz , calif , usa ) at a 1:100 dilution . sections were then rinsed with pbs and incubated with the secondary antibody for 30 minutes at room temperature . detection was performed using the vectastain abc kit , rabbit igg , elite series ( vector laboratories , burlingame , calif , usa ) . tissue sections , known to express the protein , were used as positive controls and negative controls were incubated with pbs instead of the primary antibody . expression of tr - i was evaluated in the tumor and in adjacent nonneoplastic epithelium . quantitation was performed , following the method recommended by the college of american pathologists , as follows : 0 = no expression ; 1 + = < 25% cells ; 2 + = 2650% cells ; 3 + = > 50% cells . fresh - frozen tissue samples were macrodissected to obtain a 9095% purity of nonnecrotic tumor and noninvolved adjacent nonneoplastic epithelium . genomic dna was isolated from both the tumor and adjacent nonneoplastic tissue using the dna isolating kit for cells and tissues ( roche applied science , hague road , ind , usa ) . dna from peripheral blood lymphocytes was isolated using the qiamp blood dna maxi kit from qiagen inc . total rna was isolated from frozen tumor tissue , using the rneasy midi kit ( qiagen ) and following manufacturer 's specifications . the methylation status of the promoter region of tr - i was assessed by restriction enzyme - mediated pcr ( msre ) and methylation - specific pcr ( msp ) . genomic dna , isolated from peripheral blood lymphocytes ( pbl ) , served as normal control . the dna from both tumor and nonneoplastic epithelium ( 150200 ng ) was digested for 6 hours with bstui ( new england biolabs , ipswich , mass , usa ) according to conditions specified by the manufacturer . pcr amplification of unmodified dna and restriction digests were performed in a total volume of 25 l containing 1u faststart taq dna polymerase using the pcr buffer supplied by the manufacturer ( roche applied science , indianapolis , ind , usa ) with the addition of gc - rich resolution solution as recommended ( 200 m dntp , 200 ng of dna template , 2 mm mgcl2 , and 0.4 m of each primer ) . the sequences of sense and antisense primers have been reported previously . this was followed by 35 cycles of 30 seconds at 95c , 90 seconds at 55c , and 90 seconds at 72c . amplification products were separated in a 2% agarose gel , stained with ethidium bromide , and documented using the gel doc 1000 system with molecular analysis software ( biorad , hercules , calif , usa ) . amplification products were detected when digestion of the tumor genomic dna with methylation - sensitive restriction endonuclease bstui was inhibited by the presence of the methylated cpg motifs . since incomplete digestion of genomic dna with bstu1 could result in false positives , the procedure was performed twice to ensure full digestion and reproducibility of results . genomic dna ( 1 g ) was modified by bisulfite treatment using the cpgenome dna modification kit following manufacturer 's instructions ( intergen co. , purchase , ny , usa ) . the primer sets used anneal specifically to the methylated bisulfite - modified dna and are described elsewhere . pcr was performed using 5 l of each bisulfite - modified dna as template in a 25 l volume containing 1u faststart taq dna polymerase in the buffer supplied by the manufacturer ( roche applied science ) , with the addition of gc - rich resolution solution as recommended ( 200 m dntp , and 0.4 m of each primer , 2 mm mgcl2 and 5% dmso ) . the reaction mixture was incubated at 95c for 5 minutes and then subjected to 35 cycles of amplification consisting of 1 minute at 95c , 90 seconds at 55c , and 90 seconds at 72c and a final extension of 7 minutes at 72c . the amplified fragments were subjected to electrophoresis in a 3% agarose gel , stained with ethidium bromide and documented using the gel doc 1000 system with molecular analysis software ( biorad ) . for semiquantitative rt - pcr analysis , total rna was isolated from macrodissected ( enriched ) fresh frozen tumor tissue , suitable for mrna analysis , using the rneasy midi kit ( qiagen , valencia , calif , usa ) following manufacturer 's specifications . complimentary dna ( cdna ) was prepared from each sample using m - mlv reverse transcriptase ( gibco brl , life technologies , carlsbad , calif , usa ) . primers for the tr - i gene were designed using the primer 3 program at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi and tested for uniqueness in blast . cdna was amplified with the tr - i gene primers 5-ggtcttgcccatcttcacat-3 ( sense ) and 5-ttgctccaaaccacagagtg-3 ( antisense ) . primer amplification was performed by adding 2 l of each cdna sample to a final reaction mixture of 25 l containing 1u faststart taq dna polymerase in the buffer supplied by the manufacturer ( roche applied science ) , with the addition of gc - rich resolution solution as recommended ( 200 m dntp , 0.4 m of each primer , and 2 mm mgcl2 ) . the pcr conditions were : 95c for 5 minutes followed by 30 cycles of amplification consisting of 1 minute at 95c , 45 seconds at 59c , 1 minute at 72c and a final extension of 7 minutes at 72c . the pcr products were separated in a 2.5% agarose gel , stained with ethidium bromide , and documented with the gel doc 1000 system and molecular analysis software ( biorad ) . the levels of gene transcripts were quantified as the ratio of intensity of target signal to the intensity of actb signal , using the bio - rad 's quantity one software package . genomic dna ( gdna ) was amplified using primers specific for exons 1 and 7 . exon 1 ( > 70% gc rich ) was amplified using the primers 5-gaggcgaggtttgctgggtgaggca-3 ; 5-catgtttgagaaagagcaggagcgag-3 , and the advantage - gc genomic pcr kit from clonetech laboratories ( mountain view , calif , usa ) . exon 7 was amplified using the primers : 5-aaaggaggttcatccaaata-3 ; 5caacttctgatgctcatgacaaa-3. pcr products were generated in a volume of 50 l containing 500 ng of genomic dna , 10x pcr buffer ( 100 mm tris - hcl [ ph 8.3 ] , 500 mm kcl , 15 mm mgcl2 , 0.1% gelatin ) , 0.25 mm each of dntp , 100 ng of each primer , 0.056 m taqstart antibody ( clontech laboratories , palo alto , calif , usa ) and 2.5 units of taq ( gibco / brl , gaithersburg , md , usa ) . the pcr parameters were as follows : initial denaturing at 60c for 3 minutes and 94c for 5 minutes , followed by 30 cycles of 94c , 1 minute ; 55c , 1 minute ; 72c , 1 minute , followed by one extension cycle of 94c , 1 minute ; 55c , 2 minutes ; 72c , 5 minutes . the pcr fragments were purified using the freeze and squeeze dna purification kit ( biorad ) . dna sequencing was performed at the university of pennsylvania dna sequencing facility on an abi ( applied biosystems ) sequencer 3730xl with bigdye taq fs terminator v 3.1 . in order to investigate potential mechanisms of inactivation of the tgf- signaling pathway in squamous cell carcinomas of the head and neck ( hnscc ) , we examined the methylation and mutation status of the tr - igene in hnscc samples from 50 patients . of these , 42 hnsccs ( 84% ) were analyzed , by ihc , in archived formalin - fixed , paraffin - embedded tissue sections . twenty - five ( 50% ) fresh frozen samples , suitable for mrna analysis , were tested for rna expression by semiquantitative rt - pcr . the frequency of tr - i promoter aberrant methylation was detected using restriction enzyme - mediated ( bstui ) pcr and methylation specific pcr ( msp ) . the results of all three methods , immunohistochemistry , gene expression , and methylation analyses , are summarized , in the context of clinical - pathological features , in tables 1 and 2 . we observed no statistically significant associations between the results obtained by any of the three methods and patient 's sex , tumor anatomical location , degree of differentiation , or tumor stage ( table 1 ) . the presence of amplified products in bstui - digested dna ( lanes with the + sign ) indicates that the tr - i promoter is methylated in the tumor ( figure 1(a ) ; samples 6 , 8 , 30 , 37 , and 46 ) . lack of tr - i pcr product in normal lymphocytes , treated with methylation - sensitive bstui , is indicative of an unmethylated promoter cleaved by the restriction enzyme ( figure 1(a ) , lane h ) . in our series , 31 samples ( 62% ) showed aberrant methylation of the tr - i gene promoter . both methods detected hypermethylation in 30 hnsccs and only by msp in one additional sample [ figure 1(b ) , no . msp is the most sensitive of the two methods and can detect one copy of methylated dna in 1000 ( 0.1% ) unmethylated copies of genomic dna . the frequency of tr - i hypermethylation was highest in the oropharynx ( 80% ) and lowest in the hypopharynx ( 50% ) . to establish if there was a relationship between methylation and expression of mrna or protein , we simultaneously analyzed the hnsccs by rt - pcr ( figure 1(c ) ) and ihc ( figure 2 ) . of the 42 tumors tested by ihc , 35 ( 83% ) completely lost protein expression , 5 ( 12% ) showed a reduction of expression compared with adjacent nonneoplastic tissue and in two cases ( 5% ) the tumor showed no reduction of protein expression ( table 2 ; figure 2 ) . of the 7 ihc - positive cases , 3 ( 49% ) showed abnormal methylation and 4 ( 57% ) did not ( table 3 ) . on the other hand , of the 35 ihc - negative tumors , 24 ( 69% ) were aberrantly methylated and 11 ( 31% ) were not . methylation was in agreement with ihc in 64% of the cases but no strong correlation ( p = .389 ) was observed ( table 3 ) . this lack of agreement has been reported in previous studies and thought to be the result of subjective interpretation of ihc results with no uniformly accepted threshold for positivity . with regard to gene expression , of the total of 21 tumors tested , 18 ( 90% ) showed complete loss or downregulation of mrna expression and 3 ( 14% ) were fully expressed . of the 18 with altered mrna expression 17 ( 94% ) lost protein expression and 1 ( 6% ) did not ( table 3 ) . this suggests that decreased protein expression was likely due to downregulation of gene expression . in these 21 cases , an agreement with ihc results was observed on 90% of the cases , and this correlation was statistically significant ( p = .042 ) . also , a strong correlation was found between methylation status and tr - i mrna expression detected by comparative rt - pcr analysis using the actb transcript as an internal standard ( figure 1(c ) ) . a 186 bp fragment of the tr - i gene transcript was generated and compared with a 153 bp transcript of the actb gene . complete expression of tr - i transcripts was observed in four samples ( no . 25 , 34 , 36 , and 39 ) in concordance with lack of hypermethylation of the gene promoters ( figure 3 , lanes 9 , 17 , 19 , and 20 ) . tr - i mrna expression was reduced or absent in 21 of the 25 tumors tested ( 84% ) and , in these samples , a concordance ( p = .003 ) between tr - i gene promoter hypermethylation and tr - i gene expression was observed ( table 4 ) . 33 , 39 , and 40 ( figure 3 , lanes 16 , 22 , 23 ) , which lack tr - i aberrant methylation , could be explained by other mechanisms such as epigenetic histone alterations . of the 25 tumors in which aberrant methylation and gene expression were simultaneously studied ( table 4 ) , 18 ( 72% ) are methylated and 7 ( 28% ) are not methylated . also , of the 25 tumors , 4 ( 16% ) show normal gene expression , 12 ( 48% ) had partial loss of gene expression , and 9 ( 36% ) show complete loss of gene expression . of the 18 that are methylated 10 ( 55% ) show downregulation of the gene and 8 ( 44% ) have completely lost gene expression . in the 4 tumors in which the promoter is not methylated the gene is fully expressed indicating that lack of methylation correlates with normal gene expression . of the 3 remaining tumors in which the promoter was not methylated , 1 ( 33% ) showed no gene expression . later , we found that in this case ( no 33 ) the gene has a mutation in exon 1 . another tumor not expressing the gene ( no 40 ) showed a mutation in exon 7 . finally , in another tumor ( no 41 ) the promoter is not methylated and there are no detectable mutations but the gene is downregulated and protein expression is lost . mutations in the tr - i gene have been identified in ovarian , pancreatic , lung , and breast carcinomas [ 2025 ] . previous studies , however , showed that mutations within the coding sequence of tr - i are rare in hnscc . we examined twenty - five hnscc for mutations in the tr - i gene by pcr and direct sequencing of the pcr products . 10 , 11 , 12 , 22 , 24 , 26 , 27 , 29 , 31 , 33 , 38 , 40 , and 50 ) belonged to this cohort of hnscc patients and the other 12 samples ( data not shown ) were from hnscc patients treated at moffitt cancer center . we confirmed that mutations of the tr - i gene are , indeed , rare . an intronic g / a variant , 24 bp downstream of the exon / intron 7 boundary , was detected in sample no . in addition , a nine - base pair deletion in exon-1 , [ del(ggc)3 ] , was identified in sample no . epigenetic mechanisms ( dna methylation , histone modifications , and chromatin remodeling ) are altered in cancer and play a central role in the initiation and progression of the disease [ 11 , 12 , 37 ] . our results implicate , for the first time , the tr - igene as a target for inactivation by aberrant methylation in head and neck squamous cell carcinoma . disruption of the tgf- signaling transduction pathway has been shown in a significant subset of human cancers . key steps are the formation of a heterodimeric complex between receptors type ii and type i , phosphorylation of type i receptor and activation of the downstream targets . the fact that aberrant methylation of tr - i is likely to be an important step in cancer progression is supported by a similar observation in gastric cancer cell lines and in primary gastric adenocarcinomas [ 32 , 33 ] . our studies confirm previous studies by pinto et al . who demonstrated that aberrant methylation of the tr - i gene , in gastric tumors , gene inactivation resulted on loss of rna and protein expression ( tables 3 and 4 ) . our study also reveals a significant association between promoter hypermethylation and loss of gene expression . however a strong association with reduction or loss of protein expression could not be established . loss of protein expression , measured by ihc , appears not to be a good predictor of dna methylation - dependent gene silencing [ 36 , 39 ] suggesting that different gene silencing mechanisms such as histone modifications are likely to occur . recently , inactivation of tr - ii in lung cancer cell lines has been associated with alterations in the chromatin structure of the promoter region , most probably by histone deacetylation . dna methylation at the tr - ii promoter of exon 1 was also detected in a group of cells suggesting that aberrant methylation also played a role in the loss of tr - ii expression . it would be of interest to determine whether , in these tumors , alterations of the chromatin structure contribute to the inactivation of tr - i . on the other hand , the aberrant methylation , detected in one sample , only by msp , can be explained by the inherent sensitivity of the method which can detect methylated alleles in 0.1% of a total dna sample . mutations of tr - i have been detected in metastatic hnsccs . in our series , we found two tumors with mutations in the coding region of tr - i . in hnscc the int7g24a variant in tr - i has been detected more frequently in patients with carcinomas of kidney and bladder than in normal age - matched controls . in a study of hnsccs , this is consistent with our data , since we detected this alteration in only one of the tumors examined . also , we detected a common polymorphism of tr - i , tgfr1 * 6a , consisting of a deletion of 3 alanines within a 9-alanine repeat at the 3 end of the exon 1 coding sequence [ 38 , 41 ] . previously , pasche et al . showed that tgfr1 * 6a is somatically altered in cancer and functions as a tumor susceptibility allele . more recently , pasche et al . reported that the tgfr1 * 6a variant is rarely found ( 1.8% ) in primary hnscc . this alteration , found in one of our samples ( no . 33 ) , has been described in many cancer types . a recent meta - analysis of several large cohorts , which included a total of 13 113 individuals , supports the hypothesis , proposed by pasche , that tgfr1 * 6a is associated with increased cancer risk . more recently , bian et al . demonstrated that somatic acquisition is a critical event in the early stages of cancer development associated with field cancerization . studies by mi et al . showed that tgf- resistance , at late stages of hpv16-mediated transformation of human keratinocytes , is the result of a loss of expression of tr - i . this significant decrease in mrna levels can be explained by hypermethylation of the tr - i promoter region . similarly , marsit et al . found that promoter methylation in the secreted frizzled - related protein 4 ( sfrp4 ) gene was independently associated with the presence of hpv16 viral dna in hnscc . sfrps are antagonists of wnt signaling that inhibit wnt receptor binding and downregulate pathway signaling in development . sfrp4 has been found frequently methylated in colorectal cancer and in chronic lymphocytic leukemia [ 46 , 47 ] . we have previously shown a high frequency of hpv16 infection in puerto ricans with hnsccs . studies are under way to ascertain if infection with hpv16 facilitates hypermethylation of genes associated with cancer in hnsccs . analysis of the association between tr - i aberrant methylation and prognostic factors ( table 1 ) such as age , gender , stage , and tumor site showed no statistically significant correlations . however , tr - i aberrant methylation was shown in early ( i and ii ) and advanced ( iii and iv ) tumor stages suggesting that epigenetic disruption of tgf- signaling by aberrant methylation might contribute to the progression of hnsccs . our findings indicate that epigenetic silencing is the main mechanism of inactivation of tr - i in hnsccs . several studies have shown that promoter methylation of cancer genes is specific to preneoplastic and neoplastic cells . dna methylation may be present before the cancer is detected by conventional methods and , thus , can simultaneously provide diagnostic and prognostic information . pcr - based detection of hypermethylated genes both in tissue and in body fluids such as urine or blood can be useful in cancer diagnosis . for a biomarker to be useful in the detection of early cancer , however , it has to discriminate between neoplastic and nonneoplastic cells . more comprehensive studies , including tumors and matched controls , are needed to address the sensitivity , specificity , and predictive value of tr - i methylation - based cancer detection . nevertheless , tr - i hypermethylation has already shown to have a significant degree of specificity in gastric cancer , and it appears that the same is very likely for head and neck cancer . different frequencies of a variety of methylated cancer genes are reported in different cancer types suggesting that accurate diagnosis of a specific cancer type may require the detection of a panel of hypermethylated genes present at high frequency in the tumor cells . furthermore , gene methylation can potentially be evaluated in the patients sera to detect early recurrences in those primary tumors that display a given methylation pattern . thus , in addition to cdkn2a , tr - i gene could be added to the list of cancer genes that must be tested for methylation - based detection of head and neck cancer .

background . alterations in tgf- signaling are common in head and neck cancer ( hnscc ) . mutations in tgf- type ii receptor ( tr - ii ) occur frequently in hnscc while tgf- type i receptor ( tr - i ) mutations are rare , suggesting that other molecular alterations in the tgf- pathway are likely . to identify abnormalities in tr - i expression we analyzed 50 hnsccs and correlated the results with clinical - pathologic features . methods . hypermethylation of tr - i was evaluated via methylation - specific pcr ( msp ) and restriction enzyme - mediated pcr ( msre ) . mutations in exons 1 and 7 , mrna and protein expression were analyzed by direct sequencing , semiquantitative rt pcr and immunohistochemistry , respectively . results . tr - i expression was lost in 83% hnsccs and was linked to dna hypermethylation of the cpg - rich promoter region in 62% of the tumors . the variants 9a/6a and int7g24a were found in two patients . conclusions . this study shows that suppression of tr - i expression in hnscc is associated with dna hypermethylation .

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

over 90 percent of head and neck cancers are squamous cell carcinomas ( hnsccs ) that arise from the mucosal lining of the upper aerodigestive tract . hnscc is the fifth most common malignancy worldwide , with more than 500 000 new cases diagnosed each year . it is estimated that these tumors accounted for 45 700 new cases and 11 210 deaths in 2007 in the united states . tumors are typically staged by combining clinical and pathological parameters of the primary tumor and its metastases . the major risk factor is epithelial exposure to tobacco and alcohol but , more recently , human papillomavirus ( hpv ) , an etiological agent in cervical cancer , has been linked to hnscc , especially in the oropharynx [ 4 , 5 ] . in the majority of cases , defects in the tgf- type ii receptor ( tr - ii ) have been shown to play an important role in this resistance . the tgf- superfamily is a set of multifunctional cytokines that regulate numerous cellular functions including proliferation , differentiation , organ development , wound healing , and immunity . tgf- effects are mediated by a membrane - bound serine / threonine kinase receptor complex , consisting of type i and type ii receptors [ 8 , 9 ] and their downstream signal transducers , the smad proteins . since these abnormalities can result in unregulated cell growth , various components of the tgf- signaling pathway are considered tumor suppressor genes . genetic alterations and alterations of epigenetic information are associated with malignant transformation and progression in most cancers . it has been suggested that methylation silencing is as important as loss of heterogeneity or mutations in cancer development and that each tumor appears to have a characteristic profile of methylated genes . mutations in tr - ii have been frequently found in colon and gastric cancers but are less frequent in hnsccs [ 15 , 16 ] and cancers of prostate and breast . mutations in tr - i are less frequent and have been reported in lymphoma and in ovarian [ 20 , 21 ] and pancreatic cancers . a germline mutation , int7g24a , associated with susceptibility to cancer , has been detected in carcinomas of the lung , kidney and bladder , and breast . one study found no somatic mutations in the tr - i gene in 30 primary hnsccs while another found them in 4 of 21 metastatic hnsccs . inactivating mutations of the smad2 gene have been detected in a small group of human colorectal , lung , hepatocellular , and cervical cancers . moreover , smad4/dpc4 is inactivated by somatic mutations in pancreatic , colonic , and pulmonary carcinomas . while methylation of the tr - ii promoter region has been reported in esophageal and nonsmall cell pulmonary carcinomas , aberrant methylation of tr - i has been reported both in gastric cancer cell lines and in primary gastric adenocarcinomas [ 32 , 33 ] . in the same study , we detected a significant reduction in the expression of tr - i in most of the hnsccs tested . in order to understand the molecular mechanisms underlying this decrease in tr - i expression in hnsccs , we investigated the possible presence of mutations and aberrant methylation of the tr - i gene promoter . fifty puerto rican patients who had undergone surgery for hnscc were included in this study . there were 42 males ( 84% ) and 8 females ( 16% ) ranging in age from 38 to 84 years with a mean of 61.5 years . blocking serum sections were then incubated overnight , at 4c in a humidified atmosphere , with a primary anti tr - i antibody ( santa cruz biotechnology , santa cruz , calif , usa ) at a 1:100 dilution . tissue sections , known to express the protein , were used as positive controls and negative controls were incubated with pbs instead of the primary antibody . expression of tr - i was evaluated in the tumor and in adjacent nonneoplastic epithelium . total rna was isolated from frozen tumor tissue , using the rneasy midi kit ( qiagen ) and following manufacturer 's specifications . the methylation status of the promoter region of tr - i was assessed by restriction enzyme - mediated pcr ( msre ) and methylation - specific pcr ( msp ) . pcr amplification of unmodified dna and restriction digests were performed in a total volume of 25 l containing 1u faststart taq dna polymerase using the pcr buffer supplied by the manufacturer ( roche applied science , indianapolis , ind , usa ) with the addition of gc - rich resolution solution as recommended ( 200 m dntp , 200 ng of dna template , 2 mm mgcl2 , and 0.4 m of each primer ) . amplification products were detected when digestion of the tumor genomic dna with methylation - sensitive restriction endonuclease bstui was inhibited by the presence of the methylated cpg motifs . pcr was performed using 5 l of each bisulfite - modified dna as template in a 25 l volume containing 1u faststart taq dna polymerase in the buffer supplied by the manufacturer ( roche applied science ) , with the addition of gc - rich resolution solution as recommended ( 200 m dntp , and 0.4 m of each primer , 2 mm mgcl2 and 5% dmso ) . for semiquantitative rt - pcr analysis , total rna was isolated from macrodissected ( enriched ) fresh frozen tumor tissue , suitable for mrna analysis , using the rneasy midi kit ( qiagen , valencia , calif , usa ) following manufacturer 's specifications . primers for the tr - i gene were designed using the primer 3 program at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi and tested for uniqueness in blast . cdna was amplified with the tr - i gene primers 5-ggtcttgcccatcttcacat-3 ( sense ) and 5-ttgctccaaaccacagagtg-3 ( antisense ) . primer amplification was performed by adding 2 l of each cdna sample to a final reaction mixture of 25 l containing 1u faststart taq dna polymerase in the buffer supplied by the manufacturer ( roche applied science ) , with the addition of gc - rich resolution solution as recommended ( 200 m dntp , 0.4 m of each primer , and 2 mm mgcl2 ) . genomic dna ( gdna ) was amplified using primers specific for exons 1 and 7 . pcr products were generated in a volume of 50 l containing 500 ng of genomic dna , 10x pcr buffer ( 100 mm tris - hcl [ ph 8.3 ] , 500 mm kcl , 15 mm mgcl2 , 0.1% gelatin ) , 0.25 mm each of dntp , 100 ng of each primer , 0.056 m taqstart antibody ( clontech laboratories , palo alto , calif , usa ) and 2.5 units of taq ( gibco / brl , gaithersburg , md , usa ) . in order to investigate potential mechanisms of inactivation of the tgf- signaling pathway in squamous cell carcinomas of the head and neck ( hnscc ) , we examined the methylation and mutation status of the tr - igene in hnscc samples from 50 patients . of these , 42 hnsccs ( 84% ) were analyzed , by ihc , in archived formalin - fixed , paraffin - embedded tissue sections . twenty - five ( 50% ) fresh frozen samples , suitable for mrna analysis , were tested for rna expression by semiquantitative rt - pcr . the frequency of tr - i promoter aberrant methylation was detected using restriction enzyme - mediated ( bstui ) pcr and methylation specific pcr ( msp ) . the results of all three methods , immunohistochemistry , gene expression , and methylation analyses , are summarized , in the context of clinical - pathological features , in tables 1 and 2 . we observed no statistically significant associations between the results obtained by any of the three methods and patient 's sex , tumor anatomical location , degree of differentiation , or tumor stage ( table 1 ) . the presence of amplified products in bstui - digested dna ( lanes with the + sign ) indicates that the tr - i promoter is methylated in the tumor ( figure 1(a ) ; samples 6 , 8 , 30 , 37 , and 46 ) . lack of tr - i pcr product in normal lymphocytes , treated with methylation - sensitive bstui , is indicative of an unmethylated promoter cleaved by the restriction enzyme ( figure 1(a ) , lane h ) . in our series , 31 samples ( 62% ) showed aberrant methylation of the tr - i gene promoter . both methods detected hypermethylation in 30 hnsccs and only by msp in one additional sample [ figure 1(b ) , no . msp is the most sensitive of the two methods and can detect one copy of methylated dna in 1000 ( 0.1% ) unmethylated copies of genomic dna . the frequency of tr - i hypermethylation was highest in the oropharynx ( 80% ) and lowest in the hypopharynx ( 50% ) . to establish if there was a relationship between methylation and expression of mrna or protein , we simultaneously analyzed the hnsccs by rt - pcr ( figure 1(c ) ) and ihc ( figure 2 ) . of the 42 tumors tested by ihc , 35 ( 83% ) completely lost protein expression , 5 ( 12% ) showed a reduction of expression compared with adjacent nonneoplastic tissue and in two cases ( 5% ) the tumor showed no reduction of protein expression ( table 2 ; figure 2 ) . of the 7 ihc - positive cases , 3 ( 49% ) showed abnormal methylation and 4 ( 57% ) did not ( table 3 ) . on the other hand , of the 35 ihc - negative tumors , 24 ( 69% ) were aberrantly methylated and 11 ( 31% ) were not . methylation was in agreement with ihc in 64% of the cases but no strong correlation ( p = .389 ) was observed ( table 3 ) . this lack of agreement has been reported in previous studies and thought to be the result of subjective interpretation of ihc results with no uniformly accepted threshold for positivity . with regard to gene expression , of the total of 21 tumors tested , 18 ( 90% ) showed complete loss or downregulation of mrna expression and 3 ( 14% ) were fully expressed . of the 18 with altered mrna expression 17 ( 94% ) lost protein expression and 1 ( 6% ) did not ( table 3 ) . this suggests that decreased protein expression was likely due to downregulation of gene expression . also , a strong correlation was found between methylation status and tr - i mrna expression detected by comparative rt - pcr analysis using the actb transcript as an internal standard ( figure 1(c ) ) . a 186 bp fragment of the tr - i gene transcript was generated and compared with a 153 bp transcript of the actb gene . complete expression of tr - i transcripts was observed in four samples ( no . 25 , 34 , 36 , and 39 ) in concordance with lack of hypermethylation of the gene promoters ( figure 3 , lanes 9 , 17 , 19 , and 20 ) . tr - i mrna expression was reduced or absent in 21 of the 25 tumors tested ( 84% ) and , in these samples , a concordance ( p = .003 ) between tr - i gene promoter hypermethylation and tr - i gene expression was observed ( table 4 ) . 33 , 39 , and 40 ( figure 3 , lanes 16 , 22 , 23 ) , which lack tr - i aberrant methylation , could be explained by other mechanisms such as epigenetic histone alterations . of the 25 tumors in which aberrant methylation and gene expression were simultaneously studied ( table 4 ) , 18 ( 72% ) are methylated and 7 ( 28% ) are not methylated . also , of the 25 tumors , 4 ( 16% ) show normal gene expression , 12 ( 48% ) had partial loss of gene expression , and 9 ( 36% ) show complete loss of gene expression . of the 18 that are methylated 10 ( 55% ) show downregulation of the gene and 8 ( 44% ) have completely lost gene expression . in the 4 tumors in which the promoter is not methylated the gene is fully expressed indicating that lack of methylation correlates with normal gene expression . of the 3 remaining tumors in which the promoter was not methylated , 1 ( 33% ) showed no gene expression . finally , in another tumor ( no 41 ) the promoter is not methylated and there are no detectable mutations but the gene is downregulated and protein expression is lost . mutations in the tr - i gene have been identified in ovarian , pancreatic , lung , and breast carcinomas [ 2025 ] . previous studies , however , showed that mutations within the coding sequence of tr - i are rare in hnscc . we examined twenty - five hnscc for mutations in the tr - i gene by pcr and direct sequencing of the pcr products . we confirmed that mutations of the tr - i gene are , indeed , rare . an intronic g / a variant , 24 bp downstream of the exon / intron 7 boundary , was detected in sample no . epigenetic mechanisms ( dna methylation , histone modifications , and chromatin remodeling ) are altered in cancer and play a central role in the initiation and progression of the disease [ 11 , 12 , 37 ] . our results implicate , for the first time , the tr - igene as a target for inactivation by aberrant methylation in head and neck squamous cell carcinoma . disruption of the tgf- signaling transduction pathway has been shown in a significant subset of human cancers . key steps are the formation of a heterodimeric complex between receptors type ii and type i , phosphorylation of type i receptor and activation of the downstream targets . the fact that aberrant methylation of tr - i is likely to be an important step in cancer progression is supported by a similar observation in gastric cancer cell lines and in primary gastric adenocarcinomas [ 32 , 33 ] . who demonstrated that aberrant methylation of the tr - i gene , in gastric tumors , gene inactivation resulted on loss of rna and protein expression ( tables 3 and 4 ) . however a strong association with reduction or loss of protein expression could not be established . loss of protein expression , measured by ihc , appears not to be a good predictor of dna methylation - dependent gene silencing [ 36 , 39 ] suggesting that different gene silencing mechanisms such as histone modifications are likely to occur . recently , inactivation of tr - ii in lung cancer cell lines has been associated with alterations in the chromatin structure of the promoter region , most probably by histone deacetylation . dna methylation at the tr - ii promoter of exon 1 was also detected in a group of cells suggesting that aberrant methylation also played a role in the loss of tr - ii expression . it would be of interest to determine whether , in these tumors , alterations of the chromatin structure contribute to the inactivation of tr - i . on the other hand , the aberrant methylation , detected in one sample , only by msp , can be explained by the inherent sensitivity of the method which can detect methylated alleles in 0.1% of a total dna sample . mutations of tr - i have been detected in metastatic hnsccs . in our series , we found two tumors with mutations in the coding region of tr - i . in hnscc the int7g24a variant in tr - i has been detected more frequently in patients with carcinomas of kidney and bladder than in normal age - matched controls . in a study of hnsccs , this is consistent with our data , since we detected this alteration in only one of the tumors examined . also , we detected a common polymorphism of tr - i , tgfr1 * 6a , consisting of a deletion of 3 alanines within a 9-alanine repeat at the 3 end of the exon 1 coding sequence [ 38 , 41 ] . this alteration , found in one of our samples ( no . a recent meta - analysis of several large cohorts , which included a total of 13 113 individuals , supports the hypothesis , proposed by pasche , that tgfr1 * 6a is associated with increased cancer risk . demonstrated that somatic acquisition is a critical event in the early stages of cancer development associated with field cancerization . showed that tgf- resistance , at late stages of hpv16-mediated transformation of human keratinocytes , is the result of a loss of expression of tr - i . this significant decrease in mrna levels can be explained by hypermethylation of the tr - i promoter region . found that promoter methylation in the secreted frizzled - related protein 4 ( sfrp4 ) gene was independently associated with the presence of hpv16 viral dna in hnscc . studies are under way to ascertain if infection with hpv16 facilitates hypermethylation of genes associated with cancer in hnsccs . analysis of the association between tr - i aberrant methylation and prognostic factors ( table 1 ) such as age , gender , stage , and tumor site showed no statistically significant correlations . however , tr - i aberrant methylation was shown in early ( i and ii ) and advanced ( iii and iv ) tumor stages suggesting that epigenetic disruption of tgf- signaling by aberrant methylation might contribute to the progression of hnsccs . our findings indicate that epigenetic silencing is the main mechanism of inactivation of tr - i in hnsccs . for a biomarker to be useful in the detection of early cancer , however , it has to discriminate between neoplastic and nonneoplastic cells . more comprehensive studies , including tumors and matched controls , are needed to address the sensitivity , specificity , and predictive value of tr - i methylation - based cancer detection . nevertheless , tr - i hypermethylation has already shown to have a significant degree of specificity in gastric cancer , and it appears that the same is very likely for head and neck cancer . different frequencies of a variety of methylated cancer genes are reported in different cancer types suggesting that accurate diagnosis of a specific cancer type may require the detection of a panel of hypermethylated genes present at high frequency in the tumor cells . furthermore , gene methylation can potentially be evaluated in the patients sera to detect early recurrences in those primary tumors that display a given methylation pattern . thus , in addition to cdkn2a , tr - i gene could be added to the list of cancer genes that must be tested for methylation - based detection of head and neck cancer .

strains and plasmids the e. coli strains and plasmids used are described in supplemental table 1 , and the primers used for construction of plasmid vectors are described in supplemental table 2 . protein overexpression and purification all the proteins used in this study were purified as fusion proteins with a six - histidine tag from e. coli overexpressing strains according to the protocols given in the supplemental material . pulldown assays for pulldown assays , proteins were overexpressed with a c - terminal s - tag for use with the cognate protein that was his - tagged . membrane pellets , dissolved in detergent , or cell supernatant containing the s - tagged prey protein were mixed with 2 mg of purified his - tagged bait - protein . the mixture was loaded onto a ni - charged hitrap chelating column ( ge healthcare ) , so that the bait - protein could be immobilized on the column along with the prey - protein if they interact . the column was then washed with 15 - 20 column volumes of tris buffer containing 75 - 100 mm imidazole and triton x-100 ( 0.2% ( v / v ) ) to eliminate any false - positive results because of nonspecific interactions . the bait - prey protein complex was eluted with 500 mm imidazole tris buffer , containing triton x-100 ( 0.2% ( v / v ) ) , and sds - page was used to visualize the bait- and prey - proteins . a western blot was performed with anti - s - tag antibodies to confirm the presence of the s - tagged prey - protein . coli cells , of strain kam3(de3 ) , harboring plasmids were grown at 37 c , with shaking at 200 rpm , until the cell density gave an a600 of 0.5 , when 0.05 - 0.1 mm isopropyl 1-thio--d - galactopyranoside was added to the cells . the cells were grown for a further 3 h , when the cells were diluted with 2 yt media containing 100 g / ml erythromycin , and the growth curve was recorded over the next 12 h. for experiments to measure the loss in growth because of erythromycin , cells were grown for 3 h in the absence and presence of 50 g / ml erythromycin , and the growth loss is the ratio of the a600 values . analytical ultracentrifugation sedimentation equilibrium measurements were performed using a beckman optima xl - a analytical ultracentrifuge equipped with both absorbance and interference optics . 100 l of macb in buffer 1 ( 20 mm tris , ph 8.0 , 150 mm nacl , 1% w / v glycerol , and 0.006% ( w / v ) ddm ) supplemented with 10 , 25 , or 50% d2o was placed in the sample compartment of a epon double - sector centerpiece , and 110 l of buffer 1 was placed in the reference compartment . the final protein concentrations used in the runs were between 0.5 and 1.0 mg / ml . the d2o was used to match the density of the solvent to the density of the detergent as described previously ( 25 ) the samples were centrifuged at 283 k ( 10 c ) and 10,000 , 15,000 , and 25,000 rpm using an an60-ti rotor . scans were acquired using the absorbance optical system 15 h after the start of the experiment and in 1-h intervals until equilibrium was attained . sedimentation velocity measurements were performed using the same hardware at 55,000 rpm at 10 c in buffer 1 . figure 1.maca interacts with both macb and tolc . a , overexpression and purification of maca , macb , and tolc . an sds - polyacrylamide gel of purified macb ( lane 1 ) , maca ( lane 2 ) , and tolc ( lane 3 ) is shown . the purified his - tagged macb and tolc proteins were used as bait , immobilized on a ni - agarose column , over which a slurry of either detergent - solubilized membranes ( e.g. from strains overexpressing s - tagged maca or tolc ) or soluble proteins ( e.g. from strains overexpressing s - tagged 20maca ) was passed to test whether the cognate proteins from the tripartite pump could be pulled out of this complex mixture of proteins . 1st and 2nd lanes , sds - polyacrylamide gel of immobilized his - tagged macb ( 1st lane ) and the detergent - solubilized membranes from cells overexpressing the s - tagged maca ( 2nd lane ) . 3rd to 11th lanes , western blot using antibodies to the s - tag ( 1:5000 dilution ) on maca . the pulldown assay was performed with his - tagged macb immobilized on a ni - agarose column , over which a slurry of detergent - solubilized membranes from cells overexpressing s - tagged maca was passed ( 7th to 9th lanes ) . the flow - through ( 9th lane ) , 100 mm imidazole wash ( 8th lane ) , and the 500 mm imidazole elution ( 7th lane ) were tested for the presence of maca , which was now also detected in the elution fraction , indicating that it was bound to macb . a negative control experiment was performed in the absence of immobilized macb in which maca was passed through a ni - agarose column ( 3rd to 5th lanes ) , and the flow - through ( 5th lane ) , 100 mm imidazole wash ( 4th lane ) , and the 500 mm imidazole elution ( 3rd lane ) were tested for the presence of maca , which was only found in the flow - through ( 5th lane ) , establishing that s - tagged maca does not bind to the column . these results indicate that macb can pull maca from a complex mixture of membrane proteins . purified his - tagged macb did not cross - react with the antibodies to the s - tag ( 10th lane ) . c , pulldown of maca by tolc . an sds - polyacrylamide gel ( lanes 1 - 8 ) for the pulldown of s - tagged maca by his - tagged tolc and the corresponding western blot ( lanes 1-8 ) probed with antibodies ( 1:5000 dilution ) to the s - tag on maca . purified his - tagged tolc was immobilized on a ni - agarose column ( lanes 1 and 1 ) ; a slurry of detergent - solubilized membranes from cells overexpressing s - tagged maca was passed through the column and the proteins in the flow - through ( lane 2 and 2 ) , released by washing the column with 100 mm imidazole ( lanes 3 and 3 ) and eluted with 500 mm imidazole ( lanes 4 and 4 ) , were detected . as a negative control , s - tagged maca was passed through the column ( lanes 6 and 6 ) , in the absence of immobilized tolc , and the column was washed with 100 mm ( lanes 7 and 7 ) and 500 mm ( lanes 8 and 8 ) . comparing lane 4 and 8 demonstrates that maca is only bound to the column in the presence of tolc , indicative of its interaction with tolc . an sds - polyacrylamide gel ( lanes 1 - 4 ) for the pulldown of s - tagged tolc by his - tagged macb and the corresponding western blot ( lanes 1-4 ) probed with antibodies ( 1:5000 dilution ) to the s - tag on tolc . purified his - tagged macb was immobilized on a ni - agarose column ( lanes 1 and 1 ) , and a slurry of detergent - solubilized membranes from cells overexpressing s - tagged tolc was passed through the column ( lane 3 and 3 ) , which was then washed with 75 mm imidazole ( lanes 4 and 4 ) , and bound proteins were eluted with 500 mm imidazole ( lanes 5 and 5 ) . a weak band , which was not present in the absence of immobilized macb , was apparent in lane 5 , indicative of a weak interaction between macb and tolc . a control experiment was performed in the absence of immobilized macb in which tolc was passed through a ni - agarose column and the flow - through ( lane 6 ) , the 100 mm imidazole wash ( lane 7 ) , and the 500 mm imidazole elution ( lane 8) were tested for the presence of tolc , which was only found in the flow - through ( lane 6 ) , establishing that s - tagged tolc does not bind to the column . an sds - polyacrylamide gel shows the his - tagged macb ( lane 1 ) that was immobilized on a ni - agarose column ( lane 1 ) , a slurry of cytoplasmic proteins released by disruption of cells overexpressing s - tagged 20-maca ( lane 2 ) , which was passed through the column , over immobilized macb , and the proteins in the flow - through detected ( lane 3 ) ; the proteins were released by washing the column with 100 mm imidazole ( lane 4 ) ; and the proteins were eluted with 500 mm imidazole ( lane 5 ) . a western blot was performed on each of the corresponding protein fractions ( indicated with 1-5 ) using antibodies to the s - tag ( 1:5000 dilution ) to detect s - tagged maca . the elution of macb yields an extra , low mr , band on the sds - polyacrylamide gel that corresponds to that expected for maca ( lane 5 ) and was identified as such by western blotting ( lane 5 ) . a control experiment was performed in the absence of immobilized macb in which 20maca was passed through a ni - agarose column and the flow - through ( lane 7 ) , the first and second washes with 100 mm imidazole ( lanes 8 and 9 , respectively ) , and the 500 mm imidazole elution ( lane 10 ) were tested for the presence of maca , which was only found in the flow - through and first wash ( lane 7 and 8 , respectively ) , establishing that s - tagged maca does not bind to the column . these results indicate that maca does not require the n - terminal -helix , which anchors it to the inner membrane , to interact with macb . an sds - polyacrylamide gel of purified macb ( lane 1 ) , maca ( lane 2 ) , and tolc ( lane 3 ) is shown . the purified his - tagged macb and tolc proteins were used as bait , immobilized on a ni - agarose column , over which a slurry of either detergent - solubilized membranes ( e.g. from strains overexpressing s - tagged maca or tolc ) or soluble proteins ( e.g. from strains overexpressing s - tagged 20maca ) was passed to test whether the cognate proteins from the tripartite pump could be pulled out of this complex mixture of proteins . b , pulldown of maca by macb . 1st and 2nd lanes , sds - polyacrylamide gel of immobilized his - tagged macb ( 1st lane ) and the detergent - solubilized membranes from cells overexpressing the s - tagged maca ( 2nd lane ) . 3rd to 11th lanes , western blot using antibodies to the s - tag ( 1:5000 dilution ) on maca . the pulldown assay was performed with his - tagged macb immobilized on a ni - agarose column , over which a slurry of detergent - solubilized membranes from cells overexpressing s - tagged maca was passed ( 7th to 9th lanes ) . the flow - through ( 9th lane ) , 100 mm imidazole wash ( 8th lane ) , and the 500 mm imidazole elution ( 7th lane ) were tested for the presence of maca , which was now also detected in the elution fraction , indicating that it was bound to macb . a negative control experiment was performed in the absence of immobilized macb in which maca was passed through a ni - agarose column ( 3rd to 5th lanes ) , and the flow - through ( 5th lane ) , 100 mm imidazole wash ( 4th lane ) , and the 500 mm imidazole elution ( 3rd lane ) were tested for the presence of maca , which was only found in the flow - through ( 5th lane ) , establishing that s - tagged maca does not bind to the column . these results indicate that macb can pull maca from a complex mixture of membrane proteins . purified his - tagged macb did not cross - react with the antibodies to the s - tag ( 10th lane ) . c , pulldown of maca by tolc . an sds - polyacrylamide gel ( lanes 1 - 8 ) for the pulldown of s - tagged maca by his - tagged tolc and the corresponding western blot ( lanes 1-8 ) probed with antibodies ( 1:5000 dilution ) to the s - tag on maca . purified his - tagged tolc was immobilized on a ni - agarose column ( lanes 1 and 1 ) ; a slurry of detergent - solubilized membranes from cells overexpressing s - tagged maca was passed through the column and the proteins in the flow - through ( lane 2 and 2 ) , released by washing the column with 100 mm imidazole ( lanes 3 and 3 ) and eluted with 500 mm imidazole ( lanes 4 and 4 ) , were detected . as a negative control , s - tagged maca was passed through the column ( lanes 6 and 6 ) , in the absence of immobilized tolc , and the column was washed with 100 mm ( lanes 7 and 7 ) and 500 mm ( lanes 8 and 8 ) . comparing lane 4 and 8 demonstrates that maca is only bound to the column in the presence of tolc , indicative of its interaction with tolc . an sds - polyacrylamide gel ( lanes 1 - 4 ) for the pulldown of s - tagged tolc by his - tagged macb and the corresponding western blot ( lanes 1-4 ) probed with antibodies ( 1:5000 dilution ) to the s - tag on tolc . purified his - tagged macb was immobilized on a ni - agarose column ( lanes 1 and 1 ) , and a slurry of detergent - solubilized membranes from cells overexpressing s - tagged tolc was passed through the column ( lane 3 and 3 ) , which was then washed with 75 mm imidazole ( lanes 4 and 4 ) , and bound proteins were eluted with 500 mm imidazole ( lanes 5 and 5 ) . a weak band , which was not present in the absence of immobilized macb , was apparent in lane 5 , indicative of a weak interaction between macb and tolc . a control experiment was performed in the absence of immobilized macb in which tolc was passed through a ni - agarose column and the flow - through ( lane 6 ) , the 100 mm imidazole wash ( lane 7 ) , and the 500 mm imidazole elution ( lane 8) were tested for the presence of tolc , which was only found in the flow - through ( lane 6 ) , establishing that s - tagged tolc does not bind to the column . an sds - polyacrylamide gel shows the his - tagged macb ( lane 1 ) that was immobilized on a ni - agarose column ( lane 1 ) , a slurry of cytoplasmic proteins released by disruption of cells overexpressing s - tagged 20-maca ( lane 2 ) , which was passed through the column , over immobilized macb , and the proteins in the flow - through detected ( lane 3 ) ; the proteins were released by washing the column with 100 mm imidazole ( lane 4 ) ; and the proteins were eluted with 500 mm imidazole ( lane 5 ) . a western blot was performed on each of the corresponding protein fractions ( indicated with 1-5 ) using antibodies to the s - tag ( 1:5000 dilution ) to detect s - tagged maca . the elution of macb yields an extra , low mr , band on the sds - polyacrylamide gel that corresponds to that expected for maca ( lane 5 ) and was identified as such by western blotting ( lane 5 ) . a control experiment was performed in the absence of immobilized macb in which 20maca was passed through a ni - agarose column and the flow - through ( lane 7 ) , the first and second washes with 100 mm imidazole ( lanes 8 and 9 , respectively ) , and the 500 mm imidazole elution ( lane 10 ) were tested for the presence of maca , which was only found in the flow - through and first wash ( lane 7 and 8 , respectively ) , establishing that s - tagged maca does not bind to the column . these results indicate that maca does not require the n - terminal -helix , which anchors it to the inner membrane , to interact with macb . mass spectrometry analyses were performed in a nanoflow es mass spectrometer q - tof2 ( micromass ) . the following experimental parameters were used to record mass spectra of 2 mg / ml macb in the q - tof2 instrument : needle voltage of 1.5 kv and mcp 2350 v. atomic force microscopy macb was diluted to a final concentration of 1 g / ml , and 45 l of the sample was allowed to adsorb to freshly cleaved mica . imaging in air was performed with a multimode atomic force microscope ( digital instruments , santa barbara , ca ) in tapping mode . the silicon cantilevers containing a diamond - like extratip had a drive frequency of 300 khz and a specified spring constant of 40 newtons / m ( mikromasch , portland , or ) , and the applied imaging force was kept as low as possible ( target amplitude 1.6 - 1.8 v and amplitude set - point 1.3 - 1.5 v ) . the molecular volumes of the protein particles were determined from particle dimensions based on afm images ( see supplemental material ) . atpase assays an enzchek phosphate assay kit ( invitrogen ) was used to determine the atpase activity of macb hydrolyzing mgatp to release phosphate , when the reactants were mixed in a stopped - flow device ( see supplemental material ) . generally , 2.3 m protein was mixed with varying concentrations of atp , up to 4 mm , in the presence of 6 mm mgcl2 , to ensure that all the atp was complexed with mg . in control experiments , generally , for macb alone , the hydrolysis of atp was characterized by a pi burst , which was of near equivalence to the macb concentration , consistent with the atpase activity being attributable to macb , rather than any contaminant proteins . quantification of erythromycin binding to affinity - purified mac proteins the equilibrium binding of [ n - methyl - c]erythromycin to purified mac proteins was determined by rapid filtration and quantification of the radioactivity remaining on 0.2-m filters as outlined in the supplemental material . maca interacts with both macb and tolc via its periplasmic domain interactions between e. coli maca , macb , and tolc were tested using detergent - solubilized proteins for pulldown assays ( fig . 1b ) , which we confirmed by cross - linking the proteins ( supplemental fig . n - terminal truncated maca ( 20-maca ) interacted with macb ( fig . 1e ) , indicating that it is the periplasmic domains of these proteins that interact . the fact that in each case the cognate pump protein could be pulled out of a complex mixture of detergent - solubilized proteins from membranes or cells indicated that the interactions are specific . figure 2.macab-tolc form a tripartite complex that confers resistance to erythromycin . a , growth curves for e. coli cells , of strain kam3 ( de3 ) , harboring the plasmids petduet ( ) , petduet - macb ( ) , petduet - macb / maca ( ) , petduet - macb / tolc ( ) , petduet - macb / maca / tolc ( ) and petduet - macb / giii - ss-20maca / tolc ( ) grown in the presence of 100 g / ml erythromycin . b , bar chart showing the extent of inhibition of the growth of e. coli cells in response to 50 g / ml erythromycin , of strain kam3(de3 ) , harboring the plasmids petduet ( blank ) , petduet - macb ( macb ) , petduet - macb / maca ( macab ) or no plasmid ( wild ) . for each strain the a600 was determined after growth for 3 h in the absence and presence of erythromycin , and the growth inhibition was determined as the ratio of these measurements . cells expressing both maca and macb suffered less from erythromycin growth inhibition than those expressing only macb , suggesting that maca confers elevated resistance to erythromycin on the macb strain . macab - tolc form a tripartite complex that confers resistance to erythromycin . a , growth curves for e. coli cells , of strain kam3 ( de3 ) , harboring the plasmids petduet ( ) , petduet - macb ( ) , petduet - macb / maca ( ) , petduet - macb / tolc ( ) , petduet - macb / maca / tolc ( ) and petduet - macb / giii - ss-20maca / tolc ( ) grown in the presence of 100 g / ml erythromycin . b , bar chart showing the extent of inhibition of the growth of e. coli cells in response to 50 g / ml erythromycin , of strain kam3(de3 ) , harboring the plasmids petduet ( blank ) , petduet - macb ( macb ) , petduet - macb / maca ( macab ) or no plasmid ( wild ) . for each strain the a600 was determined after growth for 3 h in the absence and presence of erythromycin , and the growth inhibition was determined as the ratio of these measurements . cells expressing both maca and macb suffered less from erythromycin growth inhibition than those expressing only macb , suggesting that maca confers elevated resistance to erythromycin on the macb strain . e. coli maca and macb form a functional complex with tolc the simultaneous expression of maca , macb , and tolc in the e. coli acrab strain kam3 ( 26 ) conferred resistance to erythromycin , indicative of the formation of a functional complex ( fig . cells expressing macb with tolc conferred modest resistance to erythromycin in comparison with cells expressing macb , tolc , and maca , indicating that maca is required to couple macb to tolc ( fig . we sought to test whether the n terminus of maca , which incorporates an -helix that could interact with macb , is required for the functional assembly of the complex . a construct in which the giii - signal sequence was fused to truncated maca , targeting it to the periplasm , was capable of conferring resistance to erythromycin ( fig . 2a ) , indicating that the n - terminal domain is not essential for the assembly of the functional complex . this is consistent with a report that a truncated lipid - deficient acra derivative was functional as judged by resistance of the cells to erythromycin ( 27 ) . macb alone conferred elevated resistance to erythromycin , probably because of its ability to pump the antibiotic into the periplasm , but we consistently found that expressing macb with either tolc or maca conferred greater resistance . consequently , we sought to test if maca could enhance this ability . to overcome the difficulty in comparing the growth of cells overexpressing multiple proteins that tend to grow at different rates , we monitored the growth of cells in the presence and absence of erythromycin and determined the growth loss ( for cells growing in the presence of erythromycin in comparison with cells growing in the absence of erythromycin ) ( fig . this revealed a significant loss in growth of the cells expressing macb compared with those expressing macab , indicating that the simultaneous expression of maca and macb increases the resistance of the cells to erythromycin ( fig . 2b ) , suggesting that maca enhances the ability of macb to confer antibiotic resistance . similarly , a previous study reported that macab , but not macb alone , conferred resistance to macrolides ( 3 ) . macb forms dimers macb has an atypical structure for an abc transporter as it is predicted to have an n - terminal cytoplasmic nbd , which is connected to a four - helix transmembrane domain , with a large periplasmic domain formed by the loops connecting helices 1 and 2 ( 3 , 23 ) . if macb resembles other abc transporters that use a pair of nbds to bind atp , then it should function as a dimer . however , many transporters , including abc transporters , have 12 membrane - spanning helices ; macb could adopt a similar topology by forming trimers . furthermore , acrb ( 4 , 5 ) and tolc ( 9 ) , which assemble into a tripartite complex with acra , clearly form trimers . if the trimeric arrangement of the periplasmic domains in acrb forms a necessary scaffold for binding of acra , so that it can effectively interact with tolc , then by analogy the periplasmic domain of macb might also be forced into forming trimers when interacting with maca and tolc . size - exclusion chromatography indicated that it forms higher order oligomers consistent with a dimer ( data not shown ) , but such measurements are not only dependent upon the molecular weight but also the shape of the protein . furthermore , there is a need to determine the number of detergent molecules complexed by the protein . consequently , to determine whether the detergent - solubilized macb was dimeric , we added a cross - linker to trap the oligomers ; when we ran the cross - linked protein on an sds - polyacrylamide gel , the most predominant band ran between the 120- and 160-kda markers , indicative of a dimer , which has a calculated molecular mass of 145.8 kda ( supplemental fig . a representative sedimentation equilibrium profile from one of the runs ( two different velocities of the same sample ) is shown . experimental data ( dots ) and fitted model for a 162.6-kda particle ( solid line ) is shown for each . b , auc sedimentation velocity profiles of macb are consistent with the formation of a stable dimer . the upper panel shows sedimentation profile curves at different time points , and the lower panel presents a c(s ) size distribution analysis with solutions of the lamm equation . the sedimentation coefficient is 6.8 s corresponding to an apparent molecular mass of 160 kda , consistent with a dimer with about 16 detergent molecules bound . the mass spectrum indicated a molecular mass for macb of 145.96 kda , which is consistent with a dimer . a representative sedimentation equilibrium profile from one of the runs ( two different velocities of the same sample ) is shown . experimental data ( dots ) and fitted model for a 162.6-kda particle ( solid line ) is shown for each . b , auc sedimentation velocity profiles of macb are consistent with the formation of a stable dimer . the upper panel shows sedimentation profile curves at different time points , and the lower panel presents a c(s ) size distribution analysis with solutions of the lamm equation . the sedimentation coefficient is 6.8 s corresponding to an apparent molecular mass of 160 kda , consistent with a dimer with about 16 detergent molecules bound . the mass spectrum indicated a molecular mass for macb of 145.96 kda , which is consistent with a dimer . to further confirm the basic oligomeric unit as a dimer , we used two other techniques , analytical ultracentrifugation ( auc ) and electrospray mass spectrometry ( es - ms ) . for the auc experiments , we reduced the ddm concentration to just below the critical micelle concentration to avoid the formation of micelles . to determine the detergent contribution in the buoyant mass of the protein - detergent complex , we used a series of different density buffers prepared using a range of d2o concentrations . the apparent molecular mass for macb was determined from sedimentation equilibrium measurements to be 162.6 kda ( fig . 3a ) and from sedimentation velocity measurements to have a sedimentation coefficient of 6.8 s , corresponding to a molecular mass of 160.0 kda ( fig . 3b ) . this molecular mass is greater than expected for monomeric and less than expected for trimeric macb , complexed with bound detergent , but it is highly consistent with a macb dimer to which about 16 ddm molecules are bound . although the amount of detergent bound to the macb dimer appears to be lower than reported for rnd ( 28 ) and mf ( 29 ) transporters , this reflects the fact that in our experimental set - up the detergent contribution was actively suppressed using a solvent density matching technique ( 25 ) . our independent measurement of bound detergent using a calorimetric assay ( 30 ) indicated that , when the detergent concentration was close to the critical micelle concentration , the amount of bound detergent was similar to that of other membrane proteins ( e.g. macb solubilized in 0.05% w / v ddm bound 1.2 g of ddm / g of macb , which is equivalent to a ddm : macb molar ratio of 164:1 ) . electrospray - mass spectrometry ( es - ms ) was used in nontandem configuration to determine accurately the molecular mass of the protein , under conditions that would dissociate the ddm , revealing a peak with a molecular mass of 145,961.25 20.57 da that is consistent with a macb dimer ( fig . two populations were revealed with average molecular volumes of 118 and 238 nm ( fig . these would accommodate proteins of about 60 - 70 and 130 - 140 kda , respectively . furthermore , the larger particles could clearly be seen at higher resolution to consist of two protein domains that are highly suggestive of a dimer . we found that in the presence of amp - pnp , a nonhydrolysable analogue of atp , the ratio of dimers to monomers on the afm grids increased ( fig . 4c ) , which would be consistent with the nucleotide stabilizing the interaction between monomers . our findings are novel because detergent molecules tend to impair the resolution of afm studies of detergent - solubilized membrane proteins ; this implies that for macb , we may be able to get information on the topology and stoichiometry of its assemblies with maca and tolc . indeed , under coincubation of maca and macb , we could clearly distinguish a significant distribution of particles with molecular volumes larger than those corresponding to macb dimers , which is consistent with multiprotein complexes formed between both proteins ( data not shown ) . such promising data paves the way toward further characterization of membrane multiprotein complexes and could prove a powerful instrument for determining the stoichiometry of the tripartite assembly . maca regulates the atpase activity of macb macb retained atpase activity when detergent - solubilized , but this was detergent - dependent , being active in triton x-100 but not in ddm ( data not shown ) . the time course for hydrolysis of mgatp by macb was determined in a stopped - flow spectrophotometer , using the dye 2-amino-6-mercapto-7-methylpurine riboside to monitor the production of inorganic phosphate ( pi ) . the time course was characterized by a burst in pi production , during the first 20 s , followed by a slower steady - state rate ( fig . this kinetic behavior is consistent with the atp being rapidly hydrolyzed , to produce pi and adp , but further turnovers are rate - limited either by a subsequent conformational change or the slow release of products . because previous studies have established that the atpase activity of macb is inhibited by vanadate ( 24 ) , which stabilizes bound adp , this indicates that pi is released before adp , suggesting that adp release is rate - limiting . the rate constant for the hydrolysis step was determined by fitting the burst phase to an exponential function , yielding a kcat value of 0.24 s , whereas the amplitude of the burst phase was 2.2 m for 1 mm atp ( fig . when the maca and macb concentrations were increased to 3.5 m , the amplitude of the burst phase increased to 3.3 m ( data not shown ) , indicating that the burst is approximately equivalent to the macb concentration and that both nbds within the macb dimer are functional . we did not notice any deviation from a single exponential that would indicate that these nbds turn over atp differentially . the steady - state phase was characterized by a rate of pi production that increased in a hyperbolic manner with the atp concentration ( fig . 5b ) ; fitting the steady - state rate data to a hyperbolic function yielded a maximal specific activity of 8.9 nmol of atp / min / mg macb and a km of 374 m . a progressive reduction in the amplitude of the burst phase for atp concentrations below the steady - state km value hindered analyses because the pre - steady - state phase tended to merge with the steady - state phase ; consequently , we only used the steady - state rates determined at atp concentrations of 0.1 mm and above . for comparison , the lipid a transporter msba , an half - abc transporter , was characterized by a vmax of 37 nmol of atp / min / mg and a km of 878 m ( 31 ) . figure 4.afm analyses , afm imaging of macb . a , three - dimensional picture of a low magnification image of macb acquired in air in tapping mode with a diamond - like extra tip of resonant frequency 300 khz and spring constant of 40 newtons / m . m and d show particles that belong to the first and second peak in b , respectively . b , frequency distribution of molecular volumes of macb . the curve indicates a fitted gaussian function . the m and d peaks correspond to volumes of 118 1 nm ( n = 1642 ) and 238 5 nm ( n = 665 ) , consistent with the monomer and dimer , respectively . c , frequency distribution of molecular volumes of macb that had been incubated with the nonhydrolysable atp analogue amp - pnp . the peaks correspond to volumes of 112 3 nm ( n = 94 ) and 218 14 nm ( n = 116 ) . these data indicate an increase in the dimer : monomer ratio in the presence of amp - pnp . d , high resolution images of structures where two small particles ( m+m ) are attached to one another , clearly indicative of dimer formation . afm analyses , afm imaging of macb . a , three - dimensional picture of a low magnification image of macb acquired in air in tapping mode with a diamond - like extra tip of resonant frequency 300 khz and spring constant of 40 newtons / m . m and d show particles that belong to the first and second peak in b , respectively . b , frequency distribution of molecular volumes of macb . the curve indicates a fitted gaussian function . the m and d peaks correspond to volumes of 118 1 nm ( n = 1642 ) and 238 5 nm ( n = 665 ) , consistent with the monomer and dimer , respectively . c , frequency distribution of molecular volumes of macb that had been incubated with the nonhydrolysable atp analogue amp - pnp . the peaks correspond to volumes of 112 3 nm ( n = 94 ) and 218 14 nm ( n = 116 ) . these data indicate an increase in the dimer : monomer ratio in the presence of amp - pnp . d , high resolution images of structures where two small particles ( m+m ) are attached to one another , clearly indicative of dimer formation . when maca was added to macb , at an equivalent or higher concentration , with both proteins in detergent , 5a ) . considering that the detergent was present for both the atpase assays with macb and macab suggests that the burst phase is mechanistically important and can not be attributed to the detergent modifying the behavior of macb . interestingly , although there was no pi burst by macb in the presence of maca , there was a lag in pi production ; this could signify that a conformational change that precedes the hydrolysis step becomes rate - limiting for the first turnover . the steady - state rate of atp hydrolysis by macb , in the presence of maca , was enhanced ( fig . , this behavior might appear consistent with maca increasing the rate of adp dissociation from macb , so that this step was no longer rate - limiting . however , although maca increased the specific activity of macb from 8.9 to 12.3 nmol of atp / mg macb / min , which would correspond to an increase in kcat from 0.011 to 0.015 s , the steady - state rate did not exceed the rate for hydrolysis in the absence of maca ( e.g. 0.24 s ) ( fig . , the increase in the steady - state rate , which we attribute to product release , or a conformational change preceding this , would not be rapid enough to prevent the phosphate burst . it seems more likely that maca also retards the rate of atp hydrolysis , so that this process becomes slower than the rate of product release . indeed , we found that the affinity of macb for atp was increased by more than 5-fold , from 374 to 72 m , in the presence of maca ( fig . if the effect of maca was simply to enhance product release to a rate faster than that for nucleotide hydrolysis , then we would have expected a decrease in affinity for atp . on the other hand , if maca simply retarded nucleotide hydrolysis to a rate slower than product release , then the maximal steady - state rate would have decreased . our data are consistent with maca increasing the rate of product release , while decreasing the rate of hydrolysis to a similar rate to product release . in contrast to a previous study , which indicated that the atpase activity of reconstituted macb was not activated by n - terminal truncated maca ( 24 ) , we found that when macb was mixed with 20-maca no pi burst was apparent ( data not shown ) . in the same study , and consistent with our findings , n - terminal truncated maca was shown to interact with macb ; however , in contrast to our findings , when macb was expressed with n - terminal truncated maca in erythromycin - susceptible cells , there was no increase in erythromycin resistance ( 24 ) . the reason for the difference with our own findings is unclear ; however , in the previous study the signal sequence of ompa was used to target maca , truncated at position 32 , to the periplasm ( 24 ) , raising the possibility that the shorter maca sequence and/or the ompa signal sequence interfered with the ability of maca to affect the atpase activity of macb . figure 5.maca regulates the atpase activity of macb . a , time course for the change in pi concentration , corresponding to the absorbance change of the 2-amino-6-mercapto-7-methylpurine riboside in a , where 2.3 m macb was mixed with 1 mm atp in the absence ( upper trace ) and presence ( lower trace ) of an equivalent concentration of maca . in the absence of maca , macb produced a phosphate ( pi ) burst , with a rate and amplitude of 0.235 ( 0.001 ) s and 2.20 ( 0.01 ) m , respectively . b , steady - state rate of pi production by macb as a function of the atp concentration in the absence ( lower curve ) and presence ( upper curve ) of an equivalent concentration of maca . the data are characterized by vmax and km values of 8.9 ( 0.7 ) nmol of atp / mg macb / min and 374 ( 126 ) m , respectively , for macb alone ; and of 12.3 ( 0.5 ) nmol of atp / mg macb / min and 72 ( 22 ) m , respectively , for macb in the presence of an equivalent concentration of maca . maca regulates the atpase activity of macb . a , time course for the change in pi concentration , corresponding to the absorbance change of the 2-amino-6-mercapto-7-methylpurine riboside in a , where 2.3 m macb was mixed with 1 mm atp in the absence ( upper trace ) and presence ( lower trace ) of an equivalent concentration of maca . in the absence of maca , macb produced a phosphate ( pi ) burst , with a rate and amplitude of 0.235 ( 0.001 ) s and 2.20 ( 0.01 ) m , respectively . b , steady - state rate of pi production by macb as a function of the atp concentration in the absence ( lower curve ) and presence ( upper curve ) of an equivalent concentration of maca . the data are characterized by vmax and km values of 8.9 ( 0.7 ) nmol of atp / mg macb / min and 374 ( 126 ) m , respectively , for macb alone ; and of 12.3 ( 0.5 ) nmol of atp / mg macb / min and 72 ( 22 ) m , respectively , for macb in the presence of an equivalent concentration of maca . purified proteins ( 50 g of maca or macb , or 25 g of maca plus 25 g of macb ) were incubated in the presence of [ n - methyl - c]erythromycin at concentrations as indicated ( 1 , 5 , or 10 m ) , after which drug binding was measured by rapid filtration . the bars represent the erythromycin bound by maca ( left , black ) , macb ( middle , light gray ) , and macab ( right , dark gray ) . purified proteins ( 50 g of maca or macb , or 25 g of maca plus 25 g of macb ) were incubated in the presence of [ n - methyl - c]erythromycin at concentrations as indicated ( 1 , 5 , or 10 m ) , after which drug binding was measured by rapid filtration . the bars represent the erythromycin bound by maca ( left , black ) , macb ( middle , light gray ) , and macab ( right , dark gray ) . the data indicate that maca enhances the binding of erythromycin to macb . in accord with previous studies ( 24 ) , we could not detect an effect of erythromycin on the kinetics of macb atpase either in the absence or presence of maca ( data not shown ) . recent studies have established that the atpase activity of pdr5 is uncoupled from substrate binding ; this basal atpase activity might be required to constantly cycle the transporter between conformations , so as to maintain the accessibility of the cytosolic substrate - binding site ( 32 ) . in the case of macb , its atpase activity might instead be stimulated by tolc , to reset its conformation following drug transfer to tolc . maca increases the capacity of macb to bind erythromycin although we could not detect any effect of erythromycin on the atpase activity of macb , we could detect the binding of [ c]erythromycin to detergent - solubilized macb ( fig . importantly , we found that the macab complex bound more erythromycin than macb alone ( which binds considerably more than maca alone ) and that the amount bound increased in a concentration - dependent manner , as would be expected if maca increased the affinity of macb for erythromycin . however , because of the insolubility of the antibiotic in aqueous solutions , it was not possible to test a full range of erythromycin concentrations that might saturate macab . consequently , we can not exclude the possibility of the formation of additional sites within the macab complex that are not apparent in either macb or maca alone . our data indicate that maca not only modulates the atpase activity of macb but also enhances its capacity to bind erythromycin ( be this due to an increase in affinity of macb for drugs or the formation of additional drug - binding sites within the macab complex ) . gram - negative bacteria possess tripartite pumps that facilitate the extrusion of protein toxins and cytotoxic compounds , such as antibiotics , from the cell . in these tripartite assemblies the imp is coupled to an omp by a periplasmic mfp , forming a pump that can translocate molecules across both the inner and outer membranes . intriguingly , gram - positive bacteria that lack an outer membrane also possess mfps , suggesting that they play a role in addition to stabilizing the interaction of the imp with the omp . indeed , we have noted in some of these mfps a large deletion corresponding to the coiled - coil hairpin that would interact with the omp ( supplemental fig . , we sought to determine the role of maca , the mfp that couples the imp macb , an atp - driven transporter , with the omp tolc in e. coli to extrude macrolide antibiotics ( 3 ) . we established that maca interacts with both macb and tolc ( fig . 1 ) to form a functional tripartite complex ( fig . however , we also found that maca enhanced the resistance to erythromycin conferred by macb alone ( fig . 2b ) , suggesting that it modulated the transport activity of macb , which is consistent with our data demonstrating that maca regulates the drug binding and atpase activity of macb ( figs . 5 and 6 ) . the role of the mfp besa in the activity of the besabc pump in borrelia burgdorferi , the causative agent of lyme disease , has recently been highlighted ( 33 ) . it is quite remarkable that in this system besa also lacks the -helical hairpin . as such this periplasmic protein is unable to unlock the periplasmic entry site of the omp , a function that is attributed to the hairpin ( 11 ) . to compensate , the omp has evolved to be constitutively leaky . the reason for the retention of besa in the borrelia system is most likely associated with its function in the activation of the inner membrane component , in that case an rnd transporter . it is conceivable then , that a similar role is also present in the abc transporters and their associated mfps , and it could explain the preservation of the hairpin - lacking mfps in gram - positive bacteria , such as s. aureus ( supplemental fig . although e. coli macb has an nbd , which incorporates walker a and b motifs and an abc signature sequence that are characteristic features of members of the abc superfamily , it is not a classic abc transporter . it has a large periplasmic domain , reminiscent of that found in rnd transporters , which form trimers in which these domains form substantive sites of contact between the protomers . consequently , we sought to determine the oligomeric state of macb and in doing so have developed a novel es - ms approach to unambiguously establish that macb forms dimers ( fig . es - ms is now widely accepted as a powerful method to determine accurately the stoichiometry of intact protein complexes ( 34 ) . however membrane proteins , solubilized by detergent or adsorbed in micelles , have remained difficult to analyze under similar ms conditions . the development of strategies to tackle this field is challenging , primarily because the large quantities of detergent suppress the protein signal , whereas the poor solubility of membrane proteins in aqueous buffers often causes the electrospray needle to block . to date only a few ms studies have reported the observation of membrane proteins or their complexes by ms ( 35 - 39 ) . here we report on the use of a miniaturized form of es with reduced flow rates ( nano - es ) , and a high collision energy that facilitates the desolvation process and induces dissociation of detergent - proteins clusters , to determine the oligomeric state of an integral homomeric membrane protein . in our protocol we used lower quantities of ddm , than have been reported previously , without apparent detrimental effects on the stability of the protein . under our experimental conditions , we have successfully maintained the noncovalent subunit interactions , such that the oligomeric state of the protein complex could be determined without ambiguity . as would be expected for an abc transporter in which the nbds interact , our biophysical studies of macb indicate that it forms dimers ( figs . 3 and 4 and supplemental fig . . how does dimeric macb interact with trimeric tolc and the number of maca molecules needed to stabilize the tripartite complex ? extension of our afm studies will be vital for determining the stoichiometry of the interactions to provide an understanding of the assembly of the tripartite complex , which may be difficult to address by other methods , such as crystallization of the complex . our findings and previous studies , which have shown that disruption of the walker a and b motifs not only inhibits the atpase activity but also blocks the capacity of macb to confer macrolide resistance ( 24 ) , suggest that macb operates by a similar mechanism to typical abc transporters . our understanding of how abc transporters couple atp hydrolysis to transport is still rudimentary , but the determination of the crystal structures of several atpase subunits and complete abc transporters has suggested conservation of key steps in the molecular mechanism . the binding of atp to both soluble atpase subunits ( 40 ) , solubilized nbds from abc transporters ( 41 , 42 ) , and to the nbds within a complete abc transporter ( 43 , 44 ) has been shown to promote dimerization as the atp is bound at the interface of these nucleotide - binding sites , sandwiched between the walker a motif of one nbd and the abc signature motif of the other nbd . our afm studies indicated that there is a higher proportion of macb dimers in the presence of the nonhydrolysable nucleotide amp - pnp ( fig . , the binding of atp causes the transporter to adopt a conformation in which the substrate - binding site is outward - facing , because atp bridges the two nbds , closing off the inward - facing substrate - binding site ( 45 ) . conversely , the release of the hydrolysis products adp and phosphate is thought to promote an inward - facing conformation , as the structural constraint imposed by binding of the nucleotide is released . consistent with this proposal , several studies indicate that nbd dimerization can not be induced by adp ( 46 ) , because interactions between the -phosphate of atp and the signature sequence catalyze these events ( 47 ) . 5 ) , and so our kinetic data have clear implications in terms of such a mechanism . most significantly , maca increases the apparent affinity of macb for atp , while decreasing the rate of atp hydrolysis , so as to promote and stabilize the atp binding conformation , which we presume to be the conformation in which the antibiotic - binding site is outward - facing . in this manner , maca would play a direct role in driving antibiotic translocation between macb and tolc . during the course of our studies another investigation reported on the effect of maca on the steady - state atpase activity of macb ( 24 ) . in this study , macb , solubilized in triton x-100 , appeared to have a specific activity that was an order of magnitude higher than we had found ; however , a discontinuous assay was used to determine the atpase activity , and this could well have been influenced by pooling the time points from both the burst and steady - state phases . indeed , kcat was marginally slower than the hydrolysis rate determined from the pi burst phase in our experiments ( e.g. 0.17 versus 0.24 s ) . possibly for similar reasons , this assay did not detect the effect of maca on macb solubilized in triton x-100 . however , they did find that macb reconstituted into liposomes was characterized by a reduced kcat ( e.g. kcat = 0.10 s ) and decreased affinity of macb for atp ( e.g. km = 2.30 mm ) . co - reconstitution of macb with maca had the effect of increasing both kcat ( e.g. kcat = 0.78 s ) and the affinity of macb for atp ( e.g. km = 0.38 mm ) . this behavior is similar to the effect of maca on the steady - state kinetics of the atpase activity of solubilized macb in our study . to know if the reconstituted protein behaves in an identical manner to the solubilized protein would require the reconstitution of sufficient amounts of macb to define any pi burst , which is technically demanding . consequently , although these earlier studies support our conclusion that maca affects the atpase activity of macb , they do not give the detailed insight into the atpase mechanism provided by our pre - steady - state analyses . our studies established that conformational changes can be propagated from the periplasmic domain of maca to the cytoplasmic nbd of macb ( figs . 1 and 2 ) , and consequently , it is plausible that tolc , by interacting with maca , can detect the nucleotide state of macb . the binding of atp to macb would stimulate maca to interact with tolc , inducing the latter to adopt the open state . because atp and maca stabilize in the outward - facing conformation of macb , drug transfer from macb to tolc would be facilitated . tolc would then communicate with the nbd of macb ( again , such communication is probably conveyed via maca ) to stimulate atp hydrolysis , which would be required to reset macb in the inward - facing conformation . if atp hydrolysis is controlled by tolc , so that the macb conformation is only reset after the interaction with tolc and productive transfer of drugs , this would provide an explanation of the apparent insensitivity of the atpase activity of macb to drugs . indeed , without such a feedback control mechanism of drug export , it would appear that drug stimulation of the atpase activity of macb would be counter - productive because the macb conformation could be reset before drug transfer to tolc . there is an analogy in the mechanism of operation of abc transporters that work in conjunction with a periplasmic binding protein ( 47 ) . in the e. coli maltose transporter , the binding of atp to the atpase subunit malk induces conformational changes , detected by tryptic digestion , in the periplasmic loops of the membrane subunits malf and malg ( 48 ) , whilst epr studies revealed that atp binding , but not adp binding , caused an increase in affinity between the transporter and the maltose - binding protein ( mbp ) , and forcing open the bound mbp to release its substrate ( 49 ) . the transporter is reset in its original inward - facing conformation by atp hydrolysis , which is stimulated by the mbp ( 50 ) . in our model tolc replaces the mbp and , because the open state requires disruption of the second selectivity filter and subsequent twisting of the helices of the tolc channel to keep it fully open , there is a need for the maca hairpins to stabilize this conformation , which could not be achieved by interaction with macb alone . although we have interpreted our findings in terms of an alternating conformation model for macb , in which the drug - binding sites are inwardly and outwardly exposed , such a model could easily be refined to account for drug binding to a fixed periplasmic site , in analogy to acrb , with atp binding and hydrolysis coupled to conformational changes that induce tolc association and dissociation . clearly the work presented here provides an important framework for further studies that will enable the elucidation of the mechanism underlying the dynamics of assembly of the macabtolc tripartite pump in the future .

gram - negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes , consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter ( imp ) , a periplasmic membrane fusion protein , and an outer membrane channel . we have investigated the assembly and function of the macab / tolc system that confers resistance to macrolides in escherichia coli . the membrane fusion protein maca not only stabilizes the tripartite assembly by interacting with both the inner membrane protein macb and the outer membrane protein tolc , but also has a role in regulating the function of macb , apparently increasing its affinity for both erythromycin and atp . analysis of the kinetic behavior of atp hydrolysis indicated that maca promotes and stabilizes the atp - binding form of the macb transporter . for the first time , we have established unambiguously the dimeric nature of a noncanonic abc transporter , macb that has an n - terminal nucleotide binding domain , by means of nondissociating mass spectrometry , analytical ultracentrifugation , and atomic force microscopy . structural studies of abc transporters indicate that atp is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate - binding site is outward - facing . consequently , our data suggest that in the presence of atp the same conformation of macb is promoted and stabilized by maca . thus , maca would facilitate the delivery of drugs by macb to tolc by enhancing the binding of drugs to it and inducing a conformation of macb that is primed and competent for binding tolc . our structural studies are an important first step in understanding how the tripartite complex is assembled .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION Supplementary Material

the cells were grown for a further 3 h , when the cells were diluted with 2 yt media containing 100 g / ml erythromycin , and the growth curve was recorded over the next 12 h. for experiments to measure the loss in growth because of erythromycin , cells were grown for 3 h in the absence and presence of 50 g / ml erythromycin , and the growth loss is the ratio of the a600 values . the flow - through ( 9th lane ) , 100 mm imidazole wash ( 8th lane ) , and the 500 mm imidazole elution ( 7th lane ) were tested for the presence of maca , which was now also detected in the elution fraction , indicating that it was bound to macb . a negative control experiment was performed in the absence of immobilized macb in which maca was passed through a ni - agarose column ( 3rd to 5th lanes ) , and the flow - through ( 5th lane ) , 100 mm imidazole wash ( 4th lane ) , and the 500 mm imidazole elution ( 3rd lane ) were tested for the presence of maca , which was only found in the flow - through ( 5th lane ) , establishing that s - tagged maca does not bind to the column . as a negative control , s - tagged maca was passed through the column ( lanes 6 and 6 ) , in the absence of immobilized tolc , and the column was washed with 100 mm ( lanes 7 and 7 ) and 500 mm ( lanes 8 and 8 ) . a control experiment was performed in the absence of immobilized macb in which tolc was passed through a ni - agarose column and the flow - through ( lane 6 ) , the 100 mm imidazole wash ( lane 7 ) , and the 500 mm imidazole elution ( lane 8) were tested for the presence of tolc , which was only found in the flow - through ( lane 6 ) , establishing that s - tagged tolc does not bind to the column . an sds - polyacrylamide gel shows the his - tagged macb ( lane 1 ) that was immobilized on a ni - agarose column ( lane 1 ) , a slurry of cytoplasmic proteins released by disruption of cells overexpressing s - tagged 20-maca ( lane 2 ) , which was passed through the column , over immobilized macb , and the proteins in the flow - through detected ( lane 3 ) ; the proteins were released by washing the column with 100 mm imidazole ( lane 4 ) ; and the proteins were eluted with 500 mm imidazole ( lane 5 ) . a control experiment was performed in the absence of immobilized macb in which 20maca was passed through a ni - agarose column and the flow - through ( lane 7 ) , the first and second washes with 100 mm imidazole ( lanes 8 and 9 , respectively ) , and the 500 mm imidazole elution ( lane 10 ) were tested for the presence of maca , which was only found in the flow - through and first wash ( lane 7 and 8 , respectively ) , establishing that s - tagged maca does not bind to the column . these results indicate that maca does not require the n - terminal -helix , which anchors it to the inner membrane , to interact with macb . the flow - through ( 9th lane ) , 100 mm imidazole wash ( 8th lane ) , and the 500 mm imidazole elution ( 7th lane ) were tested for the presence of maca , which was now also detected in the elution fraction , indicating that it was bound to macb . a negative control experiment was performed in the absence of immobilized macb in which maca was passed through a ni - agarose column ( 3rd to 5th lanes ) , and the flow - through ( 5th lane ) , 100 mm imidazole wash ( 4th lane ) , and the 500 mm imidazole elution ( 3rd lane ) were tested for the presence of maca , which was only found in the flow - through ( 5th lane ) , establishing that s - tagged maca does not bind to the column . as a negative control , s - tagged maca was passed through the column ( lanes 6 and 6 ) , in the absence of immobilized tolc , and the column was washed with 100 mm ( lanes 7 and 7 ) and 500 mm ( lanes 8 and 8 ) . a control experiment was performed in the absence of immobilized macb in which tolc was passed through a ni - agarose column and the flow - through ( lane 6 ) , the 100 mm imidazole wash ( lane 7 ) , and the 500 mm imidazole elution ( lane 8) were tested for the presence of tolc , which was only found in the flow - through ( lane 6 ) , establishing that s - tagged tolc does not bind to the column . an sds - polyacrylamide gel shows the his - tagged macb ( lane 1 ) that was immobilized on a ni - agarose column ( lane 1 ) , a slurry of cytoplasmic proteins released by disruption of cells overexpressing s - tagged 20-maca ( lane 2 ) , which was passed through the column , over immobilized macb , and the proteins in the flow - through detected ( lane 3 ) ; the proteins were released by washing the column with 100 mm imidazole ( lane 4 ) ; and the proteins were eluted with 500 mm imidazole ( lane 5 ) . a control experiment was performed in the absence of immobilized macb in which 20maca was passed through a ni - agarose column and the flow - through ( lane 7 ) , the first and second washes with 100 mm imidazole ( lanes 8 and 9 , respectively ) , and the 500 mm imidazole elution ( lane 10 ) were tested for the presence of maca , which was only found in the flow - through and first wash ( lane 7 and 8 , respectively ) , establishing that s - tagged maca does not bind to the column . these results indicate that maca does not require the n - terminal -helix , which anchors it to the inner membrane , to interact with macb . the following experimental parameters were used to record mass spectra of 2 mg / ml macb in the q - tof2 instrument : needle voltage of 1.5 kv and mcp 2350 v. atomic force microscopy macb was diluted to a final concentration of 1 g / ml , and 45 l of the sample was allowed to adsorb to freshly cleaved mica . generally , 2.3 m protein was mixed with varying concentrations of atp , up to 4 mm , in the presence of 6 mm mgcl2 , to ensure that all the atp was complexed with mg . maca interacts with both macb and tolc via its periplasmic domain interactions between e. coli maca , macb , and tolc were tested using detergent - solubilized proteins for pulldown assays ( fig . a , growth curves for e. coli cells , of strain kam3 ( de3 ) , harboring the plasmids petduet ( ) , petduet - macb ( ) , petduet - macb / maca ( ) , petduet - macb / tolc ( ) , petduet - macb / maca / tolc ( ) and petduet - macb / giii - ss-20maca / tolc ( ) grown in the presence of 100 g / ml erythromycin . macab - tolc form a tripartite complex that confers resistance to erythromycin . e. coli maca and macb form a functional complex with tolc the simultaneous expression of maca , macb , and tolc in the e. coli acrab strain kam3 ( 26 ) conferred resistance to erythromycin , indicative of the formation of a functional complex ( fig . cells expressing macb with tolc conferred modest resistance to erythromycin in comparison with cells expressing macb , tolc , and maca , indicating that maca is required to couple macb to tolc ( fig . 2a ) , indicating that the n - terminal domain is not essential for the assembly of the functional complex . to overcome the difficulty in comparing the growth of cells overexpressing multiple proteins that tend to grow at different rates , we monitored the growth of cells in the presence and absence of erythromycin and determined the growth loss ( for cells growing in the presence of erythromycin in comparison with cells growing in the absence of erythromycin ) ( fig . macb forms dimers macb has an atypical structure for an abc transporter as it is predicted to have an n - terminal cytoplasmic nbd , which is connected to a four - helix transmembrane domain , with a large periplasmic domain formed by the loops connecting helices 1 and 2 ( 3 , 23 ) . if the trimeric arrangement of the periplasmic domains in acrb forms a necessary scaffold for binding of acra , so that it can effectively interact with tolc , then by analogy the periplasmic domain of macb might also be forced into forming trimers when interacting with maca and tolc . size - exclusion chromatography indicated that it forms higher order oligomers consistent with a dimer ( data not shown ) , but such measurements are not only dependent upon the molecular weight but also the shape of the protein . we found that in the presence of amp - pnp , a nonhydrolysable analogue of atp , the ratio of dimers to monomers on the afm grids increased ( fig . interestingly , although there was no pi burst by macb in the presence of maca , there was a lag in pi production ; this could signify that a conformational change that precedes the hydrolysis step becomes rate - limiting for the first turnover . the steady - state rate of atp hydrolysis by macb , in the presence of maca , was enhanced ( fig . in contrast to a previous study , which indicated that the atpase activity of reconstituted macb was not activated by n - terminal truncated maca ( 24 ) , we found that when macb was mixed with 20-maca no pi burst was apparent ( data not shown ) . b , steady - state rate of pi production by macb as a function of the atp concentration in the absence ( lower curve ) and presence ( upper curve ) of an equivalent concentration of maca . b , steady - state rate of pi production by macb as a function of the atp concentration in the absence ( lower curve ) and presence ( upper curve ) of an equivalent concentration of maca . purified proteins ( 50 g of maca or macb , or 25 g of maca plus 25 g of macb ) were incubated in the presence of [ n - methyl - c]erythromycin at concentrations as indicated ( 1 , 5 , or 10 m ) , after which drug binding was measured by rapid filtration . purified proteins ( 50 g of maca or macb , or 25 g of maca plus 25 g of macb ) were incubated in the presence of [ n - methyl - c]erythromycin at concentrations as indicated ( 1 , 5 , or 10 m ) , after which drug binding was measured by rapid filtration . in accord with previous studies ( 24 ) , we could not detect an effect of erythromycin on the kinetics of macb atpase either in the absence or presence of maca ( data not shown ) . recent studies have established that the atpase activity of pdr5 is uncoupled from substrate binding ; this basal atpase activity might be required to constantly cycle the transporter between conformations , so as to maintain the accessibility of the cytosolic substrate - binding site ( 32 ) . maca increases the capacity of macb to bind erythromycin although we could not detect any effect of erythromycin on the atpase activity of macb , we could detect the binding of [ c]erythromycin to detergent - solubilized macb ( fig . our data indicate that maca not only modulates the atpase activity of macb but also enhances its capacity to bind erythromycin ( be this due to an increase in affinity of macb for drugs or the formation of additional drug - binding sites within the macab complex ) . in these tripartite assemblies the imp is coupled to an omp by a periplasmic mfp , forming a pump that can translocate molecules across both the inner and outer membranes . intriguingly , gram - positive bacteria that lack an outer membrane also possess mfps , suggesting that they play a role in addition to stabilizing the interaction of the imp with the omp . here we report on the use of a miniaturized form of es with reduced flow rates ( nano - es ) , and a high collision energy that facilitates the desolvation process and induces dissociation of detergent - proteins clusters , to determine the oligomeric state of an integral homomeric membrane protein . as would be expected for an abc transporter in which the nbds interact , our biophysical studies of macb indicate that it forms dimers ( figs . our findings and previous studies , which have shown that disruption of the walker a and b motifs not only inhibits the atpase activity but also blocks the capacity of macb to confer macrolide resistance ( 24 ) , suggest that macb operates by a similar mechanism to typical abc transporters . our understanding of how abc transporters couple atp hydrolysis to transport is still rudimentary , but the determination of the crystal structures of several atpase subunits and complete abc transporters has suggested conservation of key steps in the molecular mechanism . the binding of atp to both soluble atpase subunits ( 40 ) , solubilized nbds from abc transporters ( 41 , 42 ) , and to the nbds within a complete abc transporter ( 43 , 44 ) has been shown to promote dimerization as the atp is bound at the interface of these nucleotide - binding sites , sandwiched between the walker a motif of one nbd and the abc signature motif of the other nbd . our afm studies indicated that there is a higher proportion of macb dimers in the presence of the nonhydrolysable nucleotide amp - pnp ( fig . , the binding of atp causes the transporter to adopt a conformation in which the substrate - binding site is outward - facing , because atp bridges the two nbds , closing off the inward - facing substrate - binding site ( 45 ) . most significantly , maca increases the apparent affinity of macb for atp , while decreasing the rate of atp hydrolysis , so as to promote and stabilize the atp binding conformation , which we presume to be the conformation in which the antibiotic - binding site is outward - facing . 1 and 2 ) , and consequently , it is plausible that tolc , by interacting with maca , can detect the nucleotide state of macb . because atp and maca stabilize in the outward - facing conformation of macb , drug transfer from macb to tolc would be facilitated . if atp hydrolysis is controlled by tolc , so that the macb conformation is only reset after the interaction with tolc and productive transfer of drugs , this would provide an explanation of the apparent insensitivity of the atpase activity of macb to drugs . in the e. coli maltose transporter , the binding of atp to the atpase subunit malk induces conformational changes , detected by tryptic digestion , in the periplasmic loops of the membrane subunits malf and malg ( 48 ) , whilst epr studies revealed that atp binding , but not adp binding , caused an increase in affinity between the transporter and the maltose - binding protein ( mbp ) , and forcing open the bound mbp to release its substrate ( 49 ) . although we have interpreted our findings in terms of an alternating conformation model for macb , in which the drug - binding sites are inwardly and outwardly exposed , such a model could easily be refined to account for drug binding to a fixed periplasmic site , in analogy to acrb , with atp binding and hydrolysis coupled to conformational changes that induce tolc association and dissociation .

the medical and socio - economic burden of raising people with intellectual disability ( i d ) is a complex issue all over the world . i d is a disability characterized by significant limitations both in intellectual functioning and in adaptive behavior , which affects many of the everyday social and practical skills so , this result in decreasing ability to deal with problems independently , which often begins before adulthood and has permanent effects on development and subsequently impose a lot of cost on families , health - care systems and governments annually . therefore , prevalence of i d is an important topic too that is reported in broad ranges , from 0.3 - 0.7% in sweden to 1.6 - 3% in an under developed country . overall , prevalence of between 0.5% and 2.8% has been described in various studies . since the majority of these studies have been conducted in other parts of the world , the most recently investigation that was done in south - east asia estimated the prevalence of i d across this continent at 0.06 - 1.3% except in china ( i d 's prevalence : 6.68% ) , although another survey that was carried out in that country did not confirm that estimation ( prevalence of about 0.75% so considering their affected population , ids in brief are important problems of public health because of problems those are associated with this condition such as : unexplained causes in nearly half of the cases and sometimes their impossibility to prevent , high prevalence in most communities and losing productive years of life , early onset of most of these disabilities in childhood and long - term consequences of failures throughout life and etc . besides , due to the importance of this issue , large numbers of investigations have been also performed on the specific needs of this group of individuals , cost of treatment and keeping them up . lacks of services suitable for the special conditions that confront a high proportion of this population such as psychiatric and neurologic problems , impaired social relationships and etc . , are other issues for assay . nevertheless , with developments in techniques that study various medical conditions , assessment on ids has been changed and applying the achievements of other sciences to studying the spatial patterns of disease and practices providing health facilities to these patients and other diseases is expanding . the utilization of geographical information systems ( gis ) in recent decades is an example of these accomplishments . since communication between a person 's health status , specific geographic factors , spatial distribution of a disease and other such information can be used as powerful tools , this property creates new kinds of vision to medical data by application of spatial methods that develop individual and public health . considering this actuality , medical geography usually debates two domains : geographical epidemiology and health system planning that from initial aspect , spatial epidemiology analyses cases with a recognized health condition or disease and explains spatial differentiation of them by using statistical methods . as noted before , although several studies have been performed in patients with ids and different techniques have been applied for evaluation of their related condition in asia and especially china ; limited evidence exists for using modern techniques of spatial analysis such as geographically weighted regression ( gwr ) on data related to these patients , especially in iran . forasmuch as - according to our far - reaching search in credible scientific site - , no investigation was done at the nation scale in iran and only based on local or residential area some studies were perused . however , for the 1 time , these data was an appropriate source at household level , made possible considering the prevalence of ids patient and their spatial distribution patterns across the country . hence , after preliminary statistical analysis and discovering the spatial distribution pattern of these patients , as an initial hypothesis , this assumption was also raised that the spatial pattern of patients with ids maybe associated with some socio - demographic factors such as illiteracy , migration , physician number ( pn ) and number of health - care centers ( hccs ) in the study region . illiteracy as an indicator of the tendency to receive preventive health - care services ; migration , element for seeking further therapeutic and rehabilitation services by these patient 's families ; pn and hccs as the measures for health - care delivery for prevention and thus decreasing the number of these patients were considered and for demonstrating these relationships , the spatial exploring analysis was employed in this work . for access to data of patients with i d , the statistics recorded by the iranian population and housing census ( 2006 ) were used . according to official statistics , this is the 1 time ; the data of these patients in the whole country were available for investigation and the required information were accessible on the official site of the statistical center of iran ( available from : http://www.amar.org.ir ) . information listed on the site , including data on physical and intellectual disabilities and different age groups and sex segregation and separation of various provinces and cities of each province is presented too . the population living in urban areas , rural and non - resident population in each of these sections has been separately recorded . data required for performing this research , including information related to people with i d in the whole country ( total number was 295,218 ) were extracted from these tables and entered into the excel software . based on the available frequency of patients and census of the general population in 2006 , the overall prevalence of patients with i d in accordance with the cities and provinces and the calculated values according to sex and rural and urban areas was computed [ tables 1 and 2 ] . since the prevalence of i d can be related to items such as illiteracy , immigration , the pn and the number of hccs particularly in rural areas ( providing services before and during pregnancy for prevention and help for early diagnosis ) , necessary information to evaluate the effect of these variables on the prevalence of i d were used from data of immigration and illiteracy rates listed in the statistical center of iran and information inserted in the national statistical yearbook 2007 ( available from : http://salnameh.sci.org.ir ) . prevalence of patients ( male or female , in urban or rural regions ) with intellectual disability by the counties in iran ( 2006 ) prevalence ( % ) of patients with intellectual disability by provinces in iran ( 2006 ) to display and analysis of phenomena that spatially distributed of them is important , use of gis as a decision support tool can offer much more realistic image or pattern than purely statistical methods . the gis analysis is a feasible method that furnishes opportunity for generating hypothesis and identifying the effects of different factors such as environmental , social , cultural - behavioral and genetic factors on the spatial pattern of diseases . so , for spatial analysis of the prevalence of i d patients , counties division in 2006 was needed . the prevalence of i d patients tables in urban and rural area were jointed to the table of counties map and finally attribute tables were prepared to perform spatial analysis . for estimation of spatial distribution of prevalence of i d patients in the counties , the croplet maps of spatial distribution of their total prevalence , prevalence of men and women and i d patient prevalence in rural and urban areas were provided [ figure 1 ] . after reviewing the plans , in order to obtain the patterns of spatial distribution and follow - up of prevalence of i d patients from clustering , random or disperse patterns in various provinces and cities , each province was delineated separately and then exploratory spatial data analysis ( esda ) on the scattering pattern of i d patient 's prevalence was done . spatial distribution pattern of prevalence of patient with i d ( iran-2006 ) at this stage , esda of prevalence of i d patients and their sex segregation , ids patient 's prevalence in urban and rural areas were mapped . two indicators helped us to interpret the result of this phase : global spatial autocorrelation coefficient or moran 's index spatial autocorrelation statistic was used to measure the correlation among neighboring observations in a pattern and the levels of spatial clustering among neighboring were districted . moran 's i as a device was used in esda for exploring the general model of spatial autocorrelation in the study area and the overall spatial distribution pattern of variable on the map including disperses , random or cluster could be identified as showed in figure 2 . spatial pattern of different moran 's index values local indicator of spatial association without considering the presence or absence of an overall spatial autocorrelation , another form of spatial correlation of data at the local level could be examined too . in other words , the possibility of recognition of spatial clusters in each local data sets and spatial significance of each of these indicators was assessed . usually , six categories are classified in lisa cluster and significant maps and for each category one color is allocated . lisa made it possible to know the separated regions of the whole map with low or high values of the variable that surrounded by areas with high or low values and significant level of each color was calculated . limitations of global regression models such as ordinary least squares are related to their incapability into taking consideration the spatial differences of variables . geographic regression models provide a local model of the variable or process that help to understand / predict the relations by fitting a regression equation to every feature in the regions . by entering the coordinates of location items , solve simpson paradox . however , it is also necessary to express that for considering the patterns of association between two or more variables in an area , probability of associated values for adjacent regions of space should be controlled . one of the methods to control the impact of spatial correlation that can be used is gwr , calculated using the following equation : yi = 0i + 1i x1i + 2i x2i + .+ ni xni + i where , yi : value of the dependent variable observed at the location i. x1i , x2i, xni : values of the independent variables observed at i , # 1 , # n. 0i , 1i , 2i, ni : parameters to be estimated . so as a result we can say that the spatial regression models that obtain the spatial dependence in the regression analysis prevent statistical problems such as unstable parameters and unacceptable statistical tests . spatial dependence can enter in the regression model as a relationship between predictor ( independent ) and dependent variables . gwr is a local version of spatial regression that provides non - aggregated parameters of the spatial units of analysis . these features have been provided examination of spatial heterogeneity of estimated relationships between dependent and independent variables . regarding this fact in the present study for gwr analysis , modeling spatial relationships in arc gis was used and spatial modeling relationships were performed . in order to evaluate the effect of predictor variables ( migration and illiteracy rate , hccs and pn ) and the impact of them on the prevalence of i d patients in iran , these independent variables were entered into the model simultaneously [ figure 3 ] . the spatial correlation of these four variables was assessed and the spatial correlation coefficient of prevalence of i d patients was found . separately , the effect of each independent variable on the prevalence of i d patients with gwr was studied and coefficience of determination ( r ) , adjusted r and akaike information criterion ( aicc ) in these cases were recorded in tables [ table 3 ] . geographic weighted regression analysis of prevalence of intellectual disabilities ( dependent variable ) and migration , illiteracy , health - care centers per 104 people and physicians number per 104 people ( predictor variables ) by county ( iran 2006 ) aicc , r and r adjusted of figure 3 calculated by gwr ( arc gis 9.3 software ) based on our access to hcc , pn and illiteracy data in rural areas and the higher prevalence of i d in these areas , regression modeling for these three independent variables and the prevalence of i d patients as dependent variable in rural areas was also performed and the values obtained from the gwr are included in table 4 . the overall effects of these three factors together and in the next step , each of these items separately were expressed in the form of maps [ figure 4 ] . aicc , r and r adjusted of figure 4 calculated by gwr ( arc gis 9.3 software ) geographic weighted regression analysis of prevalence of intellectual disabilities in rural area ( dependent variable ) and illiteracy , health - care centers per 104 people and physicians number per 104 people ( predictor variables ) in rural area by county ( iran 2006 ) for access to data of patients with i d , the statistics recorded by the iranian population and housing census ( 2006 ) were used . according to official statistics , this is the 1 time ; the data of these patients in the whole country were available for investigation and the required information were accessible on the official site of the statistical center of iran ( available from : http://www.amar.org.ir ) . information listed on the site , including data on physical and intellectual disabilities and different age groups and sex segregation and separation of various provinces and cities of each province is presented too . the population living in urban areas , rural and non - resident population in each of these sections has been separately recorded . data required for performing this research , including information related to people with i d in the whole country ( total number was 295,218 ) were extracted from these tables and entered into the excel software . based on the available frequency of patients and census of the general population in 2006 , the overall prevalence of patients with i d in accordance with the cities and provinces and the calculated values according to sex and rural and urban areas was computed [ tables 1 and 2 ] . since the prevalence of i d can be related to items such as illiteracy , immigration , the pn and the number of hccs particularly in rural areas ( providing services before and during pregnancy for prevention and help for early diagnosis ) , necessary information to evaluate the effect of these variables on the prevalence of i d were used from data of immigration and illiteracy rates listed in the statistical center of iran and information inserted in the national statistical yearbook 2007 ( available from : http://salnameh.sci.org.ir ) . prevalence of patients ( male or female , in urban or rural regions ) with intellectual disability by the counties in iran ( 2006 ) prevalence ( % ) of patients with intellectual disability by provinces in iran ( 2006 ) to display and analysis of phenomena that spatially distributed of them is important , use of gis as a decision support tool can offer much more realistic image or pattern than purely statistical methods . the gis analysis is a feasible method that furnishes opportunity for generating hypothesis and identifying the effects of different factors such as environmental , social , cultural - behavioral and genetic factors on the spatial pattern of diseases . so , for spatial analysis of the prevalence of i d patients , counties division in 2006 was needed . the prevalence of i d patients tables in urban and rural area were jointed to the table of counties map and finally attribute tables were prepared to perform spatial analysis . for estimation of spatial distribution of prevalence of i d patients in the counties , the croplet maps of spatial distribution of their total prevalence , prevalence of men and women and i d patient prevalence in rural and urban areas were provided [ figure 1 ] . after reviewing the plans , in order to obtain the patterns of spatial distribution and follow - up of prevalence of i d patients from clustering , random or disperse patterns in various provinces and cities , each province was delineated separately and then exploratory spatial data analysis ( esda ) on the scattering pattern of i d patient 's prevalence was done . spatial distribution pattern of prevalence of patient with i d ( iran-2006 ) at this stage , esda of prevalence of i d patients and their sex segregation , ids patient 's prevalence in urban and rural areas were mapped . two indicators helped us to interpret the result of this phase : global spatial autocorrelation coefficient or moran 's index spatial autocorrelation statistic was used to measure the correlation among neighboring observations in a pattern and the levels of spatial clustering among neighboring were districted . moran 's i as a device was used in esda for exploring the general model of spatial autocorrelation in the study area and the overall spatial distribution pattern of variable on the map including disperses , random or cluster could be identified as showed in figure 2 . spatial pattern of different moran 's index values local indicator of spatial association without considering the presence or absence of an overall spatial autocorrelation , another form of spatial correlation of data at the local level could be examined too . in other words , the possibility of recognition of spatial clusters in each local data sets and spatial significance of each of these indicators was assessed . usually , six categories are classified in lisa cluster and significant maps and for each category one color is allocated . lisa made it possible to know the separated regions of the whole map with low or high values of the variable that surrounded by areas with high or low values and significant level of each color was calculated . limitations of global regression models such as ordinary least squares are related to their incapability into taking consideration the spatial differences of variables . geographic regression models provide a local model of the variable or process that help to understand / predict the relations by fitting a regression equation to every feature in the regions . by entering the coordinates of location items , solve simpson paradox . however , it is also necessary to express that for considering the patterns of association between two or more variables in an area , probability of associated values for adjacent regions of space should be controlled . one of the methods to control the impact of spatial correlation that can be used is gwr , calculated using the following equation : yi = 0i + 1i x1i + 2i x2i + .+ ni xni + i where , yi : value of the dependent variable observed at the location i. x1i , x2i, xni : values of the independent variables observed at i , # 1 , # n. 0i , 1i , 2i, ni : parameters to be estimated . so as a result we can say that the spatial regression models that obtain the spatial dependence in the regression analysis prevent statistical problems such as unstable parameters and unacceptable statistical tests . spatial dependence can enter in the regression model as a relationship between predictor ( independent ) and dependent variables . gwr is a local version of spatial regression that provides non - aggregated parameters of the spatial units of analysis . these features have been provided examination of spatial heterogeneity of estimated relationships between dependent and independent variables . regarding this fact in the present study for gwr analysis , modeling spatial relationships in arc gis was used and spatial modeling relationships were performed . in order to evaluate the effect of predictor variables ( migration and illiteracy rate , hccs and pn ) and the impact of them on the prevalence of i d patients in iran , these independent variables were entered into the model simultaneously [ figure 3 ] . the spatial correlation of these four variables was assessed and the spatial correlation coefficient of prevalence of i d patients was found . separately , the effect of each independent variable on the prevalence of i d patients with gwr was studied and coefficience of determination ( r ) , adjusted r and akaike information criterion ( aicc ) in these cases were recorded in tables [ table 3 ] . geographic weighted regression analysis of prevalence of intellectual disabilities ( dependent variable ) and migration , illiteracy , health - care centers per 104 people and physicians number per 104 people ( predictor variables ) by county ( iran 2006 ) aicc , r and r adjusted of figure 3 calculated by gwr ( arc gis 9.3 software ) based on our access to hcc , pn and illiteracy data in rural areas and the higher prevalence of i d in these areas , regression modeling for these three independent variables and the prevalence of i d patients as dependent variable in rural areas was also performed and the values obtained from the gwr are included in table 4 . the overall effects of these three factors together and in the next step , each of these items separately were expressed in the form of maps [ figure 4 ] . aicc , r and r adjusted of figure 4 calculated by gwr ( arc gis 9.3 software ) geographic weighted regression analysis of prevalence of intellectual disabilities in rural area ( dependent variable ) and illiteracy , health - care centers per 104 people and physicians number per 104 people ( predictor variables ) in rural area by county ( iran 2006 ) for estimation of spatial distribution of prevalence of i d patients in the counties , the croplet maps of spatial distribution of their total prevalence , prevalence of men and women and i d patient prevalence in rural and urban areas were provided [ figure 1 ] . after reviewing the plans , in order to obtain the patterns of spatial distribution and follow - up of prevalence of i d patients from clustering , random or disperse patterns in various provinces and cities , each province was delineated separately and then exploratory spatial data analysis ( esda ) on the scattering pattern of i d patient 's prevalence was done . spatial distribution pattern of prevalence of patient with i d ( iran-2006 ) at this stage , esda of prevalence of i d patients and their sex segregation , ids patient 's prevalence in urban and rural areas were mapped . two indicators helped us to interpret the result of this phase : global spatial autocorrelation coefficient or moran 's index spatial autocorrelation statistic was used to measure the correlation among neighboring observations in a pattern and the levels of spatial clustering among neighboring were districted . moran 's i as a device was used in esda for exploring the general model of spatial autocorrelation in the study area and the overall spatial distribution pattern of variable on the map including disperses , random or cluster could be identified as showed in figure 2 . spatial pattern of different moran 's index values local indicator of spatial association without considering the presence or absence of an overall spatial autocorrelation , another form of spatial correlation of data at the local level could be examined too . in other words , the possibility of recognition of spatial clusters in each local data sets and spatial significance of each of these indicators was assessed . usually , six categories are classified in lisa cluster and significant maps and for each category one color is allocated . lisa made it possible to know the separated regions of the whole map with low or high values of the variable that surrounded by areas with high or low values and significant level of each color was calculated . limitations of global regression models such as ordinary least squares are related to their incapability into taking consideration the spatial differences of variables . geographic regression models provide a local model of the variable or process that help to understand / predict the relations by fitting a regression equation to every feature in the regions . by entering the coordinates of location items , solve simpson paradox . however , it is also necessary to express that for considering the patterns of association between two or more variables in an area , probability of associated values for adjacent regions of space should be controlled . one of the methods to control the impact of spatial correlation that can be used is gwr , calculated using the following equation : yi = 0i + 1i x1i + 2i x2i + .+ ni xni + i where , yi : value of the dependent variable observed at the location i. x1i , x2i, xni : values of the independent variables observed at i , # 1 , # n. 0i , 1i , 2i, ni : parameters to be estimated . so as a result we can say that the spatial regression models that obtain the spatial dependence in the regression analysis prevent statistical problems such as unstable parameters and unacceptable statistical tests . spatial dependence can enter in the regression model as a relationship between predictor ( independent ) and dependent variables . gwr is a local version of spatial regression that provides non - aggregated parameters of the spatial units of analysis . these features have been provided examination of spatial heterogeneity of estimated relationships between dependent and independent variables . regarding this fact in the present study for gwr analysis , modeling spatial relationships in arc gis was used and spatial modeling relationships were performed . in order to evaluate the effect of predictor variables ( migration and illiteracy rate , hccs and pn ) and the impact of them on the prevalence of i d patients in iran , these independent variables were entered into the model simultaneously [ figure 3 ] . the spatial correlation of these four variables was assessed and the spatial correlation coefficient of prevalence of i d patients was found . separately , the effect of each independent variable on the prevalence of i d patients with gwr was studied and coefficience of determination ( r ) , adjusted r and akaike information criterion ( aicc ) in these cases were recorded in tables [ table 3 ] . geographic weighted regression analysis of prevalence of intellectual disabilities ( dependent variable ) and migration , illiteracy , health - care centers per 104 people and physicians number per 104 people ( predictor variables ) by county ( iran 2006 ) aicc , r and r adjusted of figure 3 calculated by gwr ( arc gis 9.3 software ) based on our access to hcc , pn and illiteracy data in rural areas and the higher prevalence of i d in these areas , regression modeling for these three independent variables and the prevalence of i d patients as dependent variable in rural areas was also performed and the values obtained from the gwr are included in table 4 . the overall effects of these three factors together and in the next step , each of these items separately were expressed in the form of maps [ figure 4 ] . aicc , r and r adjusted of figure 4 calculated by gwr ( arc gis 9.3 software ) geographic weighted regression analysis of prevalence of intellectual disabilities in rural area ( dependent variable ) and illiteracy , health - care centers per 104 people and physicians number per 104 people ( predictor variables ) in rural area by county ( iran 2006 ) considering calculated moran 's i for determination the spatial distribution patterns of prevalence of i d patients [ table 5 ] , clustering model was found in total , men and women distribution of i d patients ( moran 's i = 0.36 ) and this pattern repeated in rural areas too ( moran 's i = 0.01 ) . according to moran 's i in the urban region , prevalence of i d patient in these areas followed random model ( moran 's i = 0.01 ) and p values confirmed the existence of these spatial distribution ( p = 0.38 ) . calculated moran 's i and determination of spatial pattern of prevalence of i d patients by geoda and arc gis software ( iran 2006 ) lisa showed that the highest local spatial autocorrelation in total prevalence of i d patients is in eastern provinces such as khorasan razavi ( with 27,914 patient ) , south khorasan ( with 3,639 patient ) and semnan ( with 2,378 patient ) that contain % 3.43 of total ids patient 's prevalence in iran and its predominant local spatial cluster showed clearly a high - high pattern in comparison with their neighborhoods [ figure 5 ] . geoda local indicator of spatial association significant and cluster maps , moran 's scatter plot of prevalence of intellectual disabilities by county ( iran-2006 ) ( * pr total : total ids patient 's prevalence in iran ) besides , according to analyzes for local spatial auto - correlation that was done in geoda for other variables such as prevalence of i d patients in rural and urban areas , prevalence of ids men and women that are indicated in figures 6 - 9 ( their lisa significant map , lisa cluster map and moran scatter plot are shown in these figures too ) , the comparison between prevalence of ids patients in the urban region and national scale show random distribution ( lisa moran = 0.0119 ) [ figure 6 ] but analysis indicate that ids patient 's prevalence in a rural area in some counties have significant moran lisa values [ figure 7 ] ; for instance , isfahan , semnan , south khorasan , markazi and qom counties ( lisa moran = 0.194 ) , have significant positive local spatial auto - correlation . as showed in related map , all the counties located in the central region of the country with a high rate of i d 's patient prevalence , surrounded by other high rate areas ( high - high ) . geoda local indicator of spatial association significant and cluster maps , moran 's scatter plot of prevalence of intellectual disabilities in urban area by county ( iran-2006 ) ( * pr urban : ids patient 's prevalence in urban area ) geoda local indicator of spatial association significant and cluster maps , moran 's scatter plot of prevalence of intellectual disabilities in rural area by county ( iran-2006 ) ( * pr rural : ids patient 's prevalence in rural area ) geoda local indicator of spatial association significant and cluster maps , moran 's scatter plot of prevalence of male intellectual disabilities by county ( iran-2006 ) ( * pr maletot : total prevalence of male ids patients ) geoda local indicator of spatial association significant and cluster maps , moran 's scatter plot of prevalence of female intellectual disabilities by county ( iran-2006 ) ( * pr femtot : total prevalence of female ids patient 's ) but , the other important areas that can draw attention in geoda maps are also zones with different spatial distribution pattern when compared with neighboring areas . for instance , as the distribution maps for patients with i d has been shown in urban areas , a region with high prevalence of i d is located into low prevalence areas in the southern parts of iran ( high - low pattern ) . regarding the prevalence of i d patient as dependent variables and entering pn , hcc , immigration and illiteracy rate in the model as independent variables simultaneously , coefficience of determination ( r ) are calculated . based on r , the correlation coefficient ( r ) vary across county ( total r = 0.71 ) and r values fluctuate from 0.0002 to 0.94 for total independent variables [ table 3 ] . so , according to calculated r in this section which is depicted in the form of maps [ figure 3a ] , the highest correlation in southern parts of iran where especially southern part of fars and hormozgan province is evident . however , it seems that north west and west counties , including the provinces of west and east azarbaijan and ardebil , kurdistan and kermanshah , ilam and lorestan have the least correlation in this analysis . same from is shown in the figure 3 , all of our independent variable import to gwr analysis separately for finding the relationship between the four predicted variables and i d patient 's prevalence in the country and as explained in the map legend , different r value with special interpretations can be found in investigated area [ figure 3b - e ] . maps regarding gwr analysis between three independent factors and prevalence of rural i d patients indicate an overall weak relationship in the correlation , but exceptions are shown in eastern regions with relatively strong solidarity [ figure 4a ] . as has been stated in the table 4 , it appears that when the relationship between prevalence of i d patients in rural areas and independent variables are examined , the lowest correlation existed with the number of hcc in rural populations but in assessment of this predictor variable , some exceptions are demonstrated in some parts of north khorasan , razavi khorasan and south khorasan provinces and the southern border regions , areas that have a higher correlation coefficient . the only significant observed pattern of relationship is the correlation between illiteracy rates in rural areas that show different patterns in the country . however , the high values of this interface are in southern regions ( hormozgan , bushehr and southern areas of fars ) [ figure 4b - d ] . as mentioned in the introduction , more recent studies in asia state that the prevalence across this continent is estimated between 0.06% and 1.3% . according to our official data , mean prevalence of ids patients was obtained 0.45 as a total in iran and mean prevalence in a rural area has a significant difference comparing with urban regions . these results were consistent with the results of previous studies that predict prevalence in asia and in developing countries . difference between mean prevalence of male and female at nationwide , urban and rural area was statistically significant and finding were also according to other surveys [ table 1 ] . although many examples of population based studies of specific health status of i d are available , few studies about the spatial pattern of patients with i d have been examined . however , spatial distribution pattern of disease prevalence and incidence and recognition of the potential causes of occurrences of diseases such as socio - demographic factors and environmental exposures increase the need for new methods for analyzing health data ( such as patients with i d ) . so in these situations , the maps can be the final product of analysis and for example , where there are concerns about the etiology of i d in childhood , gis can be used as a tool for generating hypotheses , particularly in the areas of environmental epidemiology , etc . results from application of gis and doing spatial analysis by using geoda and gwr enabled us to be able to answer our research questions . our initial hypothesis about the spatial distribution pattern of i d patients in iran and its spatial discipline has been proven . the results of the analysis performed in the geoda software , the distribution pattern of iranian i d patients in the whole country and in rural areas and the general patterns of male and female distribution were shown as spatial clusters . but according to the calculated moran 's i in urban regions , spatial distribution that was observed , even by considering sex segregation , was random . local tests can determine spatial clustering and aggregation in the study area and they can also identify boundaries and regions with high or low clusters and region with lower or higher cases or rates than expected . in this study , such an analysis was performed by using lisa and from recognized clusters ; areas that were affected by neighboring and showed significantly higher or lower prevalence were found too . epidemiological studies assessed the effect of some socio - economic factors such as poverty and health inequalities on life conditions of people with i d and their families . these researches gave us this idea to check the association between some of these factors with prevalence of i d patients in our country . there are many studies that use the gwr technique to assess the correlation between different dependent and predictors variables , but in this domain and especially in our country , it is the first study that emphasizes i d prevalence and the effect of some variable on it . first , as mentioned before and in nationwide we entered four independent variables in the gwr method and calculated r between these predictor variables and total i d patient 's prevalence in our analysis . according to the classification in a valid biostatistician reference that divided ( correlation coefficient ) into five categories and gave equal values to each classes ( show in the map inset ) , all correlations showed high spatial relationship in this section , while some of these results were antithetical with our first hypothesis . we assumed that more pn and access to hccs should be associated with low i d patient prevalence and have inverse effects on i d patient 's frequencies . we construed that this result may be due to more facilities or sooner and better diagnose of these patients . more often , many families migrate to states suggested by health - care system as better communities for accessibility to facilities needed for their i d patients . furthermore , it is clear that people with low illiteracy degree know less about health threatening statuses , seek less for health - care services and often have lower socio - economic conditions . each of these items can serve as a powerful risk factor for increased prevalence of i d . in this survey and analysis , as we know , the logic of using geography for studying diseases or health - care is created from conception of factors that cause uneven distribution of diseases and this logic helps the researchers for recognition of existing spatial rules . accordingly , it can be concluded that the integration of medical and spatial geography creates a new medical vision that is infrastructure of the method of using spatial data for improving population health . there for communication between an individual health status and specific geographical factors can be a powerful device that able medical specialists to utilize them for health promotion and medical geography has these potency to show how changes in the location can effect on individual exposure with health threatening risk factors . in our study , we used geographical techniques to show the spatial distribution of i d patient 's prevalence and clustered pattern in iran and analyzed the relationship between some socio - demographic factors and these patients prevalence in our country to perceive whether there is any spatial autocorrelation between them . furthermore , we decided to discuss about these patients for inviting other researchers and policy makers to investigate in this field . we really believe that with appropriate planning and adoption of good policies , health - care systems can prevent and reduce the burden of people with this disability , provide more facilities for the specific need of these patients and their families and promote their quality of life .

background : although intellectual disability ( i d ) is a common disability in iran , there is no investigation on the spatial distribution pattern of these patients in national level and the spatial maps for recognition the areas with higher prevalence of ids and local neighborhoods of these regions or effect of socio - demographic factor on this scattering is not still available . this proposition motivated us to assess the population with i d in our country.methods:in a cross - sectional study , we applied moran 's index ( moran 's i ) which includes information about the strength of the neighboring association between counties , as global univariate distribution assessment . a geographically weighted regression was used to explore relation between i d patient 's prevalence and some socio - demographic factors ( migration and illiteracy rate , physician number ( pn)/10,000 people and health - care centers ( hccs)/10,000 people).results : we found that spatial clusters of i d patients exist among iran counties ( moran 's i = 0.36 , p < 0.01 ) and in a rural area population groups ( moran 's i = 0.20 , p < 0.01 ) . further , we detected spatial associations between i d patients and all of our investigated socio - demographic factors in national scale . in rural areas , illiteracy has high association with i d especially in the south region of iran . urban area has random pattern of i d patients both within and between the iran counties ( moran 's i = 0.01 , p > 0.3).conclusions : according to the results , our initial hypothesis about the existence of spatial clusters in distribution of people with i d in iran was proven . spatial autocorrelation between migration and illiteracy rate and prevalence of patients with i d was shown and was in agreement with our hypothesis . however , our supposition that the prevalence should have inverse relationship with pn and hcc was rejected .

INTRODUCTION METHODS The data The map The analysis stage Explore on the spatial distribution of the prevalence of ID patients Spatial distribution assessment GWR RESULTS DISCUSSION CONCLUSIONS

the medical and socio - economic burden of raising people with intellectual disability ( i d ) is a complex issue all over the world . since the majority of these studies have been conducted in other parts of the world , the most recently investigation that was done in south - east asia estimated the prevalence of i d across this continent at 0.06 - 1.3% except in china ( i d 's prevalence : 6.68% ) , although another survey that was carried out in that country did not confirm that estimation ( prevalence of about 0.75% so considering their affected population , ids in brief are important problems of public health because of problems those are associated with this condition such as : unexplained causes in nearly half of the cases and sometimes their impossibility to prevent , high prevalence in most communities and losing productive years of life , early onset of most of these disabilities in childhood and long - term consequences of failures throughout life and etc . as noted before , although several studies have been performed in patients with ids and different techniques have been applied for evaluation of their related condition in asia and especially china ; limited evidence exists for using modern techniques of spatial analysis such as geographically weighted regression ( gwr ) on data related to these patients , especially in iran . hence , after preliminary statistical analysis and discovering the spatial distribution pattern of these patients , as an initial hypothesis , this assumption was also raised that the spatial pattern of patients with ids maybe associated with some socio - demographic factors such as illiteracy , migration , physician number ( pn ) and number of health - care centers ( hccs ) in the study region . illiteracy as an indicator of the tendency to receive preventive health - care services ; migration , element for seeking further therapeutic and rehabilitation services by these patient 's families ; pn and hccs as the measures for health - care delivery for prevention and thus decreasing the number of these patients were considered and for demonstrating these relationships , the spatial exploring analysis was employed in this work . according to official statistics , this is the 1 time ; the data of these patients in the whole country were available for investigation and the required information were accessible on the official site of the statistical center of iran ( available from : http://www.amar.org.ir ) . based on the available frequency of patients and census of the general population in 2006 , the overall prevalence of patients with i d in accordance with the cities and provinces and the calculated values according to sex and rural and urban areas was computed [ tables 1 and 2 ] . since the prevalence of i d can be related to items such as illiteracy , immigration , the pn and the number of hccs particularly in rural areas ( providing services before and during pregnancy for prevention and help for early diagnosis ) , necessary information to evaluate the effect of these variables on the prevalence of i d were used from data of immigration and illiteracy rates listed in the statistical center of iran and information inserted in the national statistical yearbook 2007 ( available from : http://salnameh.sci.org.ir ) . the prevalence of i d patients tables in urban and rural area were jointed to the table of counties map and finally attribute tables were prepared to perform spatial analysis . for estimation of spatial distribution of prevalence of i d patients in the counties , the croplet maps of spatial distribution of their total prevalence , prevalence of men and women and i d patient prevalence in rural and urban areas were provided [ figure 1 ] . after reviewing the plans , in order to obtain the patterns of spatial distribution and follow - up of prevalence of i d patients from clustering , random or disperse patterns in various provinces and cities , each province was delineated separately and then exploratory spatial data analysis ( esda ) on the scattering pattern of i d patient 's prevalence was done . spatial distribution pattern of prevalence of patient with i d ( iran-2006 ) at this stage , esda of prevalence of i d patients and their sex segregation , ids patient 's prevalence in urban and rural areas were mapped . two indicators helped us to interpret the result of this phase : global spatial autocorrelation coefficient or moran 's index spatial autocorrelation statistic was used to measure the correlation among neighboring observations in a pattern and the levels of spatial clustering among neighboring were districted . moran 's i as a device was used in esda for exploring the general model of spatial autocorrelation in the study area and the overall spatial distribution pattern of variable on the map including disperses , random or cluster could be identified as showed in figure 2 . in order to evaluate the effect of predictor variables ( migration and illiteracy rate , hccs and pn ) and the impact of them on the prevalence of i d patients in iran , these independent variables were entered into the model simultaneously [ figure 3 ] . separately , the effect of each independent variable on the prevalence of i d patients with gwr was studied and coefficience of determination ( r ) , adjusted r and akaike information criterion ( aicc ) in these cases were recorded in tables [ table 3 ] . geographic weighted regression analysis of prevalence of intellectual disabilities ( dependent variable ) and migration , illiteracy , health - care centers per 104 people and physicians number per 104 people ( predictor variables ) by county ( iran 2006 ) aicc , r and r adjusted of figure 3 calculated by gwr ( arc gis 9.3 software ) based on our access to hcc , pn and illiteracy data in rural areas and the higher prevalence of i d in these areas , regression modeling for these three independent variables and the prevalence of i d patients as dependent variable in rural areas was also performed and the values obtained from the gwr are included in table 4 . aicc , r and r adjusted of figure 4 calculated by gwr ( arc gis 9.3 software ) geographic weighted regression analysis of prevalence of intellectual disabilities in rural area ( dependent variable ) and illiteracy , health - care centers per 104 people and physicians number per 104 people ( predictor variables ) in rural area by county ( iran 2006 ) for access to data of patients with i d , the statistics recorded by the iranian population and housing census ( 2006 ) were used . according to official statistics , this is the 1 time ; the data of these patients in the whole country were available for investigation and the required information were accessible on the official site of the statistical center of iran ( available from : http://www.amar.org.ir ) . based on the available frequency of patients and census of the general population in 2006 , the overall prevalence of patients with i d in accordance with the cities and provinces and the calculated values according to sex and rural and urban areas was computed [ tables 1 and 2 ] . since the prevalence of i d can be related to items such as illiteracy , immigration , the pn and the number of hccs particularly in rural areas ( providing services before and during pregnancy for prevention and help for early diagnosis ) , necessary information to evaluate the effect of these variables on the prevalence of i d were used from data of immigration and illiteracy rates listed in the statistical center of iran and information inserted in the national statistical yearbook 2007 ( available from : http://salnameh.sci.org.ir ) . the prevalence of i d patients tables in urban and rural area were jointed to the table of counties map and finally attribute tables were prepared to perform spatial analysis . for estimation of spatial distribution of prevalence of i d patients in the counties , the croplet maps of spatial distribution of their total prevalence , prevalence of men and women and i d patient prevalence in rural and urban areas were provided [ figure 1 ] . after reviewing the plans , in order to obtain the patterns of spatial distribution and follow - up of prevalence of i d patients from clustering , random or disperse patterns in various provinces and cities , each province was delineated separately and then exploratory spatial data analysis ( esda ) on the scattering pattern of i d patient 's prevalence was done . spatial distribution pattern of prevalence of patient with i d ( iran-2006 ) at this stage , esda of prevalence of i d patients and their sex segregation , ids patient 's prevalence in urban and rural areas were mapped . two indicators helped us to interpret the result of this phase : global spatial autocorrelation coefficient or moran 's index spatial autocorrelation statistic was used to measure the correlation among neighboring observations in a pattern and the levels of spatial clustering among neighboring were districted . moran 's i as a device was used in esda for exploring the general model of spatial autocorrelation in the study area and the overall spatial distribution pattern of variable on the map including disperses , random or cluster could be identified as showed in figure 2 . in order to evaluate the effect of predictor variables ( migration and illiteracy rate , hccs and pn ) and the impact of them on the prevalence of i d patients in iran , these independent variables were entered into the model simultaneously [ figure 3 ] . the spatial correlation of these four variables was assessed and the spatial correlation coefficient of prevalence of i d patients was found . separately , the effect of each independent variable on the prevalence of i d patients with gwr was studied and coefficience of determination ( r ) , adjusted r and akaike information criterion ( aicc ) in these cases were recorded in tables [ table 3 ] . geographic weighted regression analysis of prevalence of intellectual disabilities ( dependent variable ) and migration , illiteracy , health - care centers per 104 people and physicians number per 104 people ( predictor variables ) by county ( iran 2006 ) aicc , r and r adjusted of figure 3 calculated by gwr ( arc gis 9.3 software ) based on our access to hcc , pn and illiteracy data in rural areas and the higher prevalence of i d in these areas , regression modeling for these three independent variables and the prevalence of i d patients as dependent variable in rural areas was also performed and the values obtained from the gwr are included in table 4 . aicc , r and r adjusted of figure 4 calculated by gwr ( arc gis 9.3 software ) geographic weighted regression analysis of prevalence of intellectual disabilities in rural area ( dependent variable ) and illiteracy , health - care centers per 104 people and physicians number per 104 people ( predictor variables ) in rural area by county ( iran 2006 ) for estimation of spatial distribution of prevalence of i d patients in the counties , the croplet maps of spatial distribution of their total prevalence , prevalence of men and women and i d patient prevalence in rural and urban areas were provided [ figure 1 ] . after reviewing the plans , in order to obtain the patterns of spatial distribution and follow - up of prevalence of i d patients from clustering , random or disperse patterns in various provinces and cities , each province was delineated separately and then exploratory spatial data analysis ( esda ) on the scattering pattern of i d patient 's prevalence was done . spatial distribution pattern of prevalence of patient with i d ( iran-2006 ) at this stage , esda of prevalence of i d patients and their sex segregation , ids patient 's prevalence in urban and rural areas were mapped . two indicators helped us to interpret the result of this phase : global spatial autocorrelation coefficient or moran 's index spatial autocorrelation statistic was used to measure the correlation among neighboring observations in a pattern and the levels of spatial clustering among neighboring were districted . moran 's i as a device was used in esda for exploring the general model of spatial autocorrelation in the study area and the overall spatial distribution pattern of variable on the map including disperses , random or cluster could be identified as showed in figure 2 . in order to evaluate the effect of predictor variables ( migration and illiteracy rate , hccs and pn ) and the impact of them on the prevalence of i d patients in iran , these independent variables were entered into the model simultaneously [ figure 3 ] . the spatial correlation of these four variables was assessed and the spatial correlation coefficient of prevalence of i d patients was found . separately , the effect of each independent variable on the prevalence of i d patients with gwr was studied and coefficience of determination ( r ) , adjusted r and akaike information criterion ( aicc ) in these cases were recorded in tables [ table 3 ] . geographic weighted regression analysis of prevalence of intellectual disabilities ( dependent variable ) and migration , illiteracy , health - care centers per 104 people and physicians number per 104 people ( predictor variables ) by county ( iran 2006 ) aicc , r and r adjusted of figure 3 calculated by gwr ( arc gis 9.3 software ) based on our access to hcc , pn and illiteracy data in rural areas and the higher prevalence of i d in these areas , regression modeling for these three independent variables and the prevalence of i d patients as dependent variable in rural areas was also performed and the values obtained from the gwr are included in table 4 . aicc , r and r adjusted of figure 4 calculated by gwr ( arc gis 9.3 software ) geographic weighted regression analysis of prevalence of intellectual disabilities in rural area ( dependent variable ) and illiteracy , health - care centers per 104 people and physicians number per 104 people ( predictor variables ) in rural area by county ( iran 2006 ) considering calculated moran 's i for determination the spatial distribution patterns of prevalence of i d patients [ table 5 ] , clustering model was found in total , men and women distribution of i d patients ( moran 's i = 0.36 ) and this pattern repeated in rural areas too ( moran 's i = 0.01 ) . according to moran 's i in the urban region , prevalence of i d patient in these areas followed random model ( moran 's i = 0.01 ) and p values confirmed the existence of these spatial distribution ( p = 0.38 ) . calculated moran 's i and determination of spatial pattern of prevalence of i d patients by geoda and arc gis software ( iran 2006 ) lisa showed that the highest local spatial autocorrelation in total prevalence of i d patients is in eastern provinces such as khorasan razavi ( with 27,914 patient ) , south khorasan ( with 3,639 patient ) and semnan ( with 2,378 patient ) that contain % 3.43 of total ids patient 's prevalence in iran and its predominant local spatial cluster showed clearly a high - high pattern in comparison with their neighborhoods [ figure 5 ] . geoda local indicator of spatial association significant and cluster maps , moran 's scatter plot of prevalence of intellectual disabilities by county ( iran-2006 ) ( * pr total : total ids patient 's prevalence in iran ) besides , according to analyzes for local spatial auto - correlation that was done in geoda for other variables such as prevalence of i d patients in rural and urban areas , prevalence of ids men and women that are indicated in figures 6 - 9 ( their lisa significant map , lisa cluster map and moran scatter plot are shown in these figures too ) , the comparison between prevalence of ids patients in the urban region and national scale show random distribution ( lisa moran = 0.0119 ) [ figure 6 ] but analysis indicate that ids patient 's prevalence in a rural area in some counties have significant moran lisa values [ figure 7 ] ; for instance , isfahan , semnan , south khorasan , markazi and qom counties ( lisa moran = 0.194 ) , have significant positive local spatial auto - correlation . geoda local indicator of spatial association significant and cluster maps , moran 's scatter plot of prevalence of intellectual disabilities in urban area by county ( iran-2006 ) ( * pr urban : ids patient 's prevalence in urban area ) geoda local indicator of spatial association significant and cluster maps , moran 's scatter plot of prevalence of intellectual disabilities in rural area by county ( iran-2006 ) ( * pr rural : ids patient 's prevalence in rural area ) geoda local indicator of spatial association significant and cluster maps , moran 's scatter plot of prevalence of male intellectual disabilities by county ( iran-2006 ) ( * pr maletot : total prevalence of male ids patients ) geoda local indicator of spatial association significant and cluster maps , moran 's scatter plot of prevalence of female intellectual disabilities by county ( iran-2006 ) ( * pr femtot : total prevalence of female ids patient 's ) but , the other important areas that can draw attention in geoda maps are also zones with different spatial distribution pattern when compared with neighboring areas . for instance , as the distribution maps for patients with i d has been shown in urban areas , a region with high prevalence of i d is located into low prevalence areas in the southern parts of iran ( high - low pattern ) . regarding the prevalence of i d patient as dependent variables and entering pn , hcc , immigration and illiteracy rate in the model as independent variables simultaneously , coefficience of determination ( r ) are calculated . same from is shown in the figure 3 , all of our independent variable import to gwr analysis separately for finding the relationship between the four predicted variables and i d patient 's prevalence in the country and as explained in the map legend , different r value with special interpretations can be found in investigated area [ figure 3b - e ] . as has been stated in the table 4 , it appears that when the relationship between prevalence of i d patients in rural areas and independent variables are examined , the lowest correlation existed with the number of hcc in rural populations but in assessment of this predictor variable , some exceptions are demonstrated in some parts of north khorasan , razavi khorasan and south khorasan provinces and the southern border regions , areas that have a higher correlation coefficient . according to our official data , mean prevalence of ids patients was obtained 0.45 as a total in iran and mean prevalence in a rural area has a significant difference comparing with urban regions . although many examples of population based studies of specific health status of i d are available , few studies about the spatial pattern of patients with i d have been examined . however , spatial distribution pattern of disease prevalence and incidence and recognition of the potential causes of occurrences of diseases such as socio - demographic factors and environmental exposures increase the need for new methods for analyzing health data ( such as patients with i d ) . our initial hypothesis about the spatial distribution pattern of i d patients in iran and its spatial discipline has been proven . the results of the analysis performed in the geoda software , the distribution pattern of iranian i d patients in the whole country and in rural areas and the general patterns of male and female distribution were shown as spatial clusters . epidemiological studies assessed the effect of some socio - economic factors such as poverty and health inequalities on life conditions of people with i d and their families . these researches gave us this idea to check the association between some of these factors with prevalence of i d patients in our country . there are many studies that use the gwr technique to assess the correlation between different dependent and predictors variables , but in this domain and especially in our country , it is the first study that emphasizes i d prevalence and the effect of some variable on it . first , as mentioned before and in nationwide we entered four independent variables in the gwr method and calculated r between these predictor variables and total i d patient 's prevalence in our analysis . in our study , we used geographical techniques to show the spatial distribution of i d patient 's prevalence and clustered pattern in iran and analyzed the relationship between some socio - demographic factors and these patients prevalence in our country to perceive whether there is any spatial autocorrelation between them . we really believe that with appropriate planning and adoption of good policies , health - care systems can prevent and reduce the burden of people with this disability , provide more facilities for the specific need of these patients and their families and promote their quality of life .

for instance , exhaustive search becomes unreasonable as the number of variables increases ; employing a multiple regression search produces over one billion possible models for data with 30 explanatory variables . in ecological studies , one of the commonly used methods for selection is stepwise regression , with forward or backward variable selection algorithms . these methods have been criticized for lacking the ability to truly pick the best model for several reasons ( boyce et al . , 1974 ; wilkinson , 1989 ) . one problem is that the choice by which the variables enter the selection algorithm is not justified theoretically . in addition , the probabilities for the selection procedure are chosen arbitrarily , which may lead to a poorly selected model . since these methods employ local search , it is unlikely that the global maximum set of variables will be found ( mantel , 1970 ; hocking , 1976 , 1983 ; moses , 1986 ) . we propose the use of genetic algorithms ( gas ) to determine the subset of variables with the highest goodness of fit for a multiple regression model . due to their global search capabilities , the ga based model building is not prone to the problems associated with local search method , hence is a wise choice for this procedure . we now explain the basics of gas briefly ; a thorough one can be found in goldberg ( 1989 ) . genetic algorithms are a set of optimization techniques inspired by biological evolution , operating under natural selection . first developed by holland ( 1975 ) , they have grown in popularity because of the ability of the algorithm to perform well on many different types of problems . in a ga , possible solutions are coded using binary strings , which are called chromosomes . each chromosome has a fitness value associated with it based on how well the string model parameters predicts the dependent variables . during each generation , which is the time step of the algorithm , a population of chromosomes compete to have their genes the selection step is used to pick the chromosomes for the next generation based on their fitness . those selected enter the mating pool , where two chromosomes mate using crossover . during this phase , parts of each parent string are swapped to form two new chromosomes that have certain aspects of their parents . mutation occurs with a small probability and is defined by a change from 0 to 1 or 1 to 0 in the binary string . mutation allows the introduction of new genes that were either lost from the population or were not there to start with . through successive generations , increasingly better chromosomes come to dominate the population , and the optimal solution ( or something very close ) is realized . a key component of a ga is the method to evaluate the fitness of a chromosome . thus , in order to use a ga for model selection in multiple regression , a way to evaluate the chromosomes is needed . more specifically the fittest chromosome is the set of parameters that maximizes the explanatory power of the model with minimum number of parameters . bozdogan ( 1988 , 2004 ) considered complexity as a measure of fitness , which can be described as follows : the complexity of a system ( of any type ) is a measure of the degree of interdependency between the whole system and a simple enumerative composition of its subsystems or parts . the concept of information complexity was first introduced by akaike ( 1973 ) as a measure of the complexity of a model : it is a relative measure of the information lost when a given model is used , and can be described as a function of the precision and accuracy of the model . the expression for aic is given as where l(k ) denotes the maximum likelihood function , ^k is the maximum likelihood estimate of parameter vector k , and m(k ) is the number of parameters in the model . the first term of aic gives the lack of fit of the model , and the second term is a penalty for the number of parameters in the model . the model with the lowest aic value is considered the best , because the model successfully determines the underlying stochastic process with the least number of parameters . although aic does take into account the problem of over - fitting , where other measures such as r - square do not , aic is not sensitive to parameter dependency , which is an important component for model selection . if a model with both low variance and low covariance can be produced , then the parameters can be better estimated , as they will not be correlated . as an alternative to aic , we consider icomp as a complexity measure which considers variance and covariance , and accounts for the problem of over - fitting the model . it is calculated by where l(k ) again denotes the maximum likelihood function , ^k is the maximum likelihood estimate of parameter vector k under the model mk , c is a real - valued complexity measure , and ^model is the estimated covariance matrix of the parameters of the model . the main difference between the two measures of complexity is that aic only considers the number of parameters as a penalty , whereas icomp considers the covariance between parameters . in predictive model building , this value for icomp is based on the inverse - fisher information matrix ( ifim ) . for multiple regression , the value of icomp can be directly calculated after regression is implemented , and is given by n is the number of parameters in the model , q is the number of observations , ^2=sse / n , tr(^2(xx)1 ) is the trace of the observation matrix multiplied by its inverse and then scaled by ^2 , and |^2(xx)1| is the determinant of the previous matrix . since the model with the lowest icomp value is considered the best , the ga chooses strings biased toward those with the lowest value . a commonly used method to form the mating pool is proportional selection , which depends on selecting strings for the mating pool with a probability proportional to their fitnesses . in proportional selection , the first step of the calculation of the fitness values is subtracting the icomp value of each string in that generation from the maximum value of icomp in the population . that is , for each i = 1,2, ,n , where n is the size of the population . then the average icomp difference ( the average fitness ) for the total population is calculated as finally , each string is given a fitness value that is the ratio of its icomp difference and the average fitness of the population : here we consider the implementation of ga 's for predictive model selection and discuss possible improvements . the first step to implementing a ga for any optimization problem is to encode the input variable into binary strings . in the case of multiple linear regression we wish to fit the data to where y is an n 1 response vector , x is an n q matrix of the data points , is a q 1 coefficient matrix , and is an n 1 error vector with entries from independent normal distributions [ n(0 , ) for all components ] . the encoding is done by creating a binary string which has n + 1 bits , where each bit represents a different parameter of the model and an intercept . the last n bits correspond to the n explanatory variables contained in the dataset , whereas the first bit is the intercept for the linear model . a parameter is included in the model if the value of the bit for that parameter is a 1 and is excluded if it is a 0 . for example , suppose we have a dataset where we are interested in predicting the reproductive fitness of a species of trees . the possible explanatory variables may include : density of trees in the surrounding area , average temperature of environment , average rainfall of environment , circumference of trunk , longitude of environment , latitude of environment , prevalence of disease in environment . in this case for example , the string 10010111101 would represent a model which includes the intercept , soil ph , average temperature of environment , average rainfall of environment , circumference of trunk , longitude of environment , and prevalence of disease in environment . similarly , the string 00001000110 is a model that has no intercept , and includes density of trees in the surrounding area , longitude of environment , and latitude of environment ( see table 1 ) . the probability that a string will be chosen for the mating pool is proportional to its fitness value . note that the string with the worst icomp value will never be picked for the mating pool , as its fitness will be 0 . now that we have a method of encoding information and a method to evaluate the fitness values , we have to determine the remaining parameters of the ga . the first one we consider is the method of creating the initial population and determining its size . unless previous knowledge about the problem is given , it is commonplace in gas to randomly generate binary strings ( goldberg , 1989 ) . however , in the case of model selection , a user may want to force a parameter(s ) to be included , even if it is not part of the model with the lowest complexity . in this case , the initial population can be generated in such a way that certain parameters are always in the model . in addition to determining the method to generate the population , the user must choose the size of the initial population . generally the size should not be too large , as this will slow the algorithm , and should not be so small that genetic drift takes over the course of evolution of the population . in typical gas , the size of the population stays the same ; however , this may not be an effective use of computation . we will see in the next section that starting with a larger size then reducing it may be more effective . finally , we discuss the genetic operators which allow the algorithm to find the optimal model . first , the probability of crossover , pc , is chosen . in the mating pool , a pair of strings are chosen along with a random number from [ 0 , 1 ] . if that number is less than the probability of crossover , crossover occurs . thus , if pc = 1 , then every pair will cross , and if pc = 0 , then no strings will be altered by crossover . after the choice of pc , the number of crossover points must be chosen . then the bits from the parent strings are swapped to create two new offspring strings ( see figure 1 ) . the purpose of crossover is to bring together models which have components that reduce complexity . recall the previous example about trees , where we specified two strings , which we will call parent 1 and parent 2 . applying crossover to the two parents creates two offspring ( see figure 1 ) , where offspring 1 represents a model with an intercept , soil ph , average temperature of environment , longitude of environment , latitude of environment , and prevalence of disease in environment , and offspring 2 represents a model that includes density of trees in the surrounding area , average rainfall of environment , circumference of trunk , and longitude of environment . through successive generations and application of crossover of low complexity models , the algorithm is able to find the least complex model ( or something close to it ) to explain the data . but , what happens if the actual least complex model includes a parameter that is not present in the population , that is , the position in the string that represents the parameter is fixed at 0 ? this value gives the probability that at each location in the string the bit will be flipped . flipping is defined as a change of a 0 to 1 or a 1 to a 0 . however , strings used for other applications of ga 's are usually longer than the ones used for determining least complex models . although there are ongoing studies on determining optimal crossover and mutation rates ( such as nested gas , self - adjusting parameterless gas ) , these rates can be determined by trial and error or by pilot runs before the actual data set has been used to build a model . we conclude this section with a pseudo code for a ga used to find the least complex model that sufficiently describes the data . generate initial population while ( t < max generations or the maximum number of computations have not been executed ) ( a)calculate icomp for the model each string encodes ( b)select strings for the mating pool ( c)create a new population using crossover ( d)mutate new population while the use of a typical ga for model selection already proves to be more efficient than stepwise regression , with a few modifications , the process can show a 10-fold increase in accuracy given the same amount of computation . first , we discuss the modifications , and then we explain the study done to determine the effectiveness of these modifications . the first modification is changing how the initial population was created . according to fisher 's fundamental theorem of natural selection ( fisher , 1930 ) , the increase in mean fitness is equal to the variance in fitness . for model selection using gas , the easiest way to increase variance in fitness would be to allow every model to be represented in the population . of course , this is impossible for a model with a large number of possible explanatory variables , and would amount to doing an exhaustive search . we believe that the next best procedure is to force the population to start with the highest variance in each position of the chromosome . since each position is either a 0 or 1 , this would imply that at each position there are the same amount of 0 s and 1 s across the entire population . to implement this procedure , the other half is generated by taking each of the chromosomes in the first half and changing each bit from 1 to 0 or 0 to 1 . in addition to increasing variance at each position , this procedure guarantees that within one generation , recombination alone could generate the best model . this does not imply that mutation is not necessary , as selection acts on the entire string , not individual positions . since selection will reduce variance at each position , mutation is still required to maintain some variance . the second modification is starting with a larger initial population and then reducing it in size . we have used a reduction method that adapts to the changes in the algorithm in this study . adaptively reducing the population is done by calculating the change in the best fitness between two consecutive generations and then reducing the population based on this change . more specifically , the population is reduced by the percentage increase in best fitness up to some limit . clearly , there must be a limit to the percentage of reduction , since the population should not be reduced too much , and also because the percent change can be more than 100 . here the amount of population reduction depends on the complexity of the problem , that is the type of the fitness function ( such as mse , aic , icom , mallow 's cp and so on ) used . this limit on the reduction may be determined by pilot studies . the percent change in fitness at generation t the change is calculated by the formula ftbest=|ft1bestft2best|/|ft2best| . the population size n(t ) at each generation is given by the recursive relation when using the adaptive method , elitism was also implemented . elitism is a procedure commonly used in gas in order to pass the best chromosome , or a group of the best chromosomes , to the next generation without any modifications . using elitism guarantees that the change in best fitness is always non - negative . as a result , since we wished to minimize icomp , we set the fitness of each chromosome to be the negative of the icomp value . the choices of these parameters should be done by considering characteristics of the problem such as the expected increase in fitness over time . this is typically a difficult characteristic to determine . generally , as the number of variables increase , the value of fmaxbest should decrease . as the number of variables increases , so does the number of possible values of icomp , and the likelihood that the population will evolve slower . the value of min_popsize should be chosen so that it is quite small ( 5 ) , regardless of the number of variables . as a side note , the ga with no population reduction is a special case of the adaptive method where fmaxbest=0 . the final modification to a ga for multiple regressions is the use of binary tournament instead of proportional selection . in this selection scheme , two chromosomes are chosen at random , and the one with the lower icomp value is selected for the mating pool . then both chromosomes are put back into the pool of contestants of the tournament . one advantage of this technique is that icomp values need only be calculated for the chromosomes that participate in the tournament . for models with few explanatory variables , this gain in computation may be negligible . on the other hand , for those models with many variables , the reduction in computation means that more generations can be used , or the initial population can be larger . when the population is being reduced , genetic drift may be amplified , since the sampling space for the next generation decreases . proportional selection may increase this effect because a few chromosomes with extremely high fitness are expected to be picked often for the mating pool . however , selection to participate in the tournament is random , avoiding the over - selection of chromosomes with extremely large fitness values . to test the benefits of these modifications , we used the data set in bozdogan ( 2004 ) where the predictive model is constructed for body fat and 13 explanatory variables . in order to determine how well the ga was performing , all subsets of the variables ( 2 1 = 16,383 subsets ) were used to generate a model , and then the icomp value was determined . testing was done to ensure the same icomp values were being generated for the matlab and java code . these cases differed in the value of fmaxbest , and as a result in the initial population size . all trials were allowed 600 computations , where a computation is the total number of chromosomes summed over every generation . each different ga scheme ran through 200 trials and the number of times the correct model was selected was recorded . parameters that were the same for all genetic algorithm schemes . the frequency of the correct model being selected over 200 trials . the first step to implementing a ga for any optimization problem is to encode the input variable into binary strings . in the case of multiple linear regression we wish to fit the data to where y is an n 1 response vector , x is an n q matrix of the data points , is a q 1 coefficient matrix , and is an n 1 error vector with entries from independent normal distributions [ n(0 , ) for all components ] . the encoding is done by creating a binary string which has n + 1 bits , where each bit represents a different parameter of the model and an intercept . the last n bits correspond to the n explanatory variables contained in the dataset , whereas the first bit is the intercept for the linear model . a parameter is included in the model if the value of the bit for that parameter is a 1 and is excluded if it is a 0 . for example , suppose we have a dataset where we are interested in predicting the reproductive fitness of a species of trees . the possible explanatory variables may include : density of trees in the surrounding area , average temperature of environment , average rainfall of environment , circumference of trunk , longitude of environment , latitude of environment , prevalence of disease in environment . in this case for example , the string 10010111101 would represent a model which includes the intercept , soil ph , average temperature of environment , average rainfall of environment , circumference of trunk , longitude of environment , and prevalence of disease in environment . similarly , the string 00001000110 is a model that has no intercept , and includes density of trees in the surrounding area , longitude of environment , and latitude of environment ( see table 1 ) . the probability that a string will be chosen for the mating pool is proportional to its fitness value . note that the string with the worst icomp value will never be picked for the mating pool , as its fitness will be 0 . now that we have a method of encoding information and a method to evaluate the fitness values , we have to determine the remaining parameters of the ga . the first one we consider is the method of creating the initial population and determining its size . unless previous knowledge about the problem is given , it is commonplace in gas to randomly generate binary strings ( goldberg , 1989 ) . however , in the case of model selection , a user may want to force a parameter(s ) to be included , even if it is not part of the model with the lowest complexity . in this case , the initial population can be generated in such a way that certain parameters are always in the model . in addition to determining the method to generate the population , the user must choose the size of the initial population . generally the size should not be too large , as this will slow the algorithm , and should not be so small that genetic drift takes over the course of evolution of the population . in typical gas , the size of the population stays the same ; however , this may not be an effective use of computation . we will see in the next section that starting with a larger size then reducing it may be more effective . finally , we discuss the genetic operators which allow the algorithm to find the optimal model . first , the probability of crossover , pc , is chosen . in the mating pool , a pair of strings are chosen along with a random number from [ 0 , 1 ] . thus , if pc = 1 , then every pair will cross , and if pc = 0 , then no strings will be altered by crossover . after the choice of pc , the number of crossover points must be chosen . then the bits from the parent strings are swapped to create two new offspring strings ( see figure 1 ) . the purpose of crossover is to bring together models which have components that reduce complexity . recall the previous example about trees , where we specified two strings , which we will call parent 1 and parent 2 . applying crossover to the two parents creates two offspring ( see figure 1 ) , where offspring 1 represents a model with an intercept , soil ph , average temperature of environment , longitude of environment , latitude of environment , and prevalence of disease in environment , and offspring 2 represents a model that includes density of trees in the surrounding area , average rainfall of environment , circumference of trunk , and longitude of environment . through successive generations and application of crossover of low complexity models , the algorithm is able to find the least complex model ( or something close to it ) to explain the data . but , what happens if the actual least complex model includes a parameter that is not present in the population , that is , the position in the string that represents the parameter is fixed at 0 ? this value gives the probability that at each location in the string the bit will be flipped . flipping is defined as a change of a 0 to 1 or a 1 to a 0 . however , strings used for other applications of ga 's are usually longer than the ones used for determining least complex models . although there are ongoing studies on determining optimal crossover and mutation rates ( such as nested gas , self - adjusting parameterless gas ) , these rates can be determined by trial and error or by pilot runs before the actual data set has been used to build a model . we conclude this section with a pseudo code for a ga used to find the least complex model that sufficiently describes the data . generate initial population while ( t < max generations or the maximum number of computations have not been executed ) ( a)calculate icomp for the model each string encodes ( b)select strings for the mating pool ( c)create a new population using crossover ( d)mutate new population while the use of a typical ga for model selection already proves to be more efficient than stepwise regression , with a few modifications , the process can show a 10-fold increase in accuracy given the same amount of computation . first , we discuss the modifications , and then we explain the study done to determine the effectiveness of these modifications . the first modification is changing how the initial population was created . according to fisher 's fundamental theorem of natural selection ( fisher , 1930 ) , the increase in mean fitness is equal to the variance in fitness . for model selection using gas , the easiest way to increase variance in fitness would be to allow every model to be represented in the population . of course , this is impossible for a model with a large number of possible explanatory variables , and would amount to doing an exhaustive search . we believe that the next best procedure is to force the population to start with the highest variance in each position of the chromosome . since each position is either a 0 or 1 , this would imply that at each position there are the same amount of 0 s and 1 s across the entire population . to implement this procedure , the other half is generated by taking each of the chromosomes in the first half and changing each bit from 1 to 0 or 0 to 1 . in addition to increasing variance at each position , this procedure guarantees that within one generation , recombination alone could generate the best model . this does not imply that mutation is not necessary , as selection acts on the entire string , not individual positions . since selection will reduce variance at each position , mutation is still required to maintain some variance . the second modification is starting with a larger initial population and then reducing it in size . we have used a reduction method that adapts to the changes in the algorithm in this study . adaptively reducing the population is done by calculating the change in the best fitness between two consecutive generations and then reducing the population based on this change . more specifically , the population is reduced by the percentage increase in best fitness up to some limit . clearly , there must be a limit to the percentage of reduction , since the population should not be reduced too much , and also because the percent change can be more than 100 . here the amount of population reduction depends on the complexity of the problem , that is the type of the fitness function ( such as mse , aic , icom , mallow 's cp and so on ) used . this limit on the reduction may be determined by pilot studies . the percent change in fitness at generation t is denoted by ftbest and the limit is denoted by fmaxbest . the population size n(t ) at each generation is given by the recursive relation when using the adaptive method , elitism was also implemented . elitism is a procedure commonly used in gas in order to pass the best chromosome , or a group of the best chromosomes , to the next generation without any modifications . using elitism guarantees that the change in best fitness is always non - negative . as a result , the population never increases in size . since we wished to minimize icomp , we set the fitness of each chromosome to be the negative of the icomp value the choices of these parameters should be done by considering characteristics of the problem such as the expected increase in fitness over time . this is typically a difficult characteristic to determine . generally , as the number of variables increase , the value of fmaxbest should decrease . as the number of variables increases , so does the number of possible values of icomp , and the likelihood that the population will evolve slower . the value of min_popsize should be chosen so that it is quite small ( 5 ) , regardless of the number of variables . as a side note , the ga with no population reduction is a special case of the adaptive method where fmaxbest=0 . the final modification to a ga for multiple regressions is the use of binary tournament instead of proportional selection . in this selection scheme , two chromosomes are chosen at random , and the one with the lower icomp value is selected for the mating pool . then both chromosomes are put back into the pool of contestants of the tournament . one advantage of this technique is that icomp values need only be calculated for the chromosomes that participate in the tournament . for models with few explanatory variables , this gain in computation may be negligible . on the other hand , for those models with many variables , the reduction in computation means that more generations can be used , or the initial population can be larger . when the population is being reduced , genetic drift may be amplified , since the sampling space for the next generation decreases . proportional selection may increase this effect because a few chromosomes with extremely high fitness are expected to be picked often for the mating pool . however , selection to participate in the tournament is random , avoiding the over - selection of chromosomes with extremely large fitness values . to test the benefits of these modifications , we used the data set in bozdogan ( 2004 ) where the predictive model is constructed for body fat and 13 explanatory variables . in order to determine how well the ga was performing , all subsets of the variables ( 2 1 = 16,383 subsets ) were used to generate a model , and then the icomp value was determined . testing was done to ensure the same icomp values were being generated for the matlab and java code . these cases differed in the value of fmaxbest , and as a result in the initial population size . all trials were allowed 600 computations , where a computation is the total number of chromosomes summed over every generation . each different ga scheme ran through 200 trials and the number of times the correct model was selected was recorded . parameters that were the same for all genetic algorithm schemes . the frequency of the correct model being selected over 200 trials . while model selection remains to be a difficult procedure in case of a large number of parameters , using a ga to find the least complex model can be quite helpful . we have shown that our modifications to the original ga for model selection can yield strong results . additionally , the ga approach ( because of the use of icomp ) is better at handling data in which collinearity exist than the traditional selection methods such as forward , backward , and stepwise selection . in particular it is clear that the modifications had a large effect on the accuracy of the ga . this seems to indicate that we may reduce computation and still get statistically the same accuracy if we employ diversification . in all trials , along with the facts presented above and the fact that diversification is easy ( and not costly ) to implement , it is our recommendation that it be used for model selection using gas . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

we implement genetic algorithm based predictive model building as an alternative to the traditional stepwise regression . we then employ the information complexity measure ( icomp ) as a measure of model fitness instead of the commonly used measure of r - square . furthermore , we propose some modifications to the genetic algorithm to increase the overall efficiency .

Introduction Genetic Algorithms Complexity of a Model A Genetic Algorithm for Multiple Linear Regression Model Selection Background Model building via accelerated genetic algorithms Conclusion Conflict of Interest Statement

in ecological studies , one of the commonly used methods for selection is stepwise regression , with forward or backward variable selection algorithms . one problem is that the choice by which the variables enter the selection algorithm is not justified theoretically . we propose the use of genetic algorithms ( gas ) to determine the subset of variables with the highest goodness of fit for a multiple regression model . due to their global search capabilities , the ga based model building is not prone to the problems associated with local search method , hence is a wise choice for this procedure . first developed by holland ( 1975 ) , they have grown in popularity because of the ability of the algorithm to perform well on many different types of problems . during each generation , which is the time step of the algorithm , a population of chromosomes compete to have their genes the selection step is used to pick the chromosomes for the next generation based on their fitness . through successive generations , increasingly better chromosomes come to dominate the population , and the optimal solution ( or something very close ) is realized . more specifically the fittest chromosome is the set of parameters that maximizes the explanatory power of the model with minimum number of parameters . bozdogan ( 1988 , 2004 ) considered complexity as a measure of fitness , which can be described as follows : the complexity of a system ( of any type ) is a measure of the degree of interdependency between the whole system and a simple enumerative composition of its subsystems or parts . the concept of information complexity was first introduced by akaike ( 1973 ) as a measure of the complexity of a model : it is a relative measure of the information lost when a given model is used , and can be described as a function of the precision and accuracy of the model . the first term of aic gives the lack of fit of the model , and the second term is a penalty for the number of parameters in the model . the model with the lowest aic value is considered the best , because the model successfully determines the underlying stochastic process with the least number of parameters . although aic does take into account the problem of over - fitting , where other measures such as r - square do not , aic is not sensitive to parameter dependency , which is an important component for model selection . if a model with both low variance and low covariance can be produced , then the parameters can be better estimated , as they will not be correlated . as an alternative to aic , we consider icomp as a complexity measure which considers variance and covariance , and accounts for the problem of over - fitting the model . it is calculated by where l(k ) again denotes the maximum likelihood function , ^k is the maximum likelihood estimate of parameter vector k under the model mk , c is a real - valued complexity measure , and ^model is the estimated covariance matrix of the parameters of the model . the main difference between the two measures of complexity is that aic only considers the number of parameters as a penalty , whereas icomp considers the covariance between parameters . in predictive model building , this value for icomp is based on the inverse - fisher information matrix ( ifim ) . for multiple regression , the value of icomp can be directly calculated after regression is implemented , and is given by n is the number of parameters in the model , q is the number of observations , ^2=sse / n , tr(^2(xx)1 ) is the trace of the observation matrix multiplied by its inverse and then scaled by ^2 , and |^2(xx)1| is the determinant of the previous matrix . a commonly used method to form the mating pool is proportional selection , which depends on selecting strings for the mating pool with a probability proportional to their fitnesses . in proportional selection , the first step of the calculation of the fitness values is subtracting the icomp value of each string in that generation from the maximum value of icomp in the population . that is , for each i = 1,2, ,n , where n is the size of the population . then the average icomp difference ( the average fitness ) for the total population is calculated as finally , each string is given a fitness value that is the ratio of its icomp difference and the average fitness of the population : here we consider the implementation of ga 's for predictive model selection and discuss possible improvements . the first step to implementing a ga for any optimization problem is to encode the input variable into binary strings . in the case of multiple linear regression we wish to fit the data to where y is an n 1 response vector , x is an n q matrix of the data points , is a q 1 coefficient matrix , and is an n 1 error vector with entries from independent normal distributions [ n(0 , ) for all components ] . the encoding is done by creating a binary string which has n + 1 bits , where each bit represents a different parameter of the model and an intercept . the last n bits correspond to the n explanatory variables contained in the dataset , whereas the first bit is the intercept for the linear model . a parameter is included in the model if the value of the bit for that parameter is a 1 and is excluded if it is a 0 . for example , suppose we have a dataset where we are interested in predicting the reproductive fitness of a species of trees . in this case for example , the string 10010111101 would represent a model which includes the intercept , soil ph , average temperature of environment , average rainfall of environment , circumference of trunk , longitude of environment , and prevalence of disease in environment . similarly , the string 00001000110 is a model that has no intercept , and includes density of trees in the surrounding area , longitude of environment , and latitude of environment ( see table 1 ) . the probability that a string will be chosen for the mating pool is proportional to its fitness value . now that we have a method of encoding information and a method to evaluate the fitness values , we have to determine the remaining parameters of the ga . the first one we consider is the method of creating the initial population and determining its size . however , in the case of model selection , a user may want to force a parameter(s ) to be included , even if it is not part of the model with the lowest complexity . in this case , the initial population can be generated in such a way that certain parameters are always in the model . in addition to determining the method to generate the population , the user must choose the size of the initial population . generally the size should not be too large , as this will slow the algorithm , and should not be so small that genetic drift takes over the course of evolution of the population . in typical gas , the size of the population stays the same ; however , this may not be an effective use of computation . finally , we discuss the genetic operators which allow the algorithm to find the optimal model . first , the probability of crossover , pc , is chosen . if that number is less than the probability of crossover , crossover occurs . after the choice of pc , the number of crossover points must be chosen . applying crossover to the two parents creates two offspring ( see figure 1 ) , where offspring 1 represents a model with an intercept , soil ph , average temperature of environment , longitude of environment , latitude of environment , and prevalence of disease in environment , and offspring 2 represents a model that includes density of trees in the surrounding area , average rainfall of environment , circumference of trunk , and longitude of environment . this value gives the probability that at each location in the string the bit will be flipped . flipping is defined as a change of a 0 to 1 or a 1 to a 0 . however , strings used for other applications of ga 's are usually longer than the ones used for determining least complex models . although there are ongoing studies on determining optimal crossover and mutation rates ( such as nested gas , self - adjusting parameterless gas ) , these rates can be determined by trial and error or by pilot runs before the actual data set has been used to build a model . we conclude this section with a pseudo code for a ga used to find the least complex model that sufficiently describes the data . generate initial population while ( t < max generations or the maximum number of computations have not been executed ) ( a)calculate icomp for the model each string encodes ( b)select strings for the mating pool ( c)create a new population using crossover ( d)mutate new population while the use of a typical ga for model selection already proves to be more efficient than stepwise regression , with a few modifications , the process can show a 10-fold increase in accuracy given the same amount of computation . first , we discuss the modifications , and then we explain the study done to determine the effectiveness of these modifications . according to fisher 's fundamental theorem of natural selection ( fisher , 1930 ) , the increase in mean fitness is equal to the variance in fitness . for model selection using gas , the easiest way to increase variance in fitness would be to allow every model to be represented in the population . we believe that the next best procedure is to force the population to start with the highest variance in each position of the chromosome . to implement this procedure , the other half is generated by taking each of the chromosomes in the first half and changing each bit from 1 to 0 or 0 to 1 . this does not imply that mutation is not necessary , as selection acts on the entire string , not individual positions . since selection will reduce variance at each position , mutation is still required to maintain some variance . the second modification is starting with a larger initial population and then reducing it in size . we have used a reduction method that adapts to the changes in the algorithm in this study . adaptively reducing the population is done by calculating the change in the best fitness between two consecutive generations and then reducing the population based on this change . clearly , there must be a limit to the percentage of reduction , since the population should not be reduced too much , and also because the percent change can be more than 100 . here the amount of population reduction depends on the complexity of the problem , that is the type of the fitness function ( such as mse , aic , icom , mallow 's cp and so on ) used . this limit on the reduction may be determined by pilot studies . elitism is a procedure commonly used in gas in order to pass the best chromosome , or a group of the best chromosomes , to the next generation without any modifications . using elitism guarantees that the change in best fitness is always non - negative . as a result , since we wished to minimize icomp , we set the fitness of each chromosome to be the negative of the icomp value . the choices of these parameters should be done by considering characteristics of the problem such as the expected increase in fitness over time . this is typically a difficult characteristic to determine . generally , as the number of variables increase , the value of fmaxbest should decrease . as the number of variables increases , so does the number of possible values of icomp , and the likelihood that the population will evolve slower . the value of min_popsize should be chosen so that it is quite small ( 5 ) , regardless of the number of variables . as a side note , the ga with no population reduction is a special case of the adaptive method where fmaxbest=0 . the final modification to a ga for multiple regressions is the use of binary tournament instead of proportional selection . then both chromosomes are put back into the pool of contestants of the tournament . proportional selection may increase this effect because a few chromosomes with extremely high fitness are expected to be picked often for the mating pool . to test the benefits of these modifications , we used the data set in bozdogan ( 2004 ) where the predictive model is constructed for body fat and 13 explanatory variables . in order to determine how well the ga was performing , all subsets of the variables ( 2 1 = 16,383 subsets ) were used to generate a model , and then the icomp value was determined . these cases differed in the value of fmaxbest , and as a result in the initial population size . parameters that were the same for all genetic algorithm schemes . the frequency of the correct model being selected over 200 trials . in the case of multiple linear regression we wish to fit the data to where y is an n 1 response vector , x is an n q matrix of the data points , is a q 1 coefficient matrix , and is an n 1 error vector with entries from independent normal distributions [ n(0 , ) for all components ] . the encoding is done by creating a binary string which has n + 1 bits , where each bit represents a different parameter of the model and an intercept . the last n bits correspond to the n explanatory variables contained in the dataset , whereas the first bit is the intercept for the linear model . a parameter is included in the model if the value of the bit for that parameter is a 1 and is excluded if it is a 0 . now that we have a method of encoding information and a method to evaluate the fitness values , we have to determine the remaining parameters of the ga . unless previous knowledge about the problem is given , it is commonplace in gas to randomly generate binary strings ( goldberg , 1989 ) . however , in the case of model selection , a user may want to force a parameter(s ) to be included , even if it is not part of the model with the lowest complexity . in addition to determining the method to generate the population , the user must choose the size of the initial population . generally the size should not be too large , as this will slow the algorithm , and should not be so small that genetic drift takes over the course of evolution of the population . in typical gas , the size of the population stays the same ; however , this may not be an effective use of computation . finally , we discuss the genetic operators which allow the algorithm to find the optimal model . thus , if pc = 1 , then every pair will cross , and if pc = 0 , then no strings will be altered by crossover . after the choice of pc , the number of crossover points must be chosen . the purpose of crossover is to bring together models which have components that reduce complexity . applying crossover to the two parents creates two offspring ( see figure 1 ) , where offspring 1 represents a model with an intercept , soil ph , average temperature of environment , longitude of environment , latitude of environment , and prevalence of disease in environment , and offspring 2 represents a model that includes density of trees in the surrounding area , average rainfall of environment , circumference of trunk , and longitude of environment . but , what happens if the actual least complex model includes a parameter that is not present in the population , that is , the position in the string that represents the parameter is fixed at 0 ? flipping is defined as a change of a 0 to 1 or a 1 to a 0 . generate initial population while ( t < max generations or the maximum number of computations have not been executed ) ( a)calculate icomp for the model each string encodes ( b)select strings for the mating pool ( c)create a new population using crossover ( d)mutate new population while the use of a typical ga for model selection already proves to be more efficient than stepwise regression , with a few modifications , the process can show a 10-fold increase in accuracy given the same amount of computation . first , we discuss the modifications , and then we explain the study done to determine the effectiveness of these modifications . the first modification is changing how the initial population was created . according to fisher 's fundamental theorem of natural selection ( fisher , 1930 ) , the increase in mean fitness is equal to the variance in fitness . for model selection using gas , the easiest way to increase variance in fitness would be to allow every model to be represented in the population . of course , this is impossible for a model with a large number of possible explanatory variables , and would amount to doing an exhaustive search . we believe that the next best procedure is to force the population to start with the highest variance in each position of the chromosome . to implement this procedure , the other half is generated by taking each of the chromosomes in the first half and changing each bit from 1 to 0 or 0 to 1 . we have used a reduction method that adapts to the changes in the algorithm in this study . adaptively reducing the population is done by calculating the change in the best fitness between two consecutive generations and then reducing the population based on this change . more specifically , the population is reduced by the percentage increase in best fitness up to some limit . clearly , there must be a limit to the percentage of reduction , since the population should not be reduced too much , and also because the percent change can be more than 100 . here the amount of population reduction depends on the complexity of the problem , that is the type of the fitness function ( such as mse , aic , icom , mallow 's cp and so on ) used . elitism is a procedure commonly used in gas in order to pass the best chromosome , or a group of the best chromosomes , to the next generation without any modifications . as a result , the population never increases in size . since we wished to minimize icomp , we set the fitness of each chromosome to be the negative of the icomp value the choices of these parameters should be done by considering characteristics of the problem such as the expected increase in fitness over time . generally , as the number of variables increase , the value of fmaxbest should decrease . as the number of variables increases , so does the number of possible values of icomp , and the likelihood that the population will evolve slower . the value of min_popsize should be chosen so that it is quite small ( 5 ) , regardless of the number of variables . as a side note , the ga with no population reduction is a special case of the adaptive method where fmaxbest=0 . the final modification to a ga for multiple regressions is the use of binary tournament instead of proportional selection . then both chromosomes are put back into the pool of contestants of the tournament . for models with few explanatory variables , this gain in computation may be negligible . when the population is being reduced , genetic drift may be amplified , since the sampling space for the next generation decreases . to test the benefits of these modifications , we used the data set in bozdogan ( 2004 ) where the predictive model is constructed for body fat and 13 explanatory variables . in order to determine how well the ga was performing , all subsets of the variables ( 2 1 = 16,383 subsets ) were used to generate a model , and then the icomp value was determined . these cases differed in the value of fmaxbest , and as a result in the initial population size . parameters that were the same for all genetic algorithm schemes . the frequency of the correct model being selected over 200 trials . while model selection remains to be a difficult procedure in case of a large number of parameters , using a ga to find the least complex model can be quite helpful . we have shown that our modifications to the original ga for model selection can yield strong results . additionally , the ga approach ( because of the use of icomp ) is better at handling data in which collinearity exist than the traditional selection methods such as forward , backward , and stepwise selection . in particular it is clear that the modifications had a large effect on the accuracy of the ga . this seems to indicate that we may reduce computation and still get statistically the same accuracy if we employ diversification . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

although diabetes is highly prevalent worldwide , its presence among american indians and alaska natives ( ai / ans ) is particularly alarming . adjusting for age , ai / ans suffer from type 2 diabetes mellitus at rates greater than two times those of non - hispanic whites and exhibit the highest prevalence of this disease of any racial group in the united states [ 1 , 2 ] . given the sharp increase in incident diabetes among ai / ans over the last 20 years , these circumstances seem unlikely to change without substantial intervention [ 35 ] . the special diabetes program for indians diabetes prevention ( sdpi - dp ) demonstration project has been implemented over the past decade to address this problem using a well - established , evidence - based preventive intervention . the sdpi - dp initiative was developed based upon the national institute of diabetes , digestive and kidney disease 's ( niddk ) diabetes prevention program ( dpp ) , which was a large - scale clinical trial that demonstrated that lifestyle interventions ( e.g. , changing diet and exercise habits ) can be effective in delaying or preventing the onset of diabetes in individuals who are at increased risk for developing this disease . the dpp outcomes did not differ significantly for various ethnic groups , including american indians ; however , the dpp was conducted as a highly controlled clinical trial , which did not allow for evaluating the effectiveness of lifestyle interventions in preventing the onset of diabetes in community - based settings with underserved populations . ai / an communities often face a lack of health care resources and a highly mobile population , thereby making it particularly difficult to implement large - scale prevention programs . therefore , the sdpi - dp worked with experts in a variety of ai / an communities to implement cultural adaptations to the original dpp lifestyle curriculum ( e.g. , the use of indigenous foods , drumming during class sessions ) , in order to make the program more relevant to ai / an individuals and more transferrable to a geographically , culturally , and organizationally diverse array of settings in tribal communities . the sdpi - dp demonstration program resulted in reduced diabetes incidence among high risk ai / ans at a rate comparable to the results for ai / ans in the original dpp study . in addition , improvements in weight , blood pressure , and lipid levels were detected following the intervention . however , despite the overall effectiveness with which the intervention was delivered to sdpi - dp participants , several participant characteristics were related to retention in the program ; participants who were younger , were male , had less education , and had lower income were more likely not to complete the core intervention . these initial findings regarding the relationship between sociodemographic factors and retention led program staff to question the potential additional impact of individual - level psychosocial factors on participant engagement , ability to grasp the knowledge conveyed , and mastery of skills related to the behavioral changes associated with the desired outcomes . therefore , it was determined that further analyses were warranted in order to evaluate the extent to which program outcomes were related to individual - level psychosocial characteristics . the observation of a potential impact of psychosocial factors on self - management of medical illnesses is not unique . for example , the influence of depression and anxiety on intervention outcomes for individuals with prediabetes was examined in at least one previous study , and more positive baseline mood was correlated with increased physical activity . specifically , there is evidence that diabetes may increase the likelihood of depressive episodes and that depression may increase the risk of developing diabetes [ 1013 ] . furthermore , psychological distress in general has also been shown to be associated with many chronic health conditions , including obesity [ 14 , 15 ] , which is a significant risk factor for the development of type 2 diabetes . other studies have identified increased odds of diabetes among ai / ans with a history of trauma and significant life stressors [ 16 , 17 ] . conversely , strong coping skills and other positive emotional attributes have been found to enhance metabolic control among those with diabetes . in addition , increased spirituality has been associated with improved self - management among african americans who suffer from diabetes , lower stress and higher quality of life in persons afflicted by chronic illness , and decreased likelihood of developing depression . although the relationship between spirituality and diabetes has not been studied specifically in ai / an populations , previous research has highlighted the importance of religious and spiritual practices for ai / an individuals struggling to overcome other health issues , such as the problematic use of alcohol . additionally , family support has been correlated with increased weight loss in the prevention of diabetes among arab americans . similarly , positive family support was correlated with improved diet in a study of older hispanic adults with diabetes . furthermore , active family nutritional support was linked to improved control of diabetes - related factors ( i.e. , triglycerides , cholesterol , and hba1c ) among navajo tribal members . finally , several psychological and behavioral factors , including increased self - efficacy , were associated with improved weight loss for dpp participants . given this prior body of evidence supporting significant relationships between a variety of psychosocial characteristics and multiple health outcomes , the correlation of psychosocial factors ( psychological distress , trauma exposure , coping skills , spirituality , and family support ) with a key clinical indicator of diabetes risk ( weight ) among ai / ans participating in the sdpi - dp demonstration project was assessed in the present study . resulting insights could suggest enhancements targeting such factors in the core components of sdpi - dp that hold promise for increasing its effectiveness . eligibility criteria for participating in the sdpi - dp demonstration projects were being ai / an ( based on eligibility to receive ihs services ) , being at least 18 years of age , and having either impaired fasting glucose ( ifg ) ( i.e. , a fasting blood glucose ( fbg ) level of 100125 mg / dl and an oral glucose tolerance test ( ogtt ) result < 200 mg / dl ) or impaired glucose tolerance ( igt ) ( i.e. , an ogtt result of 140199 mg / dl two hours after a 75 g oral glucose load and an fbg level < 126 mg / dl ) . exclusion criteria included a previous diagnosis of diabetes ( not including those who only have had gestational diabetes ) , pregnancy , end - stage renal disease on dialysis , and any condition that would affect successful participation based on provider judgment ( e.g. , cardiac concerns given the physical activity element of the program , severe substance use , and undergoing treatment for cancer ) . participants attended a 16-session educational curriculum , a series of lifestyle coaching sessions , and community - based exercise programs focused on reducing the risk of developing type 2 diabetes through moderate weight loss , increased physical activity , and healthy eating habits . clinical measurements and participant surveys were obtained at baseline , within 30 days of completing the 16-session curriculum , and annually thereafter . participants were enrolled at one of 36 tribal , indian health service ( ihs ) or urban indian health care programs serving 80 tribes between 2006 and 2010 . seventy - eight percent of participants were from a rural geographic setting , and 22% were from an urban area . to be included in the current study , participants minimally completed a baseline clinical assessment and a baseline survey ( n = 3,135 ) . the 193 individuals who completed a baseline clinical assessment but did not complete any participant surveys were excluded from these analyses . these individuals did not differ significantly from those included in the study with regard to age , gender , and baseline weight . the sdpi - dp protocol was approved by the institutional review board of the university of colorado denver and the national ihs institutional review board . when required , grantees obtained approval from other entities overseeing research in their programs ( e.g. , tribal review boards ) . the study sample was 74% female and had a mean age at baseline of 46.7 years . sixty - three percent of participants attended at least some college courses ; 72% of participants reported annual household incomes of less than $ 50,000 . sociodemographic variables including participant gender , age , educational status , and annual household income were collected through a survey at baseline . frequency of participants ' experience of various symptoms of depression and anxiety during the previous 30 days was assessed using this scale . item scores ranged from 1 ( none of the time ) to 5 ( all the time ) . participants ' ability to cope with life stressors was measured using the brief resilient coping scale . this 4-item scale asked participants to rate descriptions of coping reactions ( e.g. , approaching difficult situations in creative ways , focusing on the positive growth that can come from dealing with adversity ) , using a scale ranging from 1 ( does not describe me at all ) to 5 ( describes me exactly ) . a modified 6-item version of the diabetes family behavior checklist was used to measure participants ' perceptions of positive and negative family support in regard to their efforts to prevent the onset of diabetes . sdpi - dp research staff modified the original checklist slightly by removing items that referred to specific activities for individuals with diabetes ( e.g. , family providing suggestions about taking insulin on time ) that would not have been relevant to a program focusing on diabetes prevention . participants rated how often their family members provided positive support on 4 items ( e.g. , exercising with them ) and negative support on 2 items ( e.g. , criticizing them for not exercising regularly ) . item scores on the six items ranged from 1 ( less than once a month ) to 5 ( at least once a day ) . no items were reverse - scored , as items were phrased in either a positive or negative manner , consistent with the two scored dimensions . two additional psychosocial variables ( trauma experience and spirituality ) were assessed by participant surveys only at baseline . these two particular variables were not collected at follow - up due to the expectation of their high stability across a relatively short period of time . a single dichotomous variable from a posttraumatic stress disorder ( ptsd ) screener captured whether participants had ever experienced a significant traumatic event ( e.g. , being the victim of a violent crime or domestic violence , being in a disaster like a flood or fire , being in combat , being seriously injured in an accident , being sexually assaulted , and witnessing someone else being seriously injured or killed ) . this variable was coded either 0 ( no trauma ) or 1 ( history of trauma ) . spirituality was assessed via a 7-item scale designed specifically to capture the culturally relevant components of spirituality for ai / ans ; item scores ranged from 1 ( strongly disagree ) to 5 ( strongly agree ) . the items on this scale were developed through consultation with tribal leaders to reflect american indian cultural views of the connectedness of humans to all other physical and transcendental entities . the seven items were as follows : ( 1 ) i am in harmony with all living things , ( 2 ) i feel connected with other people in life , ( 3 ) i follow my tribal path , ( 4 ) when i need to return to balance , i know what to do , ( 5 ) i feel like i am living the right way , ( 6 ) i give to others and receive from them in turn , and ( 7 ) i am a person of integrity . confirmatory factor analyses were conducted at the item level for each psychosocial scale in order to establish measurement invariance across the two time points [ 32 , 33 ] . subsequently , latent difference scores were created to measure change over time in the outcome variable ( weight ) and applicable psychosocial variables [ 33 , 34 ] . latent difference scores are not subject to the restrictive assumptions of traditional anova approaches and permit the measurement of change without error by including multiple indicators of each construct at each of two time points . this modeling approach decomposes the data from the second time point into two components : ( 1 ) variance associated with time 1 and ( 2 ) variance associated with the difference from time 1 . therefore , latent difference scores allow for the estimation of baseline variance as well as variance regarding change in a construct over time . following these initial steps , a series of bivariate analyses were conducted within a structural equation modeling framework , which separately evaluated the relationships between each psychosocial variable and weight . for psychosocial variables that were measured at both baseline and follow - up , three parameters of primary interest were estimated : ( 1 ) the correlation between the psychosocial characteristic and weight at baseline , ( 2 ) the predictive relationship of the baseline psychosocial characteristics on change in weight , and ( 3 ) the association of change in the psychosocial characteristic with change in weight . for psychosocial variables that were measured only at baseline ( i.e. , trauma and spirituality ) , after evaluating the bivariate relationships , a multivariate model estimated the three parameters described above simultaneously for all psychosocial variables . this model also controlled for baseline sociodemographic characteristics , including gender , age , education , and income . psychosocial variables were eliminated from the multivariate model in a stepwise manner if they reached a p value greater than 0.2 for all three primary parameters , in order to arrive at a final model . biostatisticians have suggested that a p value greater than 0.2 is a reasonable cutoff to eliminate variables that are clearly nonsignificant in regression models . an effect size measure for the final model ( r ) was computed as the proportion of variance of change in weight that was explained by the predictor variables . confirmatory factor analysis and structural equation models macs analyses allow for the inclusion of mean - level information in addition to the covariance structures information of standard structural equation modeling techniques , which is necessary for the interpretation of latent difference scores . macs analyses also provide a particular advantage over ordinary least - squares regression approaches , namely , the fact that the unreliability of instruments / scales is taken into account and that corrections are made for measurement error . when employing structural equation modeling techniques , it is important to assess the degree to which the specified model fits the actual data in order to determine the appropriateness of a particular model . in the present study , the root mean square error of approximation ( rmsea(90% confidence interval ) ; less than .08 is adequate fit and less than .05 is good fit ) , the comparative fit index ( cfi ; greater than .90 is adequate fit and greater than .95 is good fit ) , and the tucker - lewis index ( tli ; greater than .90 is adequate fit and greater than .95 is good fit ) were used as indices of model fit . in all models , full information maximum likelihood ( fiml ) was implemented in all analyses in order to address potential bias and decreased power due to missing data [ 37 , 38 ] . furthermore , although there was very little variation across programs in class attendance for the participants included in the current study ( 95% of participants who completed a follow - up assessment had completed at least 14 of the 16 recommended curriculum classes ) , other elements of the program may have varied slightly across sites . therefore , in order to control for the clustering of participants into 36 separate health care programs , standard errors that are robust to nonnormality and nonindependence of observations were computed using a sandwich estimator . descriptive statistics for all clinical and psychosocial variables are presented in table 2 . between baseline and follow - up , a significant decrease of 8.58 lbs was found with regard to average weight ( = 8.58 , p < .001 ) . with regard to the change in psychosocial factors , general psychological distress decreased over time ( = 0.14 , p < .001 ) , while coping abilities increased ( = 0.07 , p < .001 ) . furthermore , positive family support , as perceived by participants , was higher at follow - up ( = 0.27 , p < .001 ) , whereas negative family support remained stable ( = 0.03 , p = .28 ) . at baseline , 48% of sdpi - dp participants reported a lifetime history of at least one significant trauma , and the average level of reported cultural spirituality at baseline was 3.81 ( on a scale from 1 to 5 ) . confirmatory factor analyses of the kessler distress , coping , and family support measures supported invariance of factor loadings and intercepts across the two measurement time points , which indicates that the measures exhibited similar structures and measured the same constructs across time . strong factorial invariance was established for both the kessler distress and coping measures , with all factor loadings and intercepts constrained to be equal across time . partial measurement invariance was established for the family support measure , as one of the intercepts for the positive family support scale was not invariant across time . it is generally acceptable to use measures with partial invariance in further structural models , if at least two indicators ( scale items ) have an invariant factor loading and intercept [ 32 , 33 ] . in the case of the family support measure , all six items exhibited loading invariance , and all but one item exhibited intercept invariance . it was important to establish that these psychosocial measures had identical or near identical structures at both time points in order to calculate reliable and valid difference scores . bivariate analyses were performed prior to running the multivariate model in order to evaluate the strengths of individual predictor / outcome relationships ( see table 3 ) . all estimates ( = covariance ; = regression coefficient ) are provided in an unstandardized metric in order to allow meaningful interpretation based upon the original scale ranges . all bivariate models exhibited good model fit ( rmsea < .05 ; cfi > .95 ; tli > .95 ) , and several significant correlations were found between psychosocial characteristics and weight at baseline . greater psychological distress at baseline was related to higher baseline weight ( = 2.44 , p < .001 ) . in addition , greater negative family support was significantly correlated with higher baseline weight ( = 4.55 , p < .001 ) , whereas greater identification with culturally relevant spirituality was associated with lower baseline weight ( = 3.13 , p < .001 ) . baseline levels of coping , positive family support , and trauma experience were not significantly related to baseline weight . in addition to investigating the relationships between psychosocial characteristics and weight at baseline , regression analyses were conducted in order to elucidate the predictive relationships between psychosocial variables ( independent variables ) and change in weight from baseline to follow - up ( dependent variable ) . the results of these analyses underscore the importance of psychological distress and family support in predicting weight loss in ai / ans with prediabetes . greater psychological distress at baseline predicted less successful weight loss between baseline and follow - up ( = 1.85 , p < .001 ) , and an increase in psychological distress between baseline and follow - up was also significantly related to less successful weight loss ( = 3.23 , p < .001 ) . participants who reported an increase in positive family support after the intervention were more successful in losing weight ( = 3.56 , p < .001 ) . conversely , higher negative family support at baseline as well as an increase in negative family support after the intervention was significantly associated with less weight loss ( = 1.38 , p = .003 and = 3.13 , p < .001 , resp . ) . coping , trauma experience , and cultural spirituality were not significantly related to weight change . after conducting the bivariate analyses , all of the psychosocial variables were included in a single multivariate model . sociodemographic variables ( gender , age , education status , and annual household income ) that have previously been shown to be related to participant engagement and retention in the sdpi - dp program were entered as covariates in the model , in order to determine the effect of the psychosocial factors on weight change above and beyond any potential effect of sociodemographic characteristics . using the stepwise procedure described above , coping and trauma experience were dropped from the final model , as they were neither correlated with baseline weight nor predictive of weight change . results of the final multivariate model are presented in figure 1 , which mirror the results of the bivariate models , with one exception . after controlling for sociodemographic factors and other psychosocial variables , baseline psychological distress was no longer predictive of weight loss from baseline to follow - up . however , the correlations between psychological distress , negative family support , and cultural spirituality with weight at baseline remained significant . in addition , change in psychological distress , positive family support , and negative family support over the course of the intervention , as well as levels of negative family support at baseline , remained significantly associated with change in weight . overall , the final multivariate model exhibited good model fit ( rmsea = .026(.025.028 ) ; cfi = .966 ; tli = .958 ) and accounted for 11% of the variability in weight change . this proportion of variance explained is not large , but it does represent a medium effect size . the importance of psychosocial characteristics as sources of diabetes risk and resilience has been demonstrated previously among ai / ans [ 16 , 17 , 25 , 41 ] . the present study is a critical first step in moving from research focused primarily on individuals with diabetes to examining factors related to successfully preventing incident diabetes among native people at high risk of the disease . although the influence of depression and anxiety on intervention outcomes for prediabetic individuals was examined in at least one previous study , the present study is the first to focus on determining which psychosocial factors successfully predict a specific outcome of a large - scale initiative aimed at preventing the onset of diabetes in the ai / an population . moreover , the statistical approach employed in the current study made it possible to simultaneously examine the relative contributions of the various psychosocial factors to successful health changes in a single model , unlike previous studies that have analyzed psychosocial factors in isolation of one another . specifically , structural equation modeling provides the ability to simultaneously examine the relationships of the psychosocial variables to a key clinical outcome with regard to baseline levels and change over time . as expected , when analyzing such relationships in a bivariate manner , several psychosocial factors were related to baseline levels of weight . higher levels of psychological distress and negative family support were associated with higher weight , whereas greater spirituality was correlated with lower weight . the same pattern of correlations of these three psychosocial variables with weight at baseline also was supported in the multivariate model when controlling for sociodemographic factors . psychosocial factors were also related to the degree of weight change following participants ' completion of the sdpi - dp intervention . greater psychological distress at baseline and increased psychological distress over the course of the intervention both contributed to less weight loss . similarly , greater negative family support at baseline and increased negative family support over the course of the intervention were associated with a smaller reduction in weight . increased positive family support , on the other hand , predicted greater weight loss . controlling for sociodemographic factors within a multivariate model , change in psychological distress , negative family support , and positive family support , as well as baseline levels of negative family support , continued to significantly affect weight reduction . the results of the present study are consistent with prior research on psychological distress as a risk factor with regard to chronic illness [ 14 , 15 , 18 ] and with previous findings regarding the role of positive family support in both reducing the risk of and successfully managing diabetes [ 2325 ] . the results underscore the importance of regularly assessing the psychosocial status and functioning of ai / ans at high risk of diabetes . prevention programs will be well served by developing the capacity to evaluate and monitor participants ' mental health status , including the presence of depression and anxiety , the nature and extent of their spirituality , and the adequacy of their family support . these personalized assessments , combined with the knowledge of the general effects of psychosocial factors uncovered in the present study , will allow program staff to know which adjunctive interventions may maximize participant benefit with respect to the desired outcomes ( e.g. , weight loss ) . for example , by increasing the focus on mental health components within the core curriculum , one could strengthen participants ' strategies for decreasing depressive and stress - related symptoms , which then may make it more likely that the participants will be more engaged in the intervention and experience more successful weight loss . offering self - management techniques , simple cognitive - behavioral skills , and referral to local support groups or treatment options is a logical extension of the goals , process , and structure of an intensive lifestyle balance intervention . additionally , knowing that a participant has a strong preexisting spiritual focus may be helpful information for program staff who then may be able to use a participant 's connectedness to the natural world as a pathway to increase motivation to engage in a healthier lifestyle . likewise , given the strong relationship between family support and program outcomes , a greater effort should be made to incorporate close family members into various aspects of the prevention program . sdpi - dp demonstration projects have begun to do so , guided by their initial impressions of the potential gains . for example , some programs encourage participants to identify a support person to attend curriculum classes and other program - related activities with the participant . the present study has several limitations , which suggest directions for future research . data specific to the psychosocial characteristics were collected solely by self - report , thereby possibly increasing shared method variance and artificially strengthening the relationships among these variables . future studies may benefit from using a variety of methods to operationalize and assess similar constructs . for example , family support could be measured through multiple informants , including close family members , and levels of depression and anxiety could be assessed through interview - based rating scales . nevertheless , the primary relationships of interest were between self - reported psychosocial characteristics and an objective clinical measure ( weight ) , which were not subject to problems of shared method variance . the relationships between psychosocial factors and program outcomes may wax and wane over a longer follow - up period , or certain interactions may occur over time that are not evident within a relatively short follow - up period . in addition , sdpi - dp participants were more likely to be female , be older , have a higher level of education , and have a higher household income than the general ai / an adult population . though previous research has shown similar trends when comparing clinical populations to the general population [ 43 , 44 ] , the generalizability of the present findings to individuals with widely differing sociodemographic backgrounds may be limited . for example , it is possible that weight change for males may not be as strongly related to psychological distress or family support as it was for this largely female sample . in addition , individuals with less education and lower household income than the participants in the current study may be more likely to have suffered a greater number of significant traumas . future studies should attempt to enroll individuals with broader sociodemographic characteristics and should include a measure of the number of traumas experienced , which would provide the opportunity to analyze a possible additive effect of repeated trauma upon successful weight loss that was not possible with the dichotomous trauma item used in the present investigation . similarly , although both rural and urban participants were included in the study , the majority of participants lived in rural settings , which may further limit the generalizability of the results . although it would be difficult to extend these results to the mainstream american population without further research , the current findings may also be applicable to other populations that share similar structures and values with ai / an communities ( e.g. , a greater emphasis on extended family support as opposed to individualism ; a spiritual emphasis on connectedness to others and nature ) . furthermore , although a medium effect size for the prediction of weight change within the multivariate model was observed ( 11% of outcome variability explained ) , additional factors are likely at work and will need to be addressed to more comprehensively improve the effectiveness of such prevention programs . some additional factors may include lack of access to healthy food selections , high levels of family and caregiver stress that make it difficult to follow through with healthy eating and exercise routines , and lack of transportation to attend program classes . moreover , characteristics of the treatment team and health care program in general previously have been shown to be related to participant retention , which in turn predicts program outcomes [ 7 , 8 ] . therefore , it likely will be critical to incorporate a multifaceted approach to crafting additional components that promise to enhance the intervention . for example , rather than just adding a stand - alone mental health screening module , a program might consider addressing barriers to participation ( e.g. , lack of transportation ) in concert with increasing positive family support and thereby decrease the isolation that can lead to psychological distress . finally , a more precise and comprehensive assessment of mental health status would enable a program to determine the most appropriate approach for decreasing symptoms likely to interfere with participation in the preventive intervention . in light of the relationship between depression and diabetes [ 1013 , 45 , 46 ] , referral to a mental health professional is a logical option to be pursued , although other possibilities , such as a group treatment model , should also be considered given the limited numbers of mental health providers within tribal , ihs , and urban indian health care programs . the present study demonstrates the importance of psychosocial factors for maximizing the potential benefits to participants in preventive interventions such as the sdpi - dp demonstration project . the challenge now becomes how to incorporate the lessons learned into the fabric of these programs . augmentation of the current intervention may be achieved either directly by incorporating adjunctive components or indirectly through referral to relevant local resources . the overall goal of these program additions would be to maximize participants ' engagement , their ability to grasp the knowledge conveyed , and their mastery of the skills related to the behavioral changes associated with the desired outcomes .

the association of psychosocial factors ( psychological distress , coping skills , family support , trauma exposure , and spirituality ) with initial weight and weight loss among american indians and alaska natives ( ai / ans ) in a diabetes prevention translational project was investigated . participants ( n = 3,135 ) were confirmed as prediabetic and subsequently enrolled in the special diabetes program for indians diabetes prevention ( sdpi - dp ) demonstration project implemented at 36 indian health care programs . measures were obtained at baseline and after completing a 16-session educational curriculum focusing on weight loss through behavioral changes . at baseline , psychological distress and negative family support were linked to greater weight , whereas cultural spirituality was correlated with lower weight . furthermore , psychological distress and negative family support predicted less weight loss , and positive family support predicted greater weight loss , over the course of the intervention . these bivariate relationships between psychosocial factors and weight remained statistically significant within a multivariate model , after controlling for sociodemographic characteristics . conversely , coping skills and trauma exposure were not significantly associated with baseline weight or change in weight . these findings demonstrate the influence of psychosocial factors on weight loss in ai / an communities and have substantial implications for incorporating adjunctive intervention components .

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

although diabetes is highly prevalent worldwide , its presence among american indians and alaska natives ( ai / ans ) is particularly alarming . adjusting for age , ai / ans suffer from type 2 diabetes mellitus at rates greater than two times those of non - hispanic whites and exhibit the highest prevalence of this disease of any racial group in the united states [ 1 , 2 ] . given the sharp increase in incident diabetes among ai / ans over the last 20 years , these circumstances seem unlikely to change without substantial intervention [ 35 ] . the special diabetes program for indians diabetes prevention ( sdpi - dp ) demonstration project has been implemented over the past decade to address this problem using a well - established , evidence - based preventive intervention . the sdpi - dp initiative was developed based upon the national institute of diabetes , digestive and kidney disease 's ( niddk ) diabetes prevention program ( dpp ) , which was a large - scale clinical trial that demonstrated that lifestyle interventions ( e.g. ai / an communities often face a lack of health care resources and a highly mobile population , thereby making it particularly difficult to implement large - scale prevention programs . therefore , the sdpi - dp worked with experts in a variety of ai / an communities to implement cultural adaptations to the original dpp lifestyle curriculum ( e.g. , the use of indigenous foods , drumming during class sessions ) , in order to make the program more relevant to ai / an individuals and more transferrable to a geographically , culturally , and organizationally diverse array of settings in tribal communities . the sdpi - dp demonstration program resulted in reduced diabetes incidence among high risk ai / ans at a rate comparable to the results for ai / ans in the original dpp study . in addition , improvements in weight , blood pressure , and lipid levels were detected following the intervention . however , despite the overall effectiveness with which the intervention was delivered to sdpi - dp participants , several participant characteristics were related to retention in the program ; participants who were younger , were male , had less education , and had lower income were more likely not to complete the core intervention . these initial findings regarding the relationship between sociodemographic factors and retention led program staff to question the potential additional impact of individual - level psychosocial factors on participant engagement , ability to grasp the knowledge conveyed , and mastery of skills related to the behavioral changes associated with the desired outcomes . the observation of a potential impact of psychosocial factors on self - management of medical illnesses is not unique . for example , the influence of depression and anxiety on intervention outcomes for individuals with prediabetes was examined in at least one previous study , and more positive baseline mood was correlated with increased physical activity . furthermore , psychological distress in general has also been shown to be associated with many chronic health conditions , including obesity [ 14 , 15 ] , which is a significant risk factor for the development of type 2 diabetes . other studies have identified increased odds of diabetes among ai / ans with a history of trauma and significant life stressors [ 16 , 17 ] . conversely , strong coping skills and other positive emotional attributes have been found to enhance metabolic control among those with diabetes . in addition , increased spirituality has been associated with improved self - management among african americans who suffer from diabetes , lower stress and higher quality of life in persons afflicted by chronic illness , and decreased likelihood of developing depression . although the relationship between spirituality and diabetes has not been studied specifically in ai / an populations , previous research has highlighted the importance of religious and spiritual practices for ai / an individuals struggling to overcome other health issues , such as the problematic use of alcohol . additionally , family support has been correlated with increased weight loss in the prevention of diabetes among arab americans . similarly , positive family support was correlated with improved diet in a study of older hispanic adults with diabetes . furthermore , active family nutritional support was linked to improved control of diabetes - related factors ( i.e. finally , several psychological and behavioral factors , including increased self - efficacy , were associated with improved weight loss for dpp participants . given this prior body of evidence supporting significant relationships between a variety of psychosocial characteristics and multiple health outcomes , the correlation of psychosocial factors ( psychological distress , trauma exposure , coping skills , spirituality , and family support ) with a key clinical indicator of diabetes risk ( weight ) among ai / ans participating in the sdpi - dp demonstration project was assessed in the present study . resulting insights could suggest enhancements targeting such factors in the core components of sdpi - dp that hold promise for increasing its effectiveness . eligibility criteria for participating in the sdpi - dp demonstration projects were being ai / an ( based on eligibility to receive ihs services ) , being at least 18 years of age , and having either impaired fasting glucose ( ifg ) ( i.e. , cardiac concerns given the physical activity element of the program , severe substance use , and undergoing treatment for cancer ) . participants attended a 16-session educational curriculum , a series of lifestyle coaching sessions , and community - based exercise programs focused on reducing the risk of developing type 2 diabetes through moderate weight loss , increased physical activity , and healthy eating habits . clinical measurements and participant surveys were obtained at baseline , within 30 days of completing the 16-session curriculum , and annually thereafter . participants were enrolled at one of 36 tribal , indian health service ( ihs ) or urban indian health care programs serving 80 tribes between 2006 and 2010 . to be included in the current study , participants minimally completed a baseline clinical assessment and a baseline survey ( n = 3,135 ) . these individuals did not differ significantly from those included in the study with regard to age , gender , and baseline weight . the sdpi - dp protocol was approved by the institutional review board of the university of colorado denver and the national ihs institutional review board . sociodemographic variables including participant gender , age , educational status , and annual household income were collected through a survey at baseline . a modified 6-item version of the diabetes family behavior checklist was used to measure participants ' perceptions of positive and negative family support in regard to their efforts to prevent the onset of diabetes . sdpi - dp research staff modified the original checklist slightly by removing items that referred to specific activities for individuals with diabetes ( e.g. , family providing suggestions about taking insulin on time ) that would not have been relevant to a program focusing on diabetes prevention . two additional psychosocial variables ( trauma experience and spirituality ) were assessed by participant surveys only at baseline . , being the victim of a violent crime or domestic violence , being in a disaster like a flood or fire , being in combat , being seriously injured in an accident , being sexually assaulted , and witnessing someone else being seriously injured or killed ) . spirituality was assessed via a 7-item scale designed specifically to capture the culturally relevant components of spirituality for ai / ans ; item scores ranged from 1 ( strongly disagree ) to 5 ( strongly agree ) . following these initial steps , a series of bivariate analyses were conducted within a structural equation modeling framework , which separately evaluated the relationships between each psychosocial variable and weight . for psychosocial variables that were measured at both baseline and follow - up , three parameters of primary interest were estimated : ( 1 ) the correlation between the psychosocial characteristic and weight at baseline , ( 2 ) the predictive relationship of the baseline psychosocial characteristics on change in weight , and ( 3 ) the association of change in the psychosocial characteristic with change in weight . , trauma and spirituality ) , after evaluating the bivariate relationships , a multivariate model estimated the three parameters described above simultaneously for all psychosocial variables . this model also controlled for baseline sociodemographic characteristics , including gender , age , education , and income . psychosocial variables were eliminated from the multivariate model in a stepwise manner if they reached a p value greater than 0.2 for all three primary parameters , in order to arrive at a final model . an effect size measure for the final model ( r ) was computed as the proportion of variance of change in weight that was explained by the predictor variables . in the present study , the root mean square error of approximation ( rmsea(90% confidence interval ) ; less than .08 is adequate fit and less than .05 is good fit ) , the comparative fit index ( cfi ; greater than .90 is adequate fit and greater than .95 is good fit ) , and the tucker - lewis index ( tli ; greater than .90 is adequate fit and greater than .95 is good fit ) were used as indices of model fit . furthermore , although there was very little variation across programs in class attendance for the participants included in the current study ( 95% of participants who completed a follow - up assessment had completed at least 14 of the 16 recommended curriculum classes ) , other elements of the program may have varied slightly across sites . therefore , in order to control for the clustering of participants into 36 separate health care programs , standard errors that are robust to nonnormality and nonindependence of observations were computed using a sandwich estimator . with regard to the change in psychosocial factors , general psychological distress decreased over time ( = 0.14 , p < .001 ) , while coping abilities increased ( = 0.07 , p < .001 ) . furthermore , positive family support , as perceived by participants , was higher at follow - up ( = 0.27 , p < .001 ) , whereas negative family support remained stable ( = 0.03 , p = .28 ) . at baseline , 48% of sdpi - dp participants reported a lifetime history of at least one significant trauma , and the average level of reported cultural spirituality at baseline was 3.81 ( on a scale from 1 to 5 ) . confirmatory factor analyses of the kessler distress , coping , and family support measures supported invariance of factor loadings and intercepts across the two measurement time points , which indicates that the measures exhibited similar structures and measured the same constructs across time . partial measurement invariance was established for the family support measure , as one of the intercepts for the positive family support scale was not invariant across time . in the case of the family support measure , all six items exhibited loading invariance , and all but one item exhibited intercept invariance . all bivariate models exhibited good model fit ( rmsea < .05 ; cfi > .95 ; tli > .95 ) , and several significant correlations were found between psychosocial characteristics and weight at baseline . greater psychological distress at baseline was related to higher baseline weight ( = 2.44 , p < .001 ) . in addition , greater negative family support was significantly correlated with higher baseline weight ( = 4.55 , p < .001 ) , whereas greater identification with culturally relevant spirituality was associated with lower baseline weight ( = 3.13 , p < .001 ) . baseline levels of coping , positive family support , and trauma experience were not significantly related to baseline weight . in addition to investigating the relationships between psychosocial characteristics and weight at baseline , regression analyses were conducted in order to elucidate the predictive relationships between psychosocial variables ( independent variables ) and change in weight from baseline to follow - up ( dependent variable ) . the results of these analyses underscore the importance of psychological distress and family support in predicting weight loss in ai / ans with prediabetes . greater psychological distress at baseline predicted less successful weight loss between baseline and follow - up ( = 1.85 , p < .001 ) , and an increase in psychological distress between baseline and follow - up was also significantly related to less successful weight loss ( = 3.23 , p < .001 ) . participants who reported an increase in positive family support after the intervention were more successful in losing weight ( = 3.56 , p < .001 ) . conversely , higher negative family support at baseline as well as an increase in negative family support after the intervention was significantly associated with less weight loss ( = 1.38 , p = .003 and = 3.13 , p < .001 , resp . ) coping , trauma experience , and cultural spirituality were not significantly related to weight change . after conducting the bivariate analyses , all of the psychosocial variables were included in a single multivariate model . sociodemographic variables ( gender , age , education status , and annual household income ) that have previously been shown to be related to participant engagement and retention in the sdpi - dp program were entered as covariates in the model , in order to determine the effect of the psychosocial factors on weight change above and beyond any potential effect of sociodemographic characteristics . using the stepwise procedure described above , coping and trauma experience were dropped from the final model , as they were neither correlated with baseline weight nor predictive of weight change . results of the final multivariate model are presented in figure 1 , which mirror the results of the bivariate models , with one exception . after controlling for sociodemographic factors and other psychosocial variables , baseline psychological distress was no longer predictive of weight loss from baseline to follow - up . however , the correlations between psychological distress , negative family support , and cultural spirituality with weight at baseline remained significant . in addition , change in psychological distress , positive family support , and negative family support over the course of the intervention , as well as levels of negative family support at baseline , remained significantly associated with change in weight . overall , the final multivariate model exhibited good model fit ( rmsea = .026(.025.028 ) ; cfi = .966 ; tli = .958 ) and accounted for 11% of the variability in weight change . the importance of psychosocial characteristics as sources of diabetes risk and resilience has been demonstrated previously among ai / ans [ 16 , 17 , 25 , 41 ] . although the influence of depression and anxiety on intervention outcomes for prediabetic individuals was examined in at least one previous study , the present study is the first to focus on determining which psychosocial factors successfully predict a specific outcome of a large - scale initiative aimed at preventing the onset of diabetes in the ai / an population . moreover , the statistical approach employed in the current study made it possible to simultaneously examine the relative contributions of the various psychosocial factors to successful health changes in a single model , unlike previous studies that have analyzed psychosocial factors in isolation of one another . higher levels of psychological distress and negative family support were associated with higher weight , whereas greater spirituality was correlated with lower weight . the same pattern of correlations of these three psychosocial variables with weight at baseline also was supported in the multivariate model when controlling for sociodemographic factors . psychosocial factors were also related to the degree of weight change following participants ' completion of the sdpi - dp intervention . greater psychological distress at baseline and increased psychological distress over the course of the intervention both contributed to less weight loss . similarly , greater negative family support at baseline and increased negative family support over the course of the intervention were associated with a smaller reduction in weight . increased positive family support , on the other hand , predicted greater weight loss . controlling for sociodemographic factors within a multivariate model , change in psychological distress , negative family support , and positive family support , as well as baseline levels of negative family support , continued to significantly affect weight reduction . the results of the present study are consistent with prior research on psychological distress as a risk factor with regard to chronic illness [ 14 , 15 , 18 ] and with previous findings regarding the role of positive family support in both reducing the risk of and successfully managing diabetes [ 2325 ] . the results underscore the importance of regularly assessing the psychosocial status and functioning of ai / ans at high risk of diabetes . prevention programs will be well served by developing the capacity to evaluate and monitor participants ' mental health status , including the presence of depression and anxiety , the nature and extent of their spirituality , and the adequacy of their family support . these personalized assessments , combined with the knowledge of the general effects of psychosocial factors uncovered in the present study , will allow program staff to know which adjunctive interventions may maximize participant benefit with respect to the desired outcomes ( e.g. for example , by increasing the focus on mental health components within the core curriculum , one could strengthen participants ' strategies for decreasing depressive and stress - related symptoms , which then may make it more likely that the participants will be more engaged in the intervention and experience more successful weight loss . offering self - management techniques , simple cognitive - behavioral skills , and referral to local support groups or treatment options is a logical extension of the goals , process , and structure of an intensive lifestyle balance intervention . likewise , given the strong relationship between family support and program outcomes , a greater effort should be made to incorporate close family members into various aspects of the prevention program . sdpi - dp demonstration projects have begun to do so , guided by their initial impressions of the potential gains . for example , family support could be measured through multiple informants , including close family members , and levels of depression and anxiety could be assessed through interview - based rating scales . the relationships between psychosocial factors and program outcomes may wax and wane over a longer follow - up period , or certain interactions may occur over time that are not evident within a relatively short follow - up period . in addition , sdpi - dp participants were more likely to be female , be older , have a higher level of education , and have a higher household income than the general ai / an adult population . for example , it is possible that weight change for males may not be as strongly related to psychological distress or family support as it was for this largely female sample . future studies should attempt to enroll individuals with broader sociodemographic characteristics and should include a measure of the number of traumas experienced , which would provide the opportunity to analyze a possible additive effect of repeated trauma upon successful weight loss that was not possible with the dichotomous trauma item used in the present investigation . similarly , although both rural and urban participants were included in the study , the majority of participants lived in rural settings , which may further limit the generalizability of the results . although it would be difficult to extend these results to the mainstream american population without further research , the current findings may also be applicable to other populations that share similar structures and values with ai / an communities ( e.g. furthermore , although a medium effect size for the prediction of weight change within the multivariate model was observed ( 11% of outcome variability explained ) , additional factors are likely at work and will need to be addressed to more comprehensively improve the effectiveness of such prevention programs . moreover , characteristics of the treatment team and health care program in general previously have been shown to be related to participant retention , which in turn predicts program outcomes [ 7 , 8 ] . , lack of transportation ) in concert with increasing positive family support and thereby decrease the isolation that can lead to psychological distress . in light of the relationship between depression and diabetes [ 1013 , 45 , 46 ] , referral to a mental health professional is a logical option to be pursued , although other possibilities , such as a group treatment model , should also be considered given the limited numbers of mental health providers within tribal , ihs , and urban indian health care programs . the present study demonstrates the importance of psychosocial factors for maximizing the potential benefits to participants in preventive interventions such as the sdpi - dp demonstration project . augmentation of the current intervention may be achieved either directly by incorporating adjunctive components or indirectly through referral to relevant local resources . the overall goal of these program additions would be to maximize participants ' engagement , their ability to grasp the knowledge conveyed , and their mastery of the skills related to the behavioral changes associated with the desired outcomes .

despite the complexity of language and learning disorders , individual genes are being defined which appear to influence the development of abilities that are necessary in speech , language , and reading . most of the identified candidate genes involve reading disability , and although the evidence supporting some of these genes is still somewhat tenuous due to small sample sizes and limited replication , most are known to be involved in early development , particularly neuronal migration ( galaburda 2005 ; gabel et al . most of these candidate genes have been associated with several learning and language phenotypes , suggesting that they facilitate learning processes which are basic to learning reading and language . similar pleiotropic effects are seen for several genes that primarily affect autism or language but have also shown effects on reading , including cntnap2 and atp2c2 ( vernes et al . 2008 ; newbury et al . 2011 ) . however , despite replicated evidence for association of single nucleotide polymorphisms within and around the genes , very few coding mutations have been reported to account for their influence on these disorders . this has led to the hypothesis that mutations affecting reading and related disorders are likely to be in regulatory regions , controlling the quantity rather than quality of the gene product ( bates et al . alterations of gene expression can be caused by mutations in gene promoters and enhancers located near the gene , but mutations in genes that mediate epigenetic controls of gene expression have been found that affect developmental learning disorders . these mutations may be in regions located further from the target gene , making it more difficult to recognize their significance . of all of the genes that have been proposed as candidates for reading disability and language impairment , six genes have been well characterized with respect to their influence on reading and language disorders , the regions within and around the genes that appear to contain causal mutations , and the effects of the putative mutations or risk alleles on gene transcription : dyx1c1 , dcdc2 , kiaa0319 , robo1 , and the co - regulated genes mrpl1 and c2orf3 . dyx1c1 the 15q21 region was identified as a candidate region for a gene or genes influencing reading disability ( rd ) through linkage studies ( fulker et al . the dyx1c1 ( dyx1-candidate 1 ) gene was specifically targeted after a translocation t(2;15 ) ( q11;q21 ) disrupting the previously uncharacterized gene was observed in a family with rd ( taipale et al . since then , the dyx1c1 protein has been found to contain estrogen - receptor - binding sites ( massinen et al . 2009 ) and knockdown of the gene in embryonic rat brain produces delays in neuronal migration ( wang et al . 2006).sequence analysis of the dyx1c1 coding regions identified a missense mutation in some rd families : rs57809907 , 1249g > t , which results in glu417x and truncates the protein by four amino acids ( taipale et al . 2003 ) , but this variant has not been consistently associated with reading disability in other studies ( scerri et al . 2004 ; wigg et al . another missense mutation , rs17819126 ( 271g > a , val91ile ) has been associated with reading ability ( bates et al . 2010 ) , but analyses of putative effect on protein function by the sift ( ng and henikoff 2003 ) and polyphen ( adzhubei et al . 2010 ) algorithms indicate that this should be a benign change for the protein ( ensembl release 63 : www.ensembl.org ) . since studies have found association of rd with other snps within the gene , it seems likely that mutations affecting rd are in the regulatory rather than coding regions . a possible candidate is the 3g > a snp rs3743205 in the 5 untranslated region ( utr ) . in the original report by taipale et al . ( 2005 ) , the a allele was associated with rd , but subsequent reports found association with the common g allele ( scerri et al . interestingly , a study of rd in chinese children also found strong association with the g allele ( lim et al . studies of transcription factor binding in the promoter region have shown that the a allele shows decreased binding to a repressive transcription factor , resulting in increased dyx1c1 expression ( tapia - paez et al . 2008 ) , leading lim et al . to hypothesize that the a allele is actually protective compared to the downregulating g allele , and that instances where the a allele appeared to be associated with rd could be secondary to linkage disequilibrium with a second causal variant nearby . two other snps , rs12899331 and rs16787 , in the promoter region were also found to be involved in transcription factor binding ( tapia - paez et al . 2008 ) , but these were not found to be associated with rd in later studies ( dahdouh et al . thus , further studies are needed to define the role of particular regulatory regions of dyx1c1 in the cause of rd.there is also evidence for pleiotropic effects of dyx1c1 on short - term memory and mental calculation , a mathematics measure ( marino et al . linkage to the dyx1c1 region was found with speech sound disorder phenotypes ( ssd ) in one study ( smith et al . further studies are needed to determine whether the linkage signal for ssd was actually related to dyx1c1 . dcdc2 initial linkage and association analysis defined the dyx2 locus on chromosome 6p22 ( grigorenko et al . 1999 ; gayan et al . 1999 ; kaplan et al . 2002 ; deffenbacher et al . 2004 ) , and several subsequent studies have reported associations with the dcdc2 ( doublecortin-2 ) gene within the region ( newbury et al . 2011 ; meng et al . the structure of the gene is analogous to the x - linked dcx gene which is known to be involved in microtubular structure and influences neuronal migration . mutation of the dcx gene produces lissencephaly in males and cortical abnormalities in females ( des portes et al . 1998 ) ; accordingly , knockdown of dcdc2 produces delays in neuronal migration in embryonic rat brain ( meng et al . 2005b).sequence analysis of coding regions of dcdc2 in rd families has not identified causal mutations ; however , association of rd was reported with a deletion in intron 2 , termed bv677278 , which appeared to contain transcription factor binding sites ( meng et al . still , the importance of this region in gene regulation has been demonstrated by in vitro studies showing that sequences in the region act as enhancers for dcdc2 expression ( meng et al . furthermore , these differences in gene expression may have a measurable phenotypic effect on brain structure in that bv677278 variants have been associated with differences in gray matter volume in unselected individuals ( meda et al . 2008 ) , so this deletion appears to be an example of a mutation in a regulatory region that affects rd.in addition to influencing rd , snps in dcdc2 have been associated with both hyperactive and inattentive forms of adhd , indicating that this gene can affect both disorders ( couto et al . more recently , evidence has been presented that dcdc2 contributes to the risk for autism in families with both dyslexia and autism ( cuccaro et al . kiaa0319 dcdc2 and kiaa0319 coding regions are separated by only 160 kb and were both included in the candidate region defined by linkage and association analyses that identified the dyx2 locus . association of kiaa0319 with rd phenotypes as well as reading in the normal range has been supported by numerous studies ( newbury et al . although the function of the gene is not clear , knockdown of expression in embryonic rat brain results in delayed neuronal migration ( paracchini et al . 2006 ) , similar to the knockdowns of dyx1c1 and dcdc2 noted above.the snps showing association with rd tend to be located in the 5 utr , the first untranslated exon , and the first intron ( elbert et al . expression of the allele containing the rd - associated snp haplotype in this region of kiaa0319 was shown to be decreased in cell lines from individuals with rd ( paracchini et al . moreover , one associated snp , rs9461045 , has been shown to have a regulatory function . reporter assays showed that the risk allele , which was hypothesized to create a binding site for the repressor oct-1 , resulted in decreased expression of kiaa0319 in vitro , and knockdown of oct-1 restored expression ( dennis et al . 2009).in recognition of the likely influence of epigenetic mechanisms on kiaa0319 expression in the etiology of rd , regions of acetylated histones were mapped in and around the gene in a neuroblastoma cell line to identify promoter regions ( couto et al . a 2.7-kb acetylated region was found spanning the 5 utr , first exon and first intron of kiaa0319 which corresponded to the location of five snps that had been associated with rd phenotypes in other studies . in addition , snps within or very near the acetylated region have been associated with language impairment phenotypes ( newbury et al . 2011 ; rice et al . 2009 ) and linkage to the dcdc2/kiaa0319 region has been reported for ssd ( smith et al . 2005 ) . studies in an unselected population did not show effects on language in the normal range , suggesting that this gene has more of an effect on language impairment ( scerri et al . robo1 linkage analysis of a large family localized rd to a region on chromosome 3 ( 3p12-q13 ) which was designated dyx5 ( nopola - hemmi et al . 2001 ) . a translocation within this region , t(3;8)(p12;q11 ) , was found in an individual with rd and it was determined that this disrupted the robo1 gene ( hannula - jouppi et al . this gene is the human homologue of the roundabout gene in drosophila and mice and is known to affect axonal guidance through the midline of the cns and spinal cord . the coding regions of the robo1 gene were sequenced in the original dyx5 family , but no causal mutations were found . association was found with a haplotype of snps in the gene in this family , and transcription of the allele containing the risk haplotype was decreased in lymphoblasts from individuals with rd . the individuals snps were not felt to have a regulatory function since they were also noted in unaffected individuals , pointing to an unknown regulatory mutation in the individuals with the risk haplotype . although subsequent studies have not replicated the association of robo1 snps with rd , linkage has been found with ssd ( stein and schick 2004 ) and snp association has been found with phonological buffer deficits in an unselected population ( bates et al . 2011 ) indicating that the gene s primary effects could be on language abilities related to rd . mrpl19 and c2orf3 the designation of these two genes as candidates in influencing rd rests on the assumption that the causal mutation is in a regulatory region that is about 34 kb from the genes . the 2p16p12 region was first highlighted by a genome - wide microsatellite linkage study in an extended family ( fagerheim et al . 1999 ) , and subsequent linkage studies replicated these results across the region ( petryshen et al . 2002 ; fisher et al . 2002 ; francks et al . 2002 ; kaminen et al . 2003 ) . snp association studies focused on the 2p12 region , with results indicating a region that did not contain recognizable genes ( peyrard - janvid et al . the transcription products of three nearby genes , flj13391 , mrpl19 , and c2orf3 , were examined to determine if the risk haplotype of snps in that region had an effect on gene expression . there was no effect on the transcription of flj13391 , but transcripts of one allele from mrlp19 and c2orf3 were decreased in individuals who carried the risk haplotypes in the adjacent region ( as determined by heterozygous snps within the coding regions of the two genes ) . this suggested that an unknown mutation in the region of snp association has an effect on gene expression of both genes . the mrpl19 protein is a component of the mitochondrial ribosome , but the function of the c2orf3 gene is unknown.overall , there is substantial evidence for involvement of mutations in regulatory regions of the primary candidate genes influencing rd , and several of these genes also affect related language and learning disorders . further investigation of epigenetic mechanisms of gene regulation is likely to be profitable , including elements that may be quite distant from the genes they affect , or factors that regulate more than one gene . the term epigenetics refers to the controls of gene expression that are maintained through somatic cell division ( and occasionally in germline cells ) but do not involve change in the dna code itself . stable epigenetic controls are applied and subsequently maintained in cell lineages during differentiation and cell proliferation , and reversible epigenetic changes in gene expression can occur in differentiated cells in response to external signals ( jaenisch and bird 2003 ; ptashne 2007 ; day and sweatt 2011 ) . the two major methods of epigenetic regulation involve changes in methylation of cytosines in regulatory regions of dna or modification of histone proteins , primarily through acetylation and methylation . methylation of cytosines in regulatory elements or the complexing of dna around nucleosomes can block gene expression , while removal of dna methylation or relaxing of histone complexing can make dna more accessible . methylation often acts on cpg islands or shores , which are regions of cytosine guanine dinucleotide sequences in promoters of genes , thus inhibiting the binding of transcription factors . one or both strands may be methylated , which can fine - tune the degree of expression . in addition , methylation of these regions can recruit histone modifications that also block transcription machinery . in contrast , methylation within the gene exons and introns is correlated with gene expression . dna methylation is mediated by a family of dnmt enzymes which apply and maintain methylation tags ( day and sweatt 2011 ; portela and esteller 2010 ; gropman and batshaw 2010 ) . histone modification affects the wrapping of dna around nucleosomes , which are octomers composed of two each of four different histone proteins : h2a , h2b , h3 , h4 . dna complexing with nucleosomes is part of chromatin compaction into heterochromatin , which generally is less transcriptionally active . each histone protein has multiple sites that are subject to modification ( methylation , acetylation , phosphorylation , ubiquination , adp ribosylation , or sumoylation ) ( kouzarides 2007 ) . specific sites are designated by the histone type and the amino acid number , such that h4k12 designates the 14th amino acid in an h4 protein , which is a lysine ( k ) . these modifications are reversible , mediated by families of enzymes such as histone acetylases ( hats ) , histone deacetylases ( hdacs ) , histone methylases ( hmts ) , histone demethylases ( hdms ) , and so on . code or language that determines when , where , and how much a particular gene is expressed ( day and sweatt 2011 ; portela and esteller 2010 ; lee et al . since epigenetic mechanisms regulate the differential expression of genes in developing tissues , gene mutations that interfere with dna methylation or histone modification may disrupt multiple organ systems . table 1 gives several examples of developmental cognitive disorders caused by mutations in genes that disrupt epigenetic processes resulting in varying degrees of motor , craniofacial , and skeletal problems in addition to their effects on cognitive abilities . other cognitive disorders such as alzheimer disease and huntington disease develop in adulthood through gradual neurodegeneration secondary to deregulated genes.table 1developmental disorders resulting from disruption of epigenetic mechanisms ( galaburda 2005)mechanismdiseasegeneeffectconsequencesdna methylationrett syndromemecp2hypermethylation , abnormal mrna splicingtranscription repression or activationfragile x syndromefmr1promoter hypermethylationtranscription repressionprader willi syndrome / angelman syndromedel15q11-q13 , ube3aaberrant methylation in imprint control regiontranscription repression or activationimmunodeficiency , centromere instability , facial dysmorphismdnmt3bhypomethylationtranscription activationalzheimer diseasenepcpg island hypomethylationtranscription activationhistone acetylationrubenstein - taybi syndromecbp ( hat)reduced histone acetylation , hypertrimethylation of dnatranscription repressioncoffin - lowry syndromersk32hypophosphorylation of site h3s10increased transcription of map kinase genesoculofaciocardio - dentalbcordisruption of hdacstranscription activationhistone methylationsotos syndromensd1decreased methylation of sites h4k20 , h3k36transcription activation of multiple geneskleefstra syndromeehmt1decreased histone methylationtranscription activationhuntington diseasehttincreased methylation at site h3k9 and possibly h3k27transcription activationgalaburda ( 2005 ) adapted from portela and esteller ( 2010 ) ; day and sweatt ( 2011 ) ; kelly et al . ( 2010 ) ; and gropman and batshaw ( 2010 ) developmental disorders resulting from disruption of epigenetic mechanisms ( galaburda 2005 ) galaburda ( 2005 ) adapted from portela and esteller ( 2010 ) ; day and sweatt ( 2011 ) ; kelly et al . ( 2010 ) ; and gropman and batshaw ( 2010 ) while the effects of mutations of genes that affect epigenetic processes can be severe and disrupt multiple systems , other genetic effects on epigenetic modification can be much more circumscribed . the language of methylated dna and specific histone modifications can precisely control gene expression to produce and maintain tissue - specific and region - specific cellular differentiation . once differentiation is completed , the same regulatory mechanisms appear to be involved in the changes in gene expression that result from learning and memory in the hippocampus ( day and sweatt 2011 ) . for example , certain types of learning are correlated with specific patterns of histone modification in chromatin of hippocampal cells , e.g. , the learning of contextual fear responses in mice is associated with acetylation at h3k9 , h3k14 , h4k5 , h4k8 , and h4k12 , as well as changes in methylation and phosphorylation at other sites . moreover , loss of acetylation at h4k12 interferes with learning , which is normalized by introduction of an hdac inhibitor which restores actylation at that site ( day and sweatt 2011 ; peleg et al . interference with the machinery that applies histone modifications or dna methylation such as hats , hdacs , hdms , or dnmts also cause learning problems ; for example , mutation of the cbp gene in mice , or blockage of dna methylation through inhibition of dnmts will both interfere with memory and long - term potentiation in the hippocampus ( day and sweatt 2011 ; alarcon et al . 2004 ; lubin et al . 2008 ; levenson et al . 2006 ; miller and sweatt 2007 ) . the ehmt gene in humans encodes a histone demethylase and heterozygous deletion of this telomeric gene causes kleefstra syndrome , a condition with severe intellectual disability , dysmorphic features , and behavioral problems such as autistic features , aggression , and bipolar disorder that can change in expression and severity over time ( kleefstra et al . 2009 ) . in drosophila , mutation of the ehmt homologue results in disruption of a jumping reflex and courtship memory . these deficits were also rescued by expression of ehmt in adult flies ( kramer et al . 2011 ) . additional studies of mouse models of alzheimer disease and other neurodegenerative diseases have also shown rescue of learning deficits with treatment by hdacs ( fischer et al . 2007 ; guan et al . 2009 ) . most recently , there have been several reports of alterations of methylation in autism spectrum disorders . alterations in methylation of cpg islands associated with the oxtr oxytocin receptor gene have been reported in brain tissues of individuals with autism spectrum disorders ( gregory et al . abstracts at the international congress of human genetics / american society of human genetics meeting in montreal in october 2011 reported that identical twins discordant for autism had significantly different genome - wide methylation patterns ( wong et al . 2011 ) , and siblings discordant for autism had differences in 5-hydroxymethylcytosine across exonic sequences . finally , dna methylation was altered in cpg islands associated with the candidate gene shank3 in brain samples from individuals with autism spectrum disorders , resulting in an altered pattern of isoform expression ( zhu et al . the influence of more remote regulatory regions was noted in the downregulation of the chrna7 gene in autism by the prader willi imprinting center at 15q11.213.3 ( yasui et al . mouse models of human epigenetic syndromes , such as those listed in table 1 , can show severe phenotypic effects similar to their human counterparts ( unless the models are constructed such that the mutations are only expressed in selected tissues ) ; however , many of these disorders are caused by null mutations that have a significant effect on function . other models of mutations of genes affecting epigenetic regulation can show much milder changes in hippocampal neurons or dendritic spines ( lagali et al . 2010 ) . it seems possible , then , that less disruptive mutations or mutations of other genes may have much more focused effects on development and thus may be much more analogous to deficits that affect reading and language disorders . thus , while mutations affecting epigenetic mechanisms have not been described in reading disability or language impairments , the role of epigenetic changes in learning and autism and the hints of potential therapy make it especially worthwhile to look for mutations in such genes in individuals with language and learning problems . although candidate genes have been identified for reading disability and language impairment , the snps in these genes appear to account for a small portion of the phenotypic variability . in contrast , fairly substantial heritabilities have been claimed for these disorders , between 0.450.85 depending on population and definitions ( gayan and olson 2001 ; hawke et al . there are several possible explanations for this missing heritability , but one of the primary reasons appears to be inherent in the current studies of snps , particularly in the large panels that are used for genome - wide studies . the snps selected for such panels are generally common in the population , which makes them more informative in comparisons between affected and unaffected individuals , but assessment of individual common snps ignores rare variants which are likely to have more impact , and also ignores epistatic interactions between loci ( manolio et al . there are approaches that enhance the identification of causal genes from genome - wide association studies ( gwas ) data such as the simultaneous analysis of multiple variants associated with a gene ( neale and sham 2004 ; huang et al . 2011 ; li et al . 2011 ) or focus on snps associated with loci which show phenotype - based differences in expression ( eqtls or esnps ) ( innocenti et al . 2011 ; majewski and pastinen 2011 ) and next generation sequencing allows the analysis of rare as well as common variants around a gene ; however , sites involved in epigenetic control of gene expression may not be included in the set of loci in gene - based analysis , and the variation in expression of eqtls may be due in part to mutations in epigenetic regions which may be somewhat distant from the gene itself ( ernst et al . the influence of remote regulatory elements is likely to be missing in targeted screening approaches which focus on candidate genes , whether through snp analysis or sequencing . this is due in part to the lack of information on where these regions are located , and initiatives such as the nih epigenomics roadmap program ( http://nihroadmap.nih.gov/epigenomics/initiatives.asp ) and the international human epigenome consortium ( http://www.ihec-epigenomes.org/ ) . these are large collaborative efforts to map regions in the genome that are involved in epigenetic regulation , and the results will assist investigators in identifying regions for evaluation . heritable mutations that influence reading and language disorders could be in the genes that regulate epigenetic processes , analogous to the mutations in hdacs or dnmts , or in genes such as mecp2 or in the mapk signaling pathway ( day and sweatt 2011 ) . genome - wide association studies or even targeted snp analysis might be able to detect such mutations , given that the sample size is large enough , the variants are not rare , and the adjacent snps are in linkage disequilibrium . knowledge of the location of epigenetic regions could help prioritize the follow - up of snps in a gwas that otherwise might be ignored because of lack of apparent functional relevance ( ernst et al . 2011 ) , and location information would also guide the placement of snps in a targeted array . sequence analysis would detect rare variants , but until whole genome sequencing of large populations is financially feasible , targeted sequencing studies are also dependent upon the selection of candidate genes and regulatory regions . studies of epigenetic mechanisms in animal models should produce additional candidate genes for examination in cognitive disorders in humans . another approach would be to look for genomic regions of abnormal methylation or histone modification in individuals with specific forms of language or learning disorders . however , epigenetic patterns are likely to be different in different tissues , and histone modifications especially may change over time . fortunately , there are studies which indicate that methylation patterns affecting disorders can be consistent across tissues , such as lymphocyte and brain methylation patterns in individuals with psychiatric disorders , suggesting that lymphocyte tissues can be a good proxy for brain ( dempster et al . an abnormal methylation pattern in a region of dna from human tissues such as lymphocytes or fibroblasts could indicate an epigenetic process that could be pursued further by determination of the effects of that abnormality on gene expression and the impact on learning in animal models . such studies could be valuable in identifying important genes and signaling pathways involved in learning . further genetic studies such as association and sequencing could assess the influence of these new candidates at the population level . conversely , though , lack of a methylation abnormality in proxy tissues would not rule out the involvement of an epigenetic mechanism that is confined to a region of the brain . there are many approaches to the identification of genes that affect quantitative traits such as language and learning disorders , and the most effective will take advantage of simultaneous analysis of genomic and expression analyses ( charlesworth et al . the inclusion of information on epigenetic mechanisms of gene regulation may turn out to be an important consideration in gene identification and possibly even in therapy . of all of the genes that have been proposed as candidates for reading disability and language impairment , six genes have been well characterized with respect to their influence on reading and language disorders , the regions within and around the genes that appear to contain causal mutations , and the effects of the putative mutations or risk alleles on gene transcription : dyx1c1 , dcdc2 , kiaa0319 , robo1 , and the co - regulated genes mrpl1 and c2orf3 . dyx1c1 the 15q21 region was identified as a candidate region for a gene or genes influencing reading disability ( rd ) through linkage studies ( fulker et al . the dyx1c1 ( dyx1-candidate 1 ) gene was specifically targeted after a translocation t(2;15 ) ( q11;q21 ) disrupting the previously uncharacterized gene was observed in a family with rd ( taipale et al . since then , the dyx1c1 protein has been found to contain estrogen - receptor - binding sites ( massinen et al . 2009 ) and knockdown of the gene in embryonic rat brain produces delays in neuronal migration ( wang et al . 2006).sequence analysis of the dyx1c1 coding regions identified a missense mutation in some rd families : rs57809907 , 1249g > t , which results in glu417x and truncates the protein by four amino acids ( taipale et al . 2003 ) , but this variant has not been consistently associated with reading disability in other studies ( scerri et al . 2004 ; marino et al . 2005 ; meng et al . 2005a ; dahdouh et al . 2009 another missense mutation , rs17819126 ( 271g > a , val91ile ) has been associated with reading ability ( bates et al . 2010 ) , but analyses of putative effect on protein function by the sift ( ng and henikoff 2003 ) and polyphen ( adzhubei et al . 2010 ) algorithms indicate that this should be a benign change for the protein ( ensembl release 63 : www.ensembl.org ) . since studies have found association of rd with other snps within the gene , it seems likely that mutations affecting rd are in the regulatory rather than coding regions . a possible candidate is the 3g > a snp rs3743205 in the 5 untranslated region ( utr ) . in the original report by taipale et al . ( 2005 ) , the a allele was associated with rd , but subsequent reports found association with the common g allele ( scerri et al . interestingly , a study of rd in chinese children also found strong association with the g allele ( lim et al . studies of transcription factor binding in the promoter region have shown that the a allele shows decreased binding to a repressive transcription factor , resulting in increased dyx1c1 expression ( tapia - paez et al . 2008 ) , leading lim et al . to hypothesize that the a allele is actually protective compared to the downregulating g allele , and that instances where the a allele appeared to be associated with rd could be secondary to linkage disequilibrium with a second causal variant nearby two other snps , rs12899331 and rs16787 , in the promoter region were also found to be involved in transcription factor binding ( tapia - paez et al . 2008 ) , but these were not found to be associated with rd in later studies ( dahdouh et al . thus , further studies are needed to define the role of particular regulatory regions of dyx1c1 in the cause of rd.there is also evidence for pleiotropic effects of dyx1c1 on short - term memory and mental calculation , a mathematics measure ( marino et al . linkage to the dyx1c1 region was found with speech sound disorder phenotypes ( ssd ) in one study ( smith et al . 2005 ) but not in a subsequent study ( stein et al . 2006 ) , which located the ssd region more centromerically . further studies are needed to determine whether the linkage signal for ssd was actually related to dyx1c1 . dcdc2 initial linkage and association analysis defined the dyx2 locus on chromosome 6p22 ( grigorenko et al . 1994 ; cardon et al . 1995 ; fisher et al . 1999 ; gayan et al . 1999 ; kaplan et al . 2002 ; deffenbacher et al . 2004 ) , and several subsequent studies have reported associations with the dcdc2 ( doublecortin-2 ) gene within the region ( newbury et al . 2011 ; meng et al . the structure of the gene is analogous to the x - linked dcx gene which is known to be involved in microtubular structure and influences neuronal migration . mutation of the dcx gene produces lissencephaly in males and cortical abnormalities in females ( des portes et al . 1998 ) ; accordingly , knockdown of dcdc2 produces delays in neuronal migration in embryonic rat brain ( meng et al . 2005b).sequence analysis of coding regions of dcdc2 in rd families has not identified causal mutations ; however , association of rd was reported with a deletion in intron 2 , termed bv677278 , which appeared to contain transcription factor binding sites ( meng et al . 2008 ) , but other studies have replicated it ( brkanac et al . 2007 ; harold et al . 2006 ; wilcke et al . 2009 ; marino et al . still , the importance of this region in gene regulation has been demonstrated by in vitro studies showing that sequences in the region act as enhancers for dcdc2 expression ( meng et al . furthermore , these differences in gene expression may have a measurable phenotypic effect on brain structure in that bv677278 variants have been associated with differences in gray matter volume in unselected individuals ( meda et al . 2008 ) , so this deletion appears to be an example of a mutation in a regulatory region that affects rd.in addition to influencing rd , snps in dcdc2 have been associated with both hyperactive and inattentive forms of adhd , indicating that this gene can affect both disorders ( couto et al . more recently , evidence has been presented that dcdc2 contributes to the risk for autism in families with both dyslexia and autism ( cuccaro et al . kiaa0319 dcdc2 and kiaa0319 coding regions are separated by only 160 kb and were both included in the candidate region defined by linkage and association analyses that identified the dyx2 locus . association of kiaa0319 with rd phenotypes as well as reading in the normal range has been supported by numerous studies ( newbury et al . 2011 ; dennis et al . 2009 ; scerri et al . 2011 ; harold et al . 2006 ; cope et al . 2005 ; paracchini et al . 2006 ; luciano et al . 2007 ; paracchini et al . 2008 ) . although the function of the gene is not clear , knockdown of expression in embryonic rat brain results in delayed neuronal migration ( paracchini et al . 2006 ) , similar to the knockdowns of dyx1c1 and dcdc2 noted above.the snps showing association with rd tend to be located in the 5 utr , the first untranslated exon , and the first intron ( elbert et al . expression of the allele containing the rd - associated snp haplotype in this region of kiaa0319 was shown to be decreased in cell lines from individuals with rd ( paracchini et al . moreover , one associated snp , rs9461045 , has been shown to have a regulatory function . reporter assays showed that the risk allele , which was hypothesized to create a binding site for the repressor oct-1 , resulted in decreased expression of kiaa0319 in vitro , and knockdown of oct-1 restored expression ( dennis et al . 2009).in recognition of the likely influence of epigenetic mechanisms on kiaa0319 expression in the etiology of rd , regions of acetylated histones were mapped in and around the gene in a neuroblastoma cell line to identify promoter regions ( couto et al . a 2.7-kb acetylated region was found spanning the 5 utr , first exon and first intron of kiaa0319 which corresponded to the location of five snps that had been associated with rd phenotypes in other studies . in addition , snps within or very near the acetylated region have been associated with language impairment phenotypes ( newbury et al . 2011 ; rice et al . 2009 ) and linkage to the dcdc2/kiaa0319 region has been reported for ssd ( smith et al . 2005 ) . studies in an unselected population did not show effects on language in the normal range , suggesting that this gene has more of an effect on language impairment ( scerri et al . robo1 linkage analysis of a large family localized rd to a region on chromosome 3 ( 3p12-q13 ) which was designated dyx5 ( nopola - hemmi et al . 2001 ) . a translocation within this region , t(3;8)(p12;q11 ) , was found in an individual with rd and it was determined that this disrupted the robo1 gene ( hannula - jouppi et al . this gene is the human homologue of the roundabout gene in drosophila and mice and is known to affect axonal guidance through the midline of the cns and spinal cord . the coding regions of the robo1 gene were sequenced in the original dyx5 family , but no causal mutations were found . association was found with a haplotype of snps in the gene in this family , and transcription of the allele containing the risk haplotype was decreased in lymphoblasts from individuals with rd . the individuals snps were not felt to have a regulatory function since they were also noted in unaffected individuals , pointing to an unknown regulatory mutation in the individuals with the risk haplotype . although subsequent studies have not replicated the association of robo1 snps with rd , linkage has been found with ssd ( stein and schick 2004 ) and snp association has been found with phonological buffer deficits in an unselected population ( bates et al . 2011 ) indicating that the gene s primary effects could be on language abilities related to rd . mrpl19 and c2orf3 the designation of these two genes as candidates in influencing rd rests on the assumption that the causal mutation is in a regulatory region that is about 34 kb from the genes . the 2p16p12 region was first highlighted by a genome - wide microsatellite linkage study in an extended family ( fagerheim et al . 1999 ) , and subsequent linkage studies replicated these results across the region ( petryshen et al . snp association studies focused on the 2p12 region , with results indicating a region that did not contain recognizable genes ( peyrard - janvid et al . the transcription products of three nearby genes , flj13391 , mrpl19 , and c2orf3 , were examined to determine if the risk haplotype of snps in that region had an effect on gene expression . there was no effect on the transcription of flj13391 , but transcripts of one allele from mrlp19 and c2orf3 were decreased in individuals who carried the risk haplotypes in the adjacent region ( as determined by heterozygous snps within the coding regions of the two genes ) . this suggested that an unknown mutation in the region of snp association has an effect on gene expression of both genes . the mrpl19 protein is a component of the mitochondrial ribosome , but the function of the c2orf3 gene is unknown.overall , there is substantial evidence for involvement of mutations in regulatory regions of the primary candidate genes influencing rd , and several of these genes also affect related language and learning disorders . further investigation of epigenetic mechanisms of gene regulation is likely to be profitable , including elements that may be quite distant from the genes they affect , or factors that regulate more than one gene . the term epigenetics refers to the controls of gene expression that are maintained through somatic cell division ( and occasionally in germline cells ) but do not involve change in the dna code itself . stable epigenetic controls are applied and subsequently maintained in cell lineages during differentiation and cell proliferation , and reversible epigenetic changes in gene expression can occur in differentiated cells in response to external signals ( jaenisch and bird 2003 ; ptashne 2007 ; day and sweatt 2011 ) . the two major methods of epigenetic regulation involve changes in methylation of cytosines in regulatory regions of dna or modification of histone proteins , primarily through acetylation and methylation . methylation of cytosines in regulatory elements or the complexing of dna around nucleosomes can block gene expression , while removal of dna methylation or relaxing of histone complexing can make dna more accessible . methylation often acts on cpg islands or shores , which are regions of cytosine guanine dinucleotide sequences in promoters of genes , thus inhibiting the binding of transcription factors . one or both strands may be methylated , which can fine - tune the degree of expression . in addition , methylation of these regions can recruit histone modifications that also block transcription machinery . in contrast , methylation within the gene exons and introns is correlated with gene expression . dna methylation is mediated by a family of dnmt enzymes which apply and maintain methylation tags ( day and sweatt 2011 ; portela and esteller 2010 ; gropman and batshaw 2010 ) . histone modification affects the wrapping of dna around nucleosomes , which are octomers composed of two each of four different histone proteins : h2a , h2b , h3 , h4 . dna complexing with nucleosomes is part of chromatin compaction into heterochromatin , which generally is less transcriptionally active . each histone protein has multiple sites that are subject to modification ( methylation , acetylation , phosphorylation , ubiquination , adp ribosylation , or sumoylation ) ( kouzarides 2007 ) . specific sites are designated by the histone type and the amino acid number , such that h4k12 designates the 14th amino acid in an h4 protein , which is a lysine ( k ) . these modifications are reversible , mediated by families of enzymes such as histone acetylases ( hats ) , histone deacetylases ( hdacs ) , histone methylases ( hmts ) , histone demethylases ( hdms ) , and so on . code or language that determines when , where , and how much a particular gene is expressed ( day and sweatt 2011 ; portela and esteller 2010 ; lee et al . since epigenetic mechanisms regulate the differential expression of genes in developing tissues , gene mutations that interfere with dna methylation or histone modification may disrupt multiple organ systems . table 1 gives several examples of developmental cognitive disorders caused by mutations in genes that disrupt epigenetic processes resulting in varying degrees of motor , craniofacial , and skeletal problems in addition to their effects on cognitive abilities . other cognitive disorders such as alzheimer disease and huntington disease develop in adulthood through gradual neurodegeneration secondary to deregulated genes.table 1developmental disorders resulting from disruption of epigenetic mechanisms ( galaburda 2005)mechanismdiseasegeneeffectconsequencesdna methylationrett syndromemecp2hypermethylation , abnormal mrna splicingtranscription repression or activationfragile x syndromefmr1promoter hypermethylationtranscription repressionprader willi syndrome / angelman syndromedel15q11-q13 , ube3aaberrant methylation in imprint control regiontranscription repression or activationimmunodeficiency , centromere instability , facial dysmorphismdnmt3bhypomethylationtranscription activationalzheimer diseasenepcpg island hypomethylationtranscription activationhistone acetylationrubenstein - taybi syndromecbp ( hat)reduced histone acetylation , hypertrimethylation of dnatranscription repressioncoffin - lowry syndromersk32hypophosphorylation of site h3s10increased transcription of map kinase genesoculofaciocardio - dentalbcordisruption of hdacstranscription activationhistone methylationsotos syndromensd1decreased methylation of sites h4k20 , h3k36transcription activation of multiple geneskleefstra syndromeehmt1decreased histone methylationtranscription activationhuntington diseasehttincreased methylation at site h3k9 and possibly h3k27transcription activationgalaburda ( 2005 ) adapted from portela and esteller ( 2010 ) ; day and sweatt ( 2011 ) ; kelly et al . ( 2010 ) ; and gropman and batshaw ( 2010 ) developmental disorders resulting from disruption of epigenetic mechanisms ( galaburda 2005 ) galaburda ( 2005 ) adapted from portela and esteller ( 2010 ) ; day and sweatt ( 2011 ) ; kelly et al . ( 2010 ) ; and gropman and batshaw ( 2010 ) while the effects of mutations of genes that affect epigenetic processes can be severe and disrupt multiple systems , other genetic effects on epigenetic modification can be much more circumscribed . the language of methylated dna and specific histone modifications can precisely control gene expression to produce and maintain tissue - specific and region - specific cellular differentiation . once differentiation is completed , the same regulatory mechanisms appear to be involved in the changes in gene expression that result from learning and memory in the hippocampus ( day and sweatt 2011 ) . for example , certain types of learning are correlated with specific patterns of histone modification in chromatin of hippocampal cells , e.g. , the learning of contextual fear responses in mice is associated with acetylation at h3k9 , h3k14 , h4k5 , h4k8 , and h4k12 , as well as changes in methylation and phosphorylation at other sites . moreover , loss of acetylation at h4k12 interferes with learning , which is normalized by introduction of an hdac inhibitor which restores actylation at that site ( day and sweatt 2011 ; peleg et al . interference with the machinery that applies histone modifications or dna methylation such as hats , hdacs , hdms , or dnmts also cause learning problems ; for example , mutation of the cbp gene in mice , or blockage of dna methylation through inhibition of dnmts will both interfere with memory and long - term potentiation in the hippocampus ( day and sweatt 2011 ; alarcon et al . the ehmt gene in humans encodes a histone demethylase and heterozygous deletion of this telomeric gene causes kleefstra syndrome , a condition with severe intellectual disability , dysmorphic features , and behavioral problems such as autistic features , aggression , and bipolar disorder that can change in expression and severity over time ( kleefstra et al . mutation of the ehmt homologue results in disruption of a jumping reflex and courtship memory . these deficits were also rescued by expression of ehmt in adult flies ( kramer et al . 2011 ) . additional studies of mouse models of alzheimer disease and other neurodegenerative diseases have also shown rescue of learning deficits with treatment by hdacs ( fischer et al . 2007 ; guan et al . 2009 ) . most recently , there have been several reports of alterations of methylation in autism spectrum disorders . alterations in methylation of cpg islands associated with the oxtr oxytocin receptor gene have been reported in brain tissues of individuals with autism spectrum disorders ( gregory et al . abstracts at the international congress of human genetics / american society of human genetics meeting in montreal in october 2011 reported that identical twins discordant for autism had significantly different genome - wide methylation patterns ( wong et al . 2011 ) , and siblings discordant for autism had differences in 5-hydroxymethylcytosine across exonic sequences . finally , dna methylation was altered in cpg islands associated with the candidate gene shank3 in brain samples from individuals with autism spectrum disorders , resulting in an altered pattern of isoform expression ( zhu et al . the influence of more remote regulatory regions was noted in the downregulation of the chrna7 gene in autism by the prader willi imprinting center at 15q11.213.3 ( yasui et al . mouse models of human epigenetic syndromes , such as those listed in table 1 , can show severe phenotypic effects similar to their human counterparts ( unless the models are constructed such that the mutations are only expressed in selected tissues ) ; however , many of these disorders are caused by null mutations that have a significant effect on function . other models of mutations of genes affecting epigenetic regulation can show much milder changes in hippocampal neurons or dendritic spines ( lagali et al . it seems possible , then , that less disruptive mutations or mutations of other genes may have much more focused effects on development and thus may be much more analogous to deficits that affect reading and language disorders . thus , while mutations affecting epigenetic mechanisms have not been described in reading disability or language impairments , the role of epigenetic changes in learning and autism and the hints of potential therapy make it especially worthwhile to look for mutations in such genes in individuals with language and learning problems . although candidate genes have been identified for reading disability and language impairment , the snps in these genes appear to account for a small portion of the phenotypic variability . in contrast , fairly substantial heritabilities have been claimed for these disorders , between 0.450.85 depending on population and definitions ( gayan and olson 2001 ; hawke et al . there are several possible explanations for this missing heritability , but one of the primary reasons appears to be inherent in the current studies of snps , particularly in the large panels that are used for genome - wide studies . the snps selected for such panels are generally common in the population , which makes them more informative in comparisons between affected and unaffected individuals , but assessment of individual common snps ignores rare variants which are likely to have more impact , and also ignores epistatic interactions between loci ( manolio et al . 2009 ) . there are approaches that enhance the identification of causal genes from genome - wide association studies ( gwas ) data such as the simultaneous analysis of multiple variants associated with a gene ( neale and sham 2004 ; huang et al . 2011 ; li et al . 2011 ) or focus on snps associated with loci which show phenotype - based differences in expression ( eqtls or esnps ) ( innocenti et al . 2011 ; majewski and pastinen 2011 ) and next generation sequencing allows the analysis of rare as well as common variants around a gene ; however , sites involved in epigenetic control of gene expression may not be included in the set of loci in gene - based analysis , and the variation in expression of eqtls may be due in part to mutations in epigenetic regions which may be somewhat distant from the gene itself ( ernst et al . the influence of remote regulatory elements is likely to be missing in targeted screening approaches which focus on candidate genes , whether through snp analysis or sequencing . this is due in part to the lack of information on where these regions are located , and initiatives such as the nih epigenomics roadmap program ( http://nihroadmap.nih.gov/epigenomics/initiatives.asp ) and the international human epigenome consortium ( http://www.ihec-epigenomes.org/ ) . these are large collaborative efforts to map regions in the genome that are involved in epigenetic regulation , and the results will assist investigators in identifying regions for evaluation . heritable mutations that influence reading and language disorders could be in the genes that regulate epigenetic processes , analogous to the mutations in hdacs or dnmts , or in genes such as mecp2 or in the mapk signaling pathway ( day and sweatt 2011 ) . genome - wide association studies or even targeted snp analysis might be able to detect such mutations , given that the sample size is large enough , the variants are not rare , and the adjacent snps are in linkage disequilibrium . knowledge of the location of epigenetic regions could help prioritize the follow - up of snps in a gwas that otherwise might be ignored because of lack of apparent functional relevance ( ernst et al . 2011 ) , and location information would also guide the placement of snps in a targeted array . sequence analysis would detect rare variants , but until whole genome sequencing of large populations is financially feasible , targeted sequencing studies are also dependent upon the selection of candidate genes and regulatory regions . studies of epigenetic mechanisms in animal models should produce additional candidate genes for examination in cognitive disorders in humans . another approach would be to look for genomic regions of abnormal methylation or histone modification in individuals with specific forms of language or learning disorders . however , epigenetic patterns are likely to be different in different tissues , and histone modifications especially may change over time . fortunately , there are studies which indicate that methylation patterns affecting disorders can be consistent across tissues , such as lymphocyte and brain methylation patterns in individuals with psychiatric disorders , suggesting that lymphocyte tissues can be a good proxy for brain ( dempster et al . an abnormal methylation pattern in a region of dna from human tissues such as lymphocytes or fibroblasts could indicate an epigenetic process that could be pursued further by determination of the effects of that abnormality on gene expression and the impact on learning in animal models . such studies could be valuable in identifying important genes and signaling pathways involved in learning . further genetic studies such as association and sequencing could assess the influence of these new candidates at the population level . conversely , though , lack of a methylation abnormality in proxy tissues would not rule out the involvement of an epigenetic mechanism that is confined to a region of the brain . there are many approaches to the identification of genes that affect quantitative traits such as language and learning disorders , and the most effective will take advantage of simultaneous analysis of genomic and expression analyses ( charlesworth et al . the inclusion of information on epigenetic mechanisms of gene regulation may turn out to be an important consideration in gene identification and possibly even in therapy .

language and learning disorders such as reading disability and language impairment are recognized to be subject to substantial genetic influences , but few causal mutations have been identified in the coding regions of candidate genes . association analyses of single nucleotide polymorphisms have suggested the involvement of regulatory regions of these genes , and a few mutations affecting gene expression levels have been identified , indicating that the quantity rather than the quality of the gene product may be most relevant for these disorders . in addition , several of the candidate genes appear to be involved in neuronal migration , confirming the importance of early developmental processes . accordingly , alterations in epigenetic processes such as dna methylation and histone modification are likely to be important in the causes of language and learning disorders based on their functions in gene regulation . epigenetic processes direct the differentiation of cells in early development when neurological pathways are set down , and mutations in genes involved in epigenetic regulation are known to cause cognitive disorders in humans . epigenetic processes also regulate the changes in gene expression in response to learning , and alterations in histone modification are associated with learning and memory deficits in animals . genetic defects in histone modification have been reversed in animals through therapeutic interventions resulting in rescue of these deficits , making it particularly important to investigate their potential contribution to learning disorders in humans .

Introduction Regulatory regions of genes influencing language and learning disorders Mechanisms of epigenetic gene regulation Mutations of genes affecting epigenetic mechanisms in humans and animal models Approaches to the identification of epigenetic mechanisms in humans

despite the complexity of language and learning disorders , individual genes are being defined which appear to influence the development of abilities that are necessary in speech , language , and reading . most of the identified candidate genes involve reading disability , and although the evidence supporting some of these genes is still somewhat tenuous due to small sample sizes and limited replication , most are known to be involved in early development , particularly neuronal migration ( galaburda 2005 ; gabel et al . most of these candidate genes have been associated with several learning and language phenotypes , suggesting that they facilitate learning processes which are basic to learning reading and language . however , despite replicated evidence for association of single nucleotide polymorphisms within and around the genes , very few coding mutations have been reported to account for their influence on these disorders . this has led to the hypothesis that mutations affecting reading and related disorders are likely to be in regulatory regions , controlling the quantity rather than quality of the gene product ( bates et al . alterations of gene expression can be caused by mutations in gene promoters and enhancers located near the gene , but mutations in genes that mediate epigenetic controls of gene expression have been found that affect developmental learning disorders . of all of the genes that have been proposed as candidates for reading disability and language impairment , six genes have been well characterized with respect to their influence on reading and language disorders , the regions within and around the genes that appear to contain causal mutations , and the effects of the putative mutations or risk alleles on gene transcription : dyx1c1 , dcdc2 , kiaa0319 , robo1 , and the co - regulated genes mrpl1 and c2orf3 . 2009 ) and knockdown of the gene in embryonic rat brain produces delays in neuronal migration ( wang et al . since studies have found association of rd with other snps within the gene , it seems likely that mutations affecting rd are in the regulatory rather than coding regions . to hypothesize that the a allele is actually protective compared to the downregulating g allele , and that instances where the a allele appeared to be associated with rd could be secondary to linkage disequilibrium with a second causal variant nearby . two other snps , rs12899331 and rs16787 , in the promoter region were also found to be involved in transcription factor binding ( tapia - paez et al . the structure of the gene is analogous to the x - linked dcx gene which is known to be involved in microtubular structure and influences neuronal migration . 1998 ) ; accordingly , knockdown of dcdc2 produces delays in neuronal migration in embryonic rat brain ( meng et al . still , the importance of this region in gene regulation has been demonstrated by in vitro studies showing that sequences in the region act as enhancers for dcdc2 expression ( meng et al . furthermore , these differences in gene expression may have a measurable phenotypic effect on brain structure in that bv677278 variants have been associated with differences in gray matter volume in unselected individuals ( meda et al . 2008 ) , so this deletion appears to be an example of a mutation in a regulatory region that affects rd.in addition to influencing rd , snps in dcdc2 have been associated with both hyperactive and inattentive forms of adhd , indicating that this gene can affect both disorders ( couto et al . kiaa0319 dcdc2 and kiaa0319 coding regions are separated by only 160 kb and were both included in the candidate region defined by linkage and association analyses that identified the dyx2 locus . although the function of the gene is not clear , knockdown of expression in embryonic rat brain results in delayed neuronal migration ( paracchini et al . 2009).in recognition of the likely influence of epigenetic mechanisms on kiaa0319 expression in the etiology of rd , regions of acetylated histones were mapped in and around the gene in a neuroblastoma cell line to identify promoter regions ( couto et al . in addition , snps within or very near the acetylated region have been associated with language impairment phenotypes ( newbury et al . the coding regions of the robo1 gene were sequenced in the original dyx5 family , but no causal mutations were found . association was found with a haplotype of snps in the gene in this family , and transcription of the allele containing the risk haplotype was decreased in lymphoblasts from individuals with rd . there was no effect on the transcription of flj13391 , but transcripts of one allele from mrlp19 and c2orf3 were decreased in individuals who carried the risk haplotypes in the adjacent region ( as determined by heterozygous snps within the coding regions of the two genes ) . the mrpl19 protein is a component of the mitochondrial ribosome , but the function of the c2orf3 gene is unknown.overall , there is substantial evidence for involvement of mutations in regulatory regions of the primary candidate genes influencing rd , and several of these genes also affect related language and learning disorders . further investigation of epigenetic mechanisms of gene regulation is likely to be profitable , including elements that may be quite distant from the genes they affect , or factors that regulate more than one gene . stable epigenetic controls are applied and subsequently maintained in cell lineages during differentiation and cell proliferation , and reversible epigenetic changes in gene expression can occur in differentiated cells in response to external signals ( jaenisch and bird 2003 ; ptashne 2007 ; day and sweatt 2011 ) . the two major methods of epigenetic regulation involve changes in methylation of cytosines in regulatory regions of dna or modification of histone proteins , primarily through acetylation and methylation . since epigenetic mechanisms regulate the differential expression of genes in developing tissues , gene mutations that interfere with dna methylation or histone modification may disrupt multiple organ systems . table 1 gives several examples of developmental cognitive disorders caused by mutations in genes that disrupt epigenetic processes resulting in varying degrees of motor , craniofacial , and skeletal problems in addition to their effects on cognitive abilities . once differentiation is completed , the same regulatory mechanisms appear to be involved in the changes in gene expression that result from learning and memory in the hippocampus ( day and sweatt 2011 ) . , the learning of contextual fear responses in mice is associated with acetylation at h3k9 , h3k14 , h4k5 , h4k8 , and h4k12 , as well as changes in methylation and phosphorylation at other sites . interference with the machinery that applies histone modifications or dna methylation such as hats , hdacs , hdms , or dnmts also cause learning problems ; for example , mutation of the cbp gene in mice , or blockage of dna methylation through inhibition of dnmts will both interfere with memory and long - term potentiation in the hippocampus ( day and sweatt 2011 ; alarcon et al . finally , dna methylation was altered in cpg islands associated with the candidate gene shank3 in brain samples from individuals with autism spectrum disorders , resulting in an altered pattern of isoform expression ( zhu et al . mouse models of human epigenetic syndromes , such as those listed in table 1 , can show severe phenotypic effects similar to their human counterparts ( unless the models are constructed such that the mutations are only expressed in selected tissues ) ; however , many of these disorders are caused by null mutations that have a significant effect on function . thus , while mutations affecting epigenetic mechanisms have not been described in reading disability or language impairments , the role of epigenetic changes in learning and autism and the hints of potential therapy make it especially worthwhile to look for mutations in such genes in individuals with language and learning problems . although candidate genes have been identified for reading disability and language impairment , the snps in these genes appear to account for a small portion of the phenotypic variability . there are several possible explanations for this missing heritability , but one of the primary reasons appears to be inherent in the current studies of snps , particularly in the large panels that are used for genome - wide studies . the snps selected for such panels are generally common in the population , which makes them more informative in comparisons between affected and unaffected individuals , but assessment of individual common snps ignores rare variants which are likely to have more impact , and also ignores epistatic interactions between loci ( manolio et al . 2011 ; majewski and pastinen 2011 ) and next generation sequencing allows the analysis of rare as well as common variants around a gene ; however , sites involved in epigenetic control of gene expression may not be included in the set of loci in gene - based analysis , and the variation in expression of eqtls may be due in part to mutations in epigenetic regions which may be somewhat distant from the gene itself ( ernst et al . the influence of remote regulatory elements is likely to be missing in targeted screening approaches which focus on candidate genes , whether through snp analysis or sequencing . these are large collaborative efforts to map regions in the genome that are involved in epigenetic regulation , and the results will assist investigators in identifying regions for evaluation . heritable mutations that influence reading and language disorders could be in the genes that regulate epigenetic processes , analogous to the mutations in hdacs or dnmts , or in genes such as mecp2 or in the mapk signaling pathway ( day and sweatt 2011 ) . sequence analysis would detect rare variants , but until whole genome sequencing of large populations is financially feasible , targeted sequencing studies are also dependent upon the selection of candidate genes and regulatory regions . studies of epigenetic mechanisms in animal models should produce additional candidate genes for examination in cognitive disorders in humans . another approach would be to look for genomic regions of abnormal methylation or histone modification in individuals with specific forms of language or learning disorders . however , epigenetic patterns are likely to be different in different tissues , and histone modifications especially may change over time . there are many approaches to the identification of genes that affect quantitative traits such as language and learning disorders , and the most effective will take advantage of simultaneous analysis of genomic and expression analyses ( charlesworth et al . of all of the genes that have been proposed as candidates for reading disability and language impairment , six genes have been well characterized with respect to their influence on reading and language disorders , the regions within and around the genes that appear to contain causal mutations , and the effects of the putative mutations or risk alleles on gene transcription : dyx1c1 , dcdc2 , kiaa0319 , robo1 , and the co - regulated genes mrpl1 and c2orf3 . 2009 ) and knockdown of the gene in embryonic rat brain produces delays in neuronal migration ( wang et al . 2003 ) , but this variant has not been consistently associated with reading disability in other studies ( scerri et al . since studies have found association of rd with other snps within the gene , it seems likely that mutations affecting rd are in the regulatory rather than coding regions . to hypothesize that the a allele is actually protective compared to the downregulating g allele , and that instances where the a allele appeared to be associated with rd could be secondary to linkage disequilibrium with a second causal variant nearby two other snps , rs12899331 and rs16787 , in the promoter region were also found to be involved in transcription factor binding ( tapia - paez et al . the structure of the gene is analogous to the x - linked dcx gene which is known to be involved in microtubular structure and influences neuronal migration . still , the importance of this region in gene regulation has been demonstrated by in vitro studies showing that sequences in the region act as enhancers for dcdc2 expression ( meng et al . furthermore , these differences in gene expression may have a measurable phenotypic effect on brain structure in that bv677278 variants have been associated with differences in gray matter volume in unselected individuals ( meda et al . 2008 ) , so this deletion appears to be an example of a mutation in a regulatory region that affects rd.in addition to influencing rd , snps in dcdc2 have been associated with both hyperactive and inattentive forms of adhd , indicating that this gene can affect both disorders ( couto et al . kiaa0319 dcdc2 and kiaa0319 coding regions are separated by only 160 kb and were both included in the candidate region defined by linkage and association analyses that identified the dyx2 locus . although the function of the gene is not clear , knockdown of expression in embryonic rat brain results in delayed neuronal migration ( paracchini et al . 2009).in recognition of the likely influence of epigenetic mechanisms on kiaa0319 expression in the etiology of rd , regions of acetylated histones were mapped in and around the gene in a neuroblastoma cell line to identify promoter regions ( couto et al . in addition , snps within or very near the acetylated region have been associated with language impairment phenotypes ( newbury et al . the coding regions of the robo1 gene were sequenced in the original dyx5 family , but no causal mutations were found . association was found with a haplotype of snps in the gene in this family , and transcription of the allele containing the risk haplotype was decreased in lymphoblasts from individuals with rd . 2011 ) indicating that the gene s primary effects could be on language abilities related to rd . there was no effect on the transcription of flj13391 , but transcripts of one allele from mrlp19 and c2orf3 were decreased in individuals who carried the risk haplotypes in the adjacent region ( as determined by heterozygous snps within the coding regions of the two genes ) . the mrpl19 protein is a component of the mitochondrial ribosome , but the function of the c2orf3 gene is unknown.overall , there is substantial evidence for involvement of mutations in regulatory regions of the primary candidate genes influencing rd , and several of these genes also affect related language and learning disorders . further investigation of epigenetic mechanisms of gene regulation is likely to be profitable , including elements that may be quite distant from the genes they affect , or factors that regulate more than one gene . stable epigenetic controls are applied and subsequently maintained in cell lineages during differentiation and cell proliferation , and reversible epigenetic changes in gene expression can occur in differentiated cells in response to external signals ( jaenisch and bird 2003 ; ptashne 2007 ; day and sweatt 2011 ) . the two major methods of epigenetic regulation involve changes in methylation of cytosines in regulatory regions of dna or modification of histone proteins , primarily through acetylation and methylation . table 1 gives several examples of developmental cognitive disorders caused by mutations in genes that disrupt epigenetic processes resulting in varying degrees of motor , craniofacial , and skeletal problems in addition to their effects on cognitive abilities . once differentiation is completed , the same regulatory mechanisms appear to be involved in the changes in gene expression that result from learning and memory in the hippocampus ( day and sweatt 2011 ) . , the learning of contextual fear responses in mice is associated with acetylation at h3k9 , h3k14 , h4k5 , h4k8 , and h4k12 , as well as changes in methylation and phosphorylation at other sites . interference with the machinery that applies histone modifications or dna methylation such as hats , hdacs , hdms , or dnmts also cause learning problems ; for example , mutation of the cbp gene in mice , or blockage of dna methylation through inhibition of dnmts will both interfere with memory and long - term potentiation in the hippocampus ( day and sweatt 2011 ; alarcon et al . the ehmt gene in humans encodes a histone demethylase and heterozygous deletion of this telomeric gene causes kleefstra syndrome , a condition with severe intellectual disability , dysmorphic features , and behavioral problems such as autistic features , aggression , and bipolar disorder that can change in expression and severity over time ( kleefstra et al . alterations in methylation of cpg islands associated with the oxtr oxytocin receptor gene have been reported in brain tissues of individuals with autism spectrum disorders ( gregory et al . finally , dna methylation was altered in cpg islands associated with the candidate gene shank3 in brain samples from individuals with autism spectrum disorders , resulting in an altered pattern of isoform expression ( zhu et al . mouse models of human epigenetic syndromes , such as those listed in table 1 , can show severe phenotypic effects similar to their human counterparts ( unless the models are constructed such that the mutations are only expressed in selected tissues ) ; however , many of these disorders are caused by null mutations that have a significant effect on function . thus , while mutations affecting epigenetic mechanisms have not been described in reading disability or language impairments , the role of epigenetic changes in learning and autism and the hints of potential therapy make it especially worthwhile to look for mutations in such genes in individuals with language and learning problems . although candidate genes have been identified for reading disability and language impairment , the snps in these genes appear to account for a small portion of the phenotypic variability . there are several possible explanations for this missing heritability , but one of the primary reasons appears to be inherent in the current studies of snps , particularly in the large panels that are used for genome - wide studies . the snps selected for such panels are generally common in the population , which makes them more informative in comparisons between affected and unaffected individuals , but assessment of individual common snps ignores rare variants which are likely to have more impact , and also ignores epistatic interactions between loci ( manolio et al . 2011 ; majewski and pastinen 2011 ) and next generation sequencing allows the analysis of rare as well as common variants around a gene ; however , sites involved in epigenetic control of gene expression may not be included in the set of loci in gene - based analysis , and the variation in expression of eqtls may be due in part to mutations in epigenetic regions which may be somewhat distant from the gene itself ( ernst et al . the influence of remote regulatory elements is likely to be missing in targeted screening approaches which focus on candidate genes , whether through snp analysis or sequencing . these are large collaborative efforts to map regions in the genome that are involved in epigenetic regulation , and the results will assist investigators in identifying regions for evaluation . heritable mutations that influence reading and language disorders could be in the genes that regulate epigenetic processes , analogous to the mutations in hdacs or dnmts , or in genes such as mecp2 or in the mapk signaling pathway ( day and sweatt 2011 ) . sequence analysis would detect rare variants , but until whole genome sequencing of large populations is financially feasible , targeted sequencing studies are also dependent upon the selection of candidate genes and regulatory regions . studies of epigenetic mechanisms in animal models should produce additional candidate genes for examination in cognitive disorders in humans . another approach would be to look for genomic regions of abnormal methylation or histone modification in individuals with specific forms of language or learning disorders . however , epigenetic patterns are likely to be different in different tissues , and histone modifications especially may change over time . an abnormal methylation pattern in a region of dna from human tissues such as lymphocytes or fibroblasts could indicate an epigenetic process that could be pursued further by determination of the effects of that abnormality on gene expression and the impact on learning in animal models . there are many approaches to the identification of genes that affect quantitative traits such as language and learning disorders , and the most effective will take advantage of simultaneous analysis of genomic and expression analyses ( charlesworth et al .

betagene participants are mexican - american adults ( both parents and three or more grandparents are mexican or of mexican descent ) who are 1 ) women who had gestational diabetes mellitus ( gdm ) within the previous 5 years , 2 ) siblings or cousins of those with a history of gdm , or 3 ) women with normal glucose levels during pregnancy in the past 5 years . women documented with and without previous gdm were identified from the los angeles county / university of southern california medical center , kaiser permanente southern california s delivery population , and obstetrical / gynecological clinics at local southern california hospitals . women without previous gdm were frequency - matched to gdm cases by age , bmi , and parity . details regarding recruitment have been previously described ( 14 ) . all protocols for betagene were approved by the institutional review boards of participating institutions , and all participants provided written informed consent before participation . research data were collected in two separate visits to the general clinical research center at the university of southern california . the first visit consisted of a physical examination , dietary and pa questionnaires , a 2-h , 75-g oral glucose tolerance test ( ogtt ) , and fasting blood for lipid measurements ( 15 ) . participants with fasting glucose < 7.0 mmol / l were invited for a second visit , which consisted of a dual - energy x - ray absorptiometry scan for body composition and an insulin - modified intravenous glucose tolerance test ( ivgtt ) for measurement of insulin sensitivity and -cell function ( 16 ) . plasma glucose was measured on an autoanalyzer using the glucose oxidase method ( ysi model 2300 ; yellow springs instruments , yellow springs , oh ) . insulin was measured by a two - site immunoenzymometric assay ( tosoh biosciences , san francisco , ca ) that has < 0.1% cross - reactivity with proinsulin and intermediate split products . trained bilingual ( english / spanish ) interviewers administered both pa and dietary questionnaires . the amount and intensity of pa was assessed by questionnaires developed in the hawaii - los angeles multiethnic cohort study ( 17,18 ) . this questionnaire is comprised of a list of structured questions describing various types of activity ( sitting , strenuous sports , vigorous work , and moderate activities including sports and work ) during the past year . responses were then used to estimate the total minutes of moderate and vigorous activity per week . the questionnaire was validated against doubly labeled water and showed a correlation coefficient of 0.3 between hours of total activity and total energy expenditure ( unpublished data ) . the u.s . department of health and human services ( dhhs ) recommends at least 75 min / week of vigorous or 150 min / week of moderate activity for americans ( 19 ) . individuals were categorized into three mutually exclusive groups according to how their reported pa matched these dhhs guidelines : low , < 75 min / week of vigorous and < 150 min / week of moderate activity ; moderate , either 75 min / week of vigorous or 150 min / week of moderate activity ; high , reported both 75 min / week of vigorous and 150 min / week of moderate activity ( 20 ) . dietary intake was assessed using the 126-item semiquantitative harvard food - frequency questionnaire ( 21 ) . the food - frequency questionnaire consisted of a list of foods with a standardized serving size and a selection of nine frequency categories ranging from never or less than one serving per month to more than six servings per day , during the past year . an open - ended free text section was used to capture food items that did not appear on the standard list and included information on usual serving size and number of servings consumed per week for incorporation in the dietary intake calculation for each subject . insulin response to oral glucose ( 30-min insulin ) was computed as the 30-min ogtt insulin concentration minus the fasting insulin concentration . total and incremental ( above basal ) areas under ogtt glucose and insulin curves were calculated by trapezoid method . insulin sensitivity ( si ) , glucose effectiveness ( sg ) , and the incremental insulin response to glucose ( airg ) during the first 10 min of the ivgtt were determined using the millenium version of the bergman minimal model ( 22 ) . the disposition index ( di ) , a measure of -cell function , was computed as the product of airg and si . characteristics of the study cohort were presented by means , medians , and interquartile ranges . fasting insulin , postchallenge insulin , insulin response , si , di , sg , and total calories were log transformed to approximate normal distribution prior to analysis . geometric means were presented for these variables , and the associated standard errors were calculated by the delta method ( 23 ) . the relationship between diabetes - related metabolic measures and pa was assessed by testing trend association between the metabolic measures and levels of pa in categories ( low , moderate , and high ) using generalized estimating equations to account for correlations among related individuals within families . the impact of sex on the associations was assessed by including sex as a covariate and testing for significant interaction between pa group and sex in the model . if no significant interaction was detected , analyses were conducted using the entire sample with adjustment for age and sex . the impact of body composition on the association was assessed through covariate adjustment for bmi or percent body fat and/or waist - to - hip ratio . betagene participants are mexican - american adults ( both parents and three or more grandparents are mexican or of mexican descent ) who are 1 ) women who had gestational diabetes mellitus ( gdm ) within the previous 5 years , 2 ) siblings or cousins of those with a history of gdm , or 3 ) women with normal glucose levels during pregnancy in the past 5 years . women documented with and without previous gdm were identified from the los angeles county / university of southern california medical center , kaiser permanente southern california s delivery population , and obstetrical / gynecological clinics at local southern california hospitals . women without previous gdm were frequency - matched to gdm cases by age , bmi , and parity . details regarding recruitment have been previously described ( 14 ) . all protocols for betagene were approved by the institutional review boards of participating institutions , and all participants provided written informed consent before participation . research data were collected in two separate visits to the general clinical research center at the university of southern california . the first visit consisted of a physical examination , dietary and pa questionnaires , a 2-h , 75-g oral glucose tolerance test ( ogtt ) , and fasting blood for lipid measurements ( 15 ) . mmol / l were invited for a second visit , which consisted of a dual - energy x - ray absorptiometry scan for body composition and an insulin - modified intravenous glucose tolerance test ( ivgtt ) for measurement of insulin sensitivity and -cell function ( 16 ) . plasma glucose was measured on an autoanalyzer using the glucose oxidase method ( ysi model 2300 ; yellow springs instruments , yellow springs , oh ) . insulin was measured by a two - site immunoenzymometric assay ( tosoh biosciences , san francisco , ca ) that has < the amount and intensity of pa was assessed by questionnaires developed in the hawaii - los angeles multiethnic cohort study ( 17,18 ) . this questionnaire is comprised of a list of structured questions describing various types of activity ( sitting , strenuous sports , vigorous work , and moderate activities including sports and work ) during the past year . responses were then used to estimate the total minutes of moderate and vigorous activity per week . the questionnaire was validated against doubly labeled water and showed a correlation coefficient of 0.3 between hours of total activity and total energy expenditure ( unpublished data ) . the u.s . department of health and human services ( dhhs ) recommends at least 75 min / week of vigorous or 150 min / week of moderate activity for americans ( 19 ) . individuals were categorized into three mutually exclusive groups according to how their reported pa matched these dhhs guidelines : low , < 75 min / week of vigorous and < 150 min / week of moderate activity ; moderate , either 75 min / week of vigorous or 150 min / week of moderate activity ; high , reported both 75 min / week of vigorous and 150 min / week of moderate activity ( 20 ) . dietary intake was assessed using the 126-item semiquantitative harvard food - frequency questionnaire ( 21 ) . the food - frequency questionnaire consisted of a list of foods with a standardized serving size and a selection of nine frequency categories ranging from never or less than one serving per month to more than six servings per day , during the past year . an open - ended free text section was used to capture food items that did not appear on the standard list and included information on usual serving size and number of servings consumed per week for incorporation in the dietary intake calculation for each subject . insulin response to oral glucose ( 30-min insulin ) was computed as the 30-min ogtt insulin concentration minus the fasting insulin concentration . total and incremental ( above basal ) areas under ogtt glucose and insulin curves were calculated by trapezoid method . insulin sensitivity ( si ) , glucose effectiveness ( sg ) , and the incremental insulin response to glucose ( airg ) during the first 10 min of the ivgtt the disposition index ( di ) , a measure of -cell function , was computed as the product of airg and si . characteristics of the study cohort were presented by means , medians , and interquartile ranges . fasting insulin , postchallenge insulin , insulin response , si , di , sg , and total calories were log transformed to approximate normal distribution prior to analysis . geometric means were presented for these variables , and the associated standard errors were calculated by the delta method ( 23 ) . the relationship between diabetes - related metabolic measures and pa was assessed by testing trend association between the metabolic measures and levels of pa in categories ( low , moderate , and high ) using generalized estimating equations to account for correlations among related individuals within families . the impact of sex on the associations was assessed by including sex as a covariate and testing for significant interaction between pa group and sex in the model . if no significant interaction was detected , analyses were conducted using the entire sample with adjustment for age and sex . the impact of body composition on the association was assessed through covariate adjustment for bmi or percent body fat and/or waist - to - hip ratio . a total of 1,250 participants were recruited into the betagene study with completed ivgtts ; of these , 1,152 completed ogtts and pa questionnaires . the mean age was 34.7 years ( range , 17.965.6 years ) , mean bmi was 29.6 kg / m , mean percent body fat was 34.7% , and 72.3% of the cohort were female . normal glucose tolerance was present in 707 ( 61.4% ) , impaired glucose tolerance in 361 ( 31.3% ) , and diabetes by 2-h glucose 11.1 mmol / l ( 24 ) in 84 ( 7.3% ) participants , respectively . among females , the median durations for vigorous and moderate activity were both 45 min / week , considerably below the dhhs recommendation of 150 min / week of moderate activity or 75 min / week of vigorous activity for chronic disease prevention ( 19 ) . cohort characteristics ( n = 1,152 ) of the study participants , 501 ( 43% ) were classified as low in pa , 448 ( 39% ) as moderate , and 203 ( 18% ) as high . the higher activity groups were significantly younger ( mean age = 35.3 , 34.2 , 34.0 years in the low , moderate , and high groups , respectively ; p = 0.012 for trend ) . the distribution of females versus males in the three pa groups was significantly different ( p < 0.001 ) and showed a pattern that indicated lower pa among females : low = 52 vs. 22% for females vs. males , moderate = 33 vs. 55% , and high = 16 vs. 23% , respectively . table 2 presents the comparison of the age - adjusted means for the metabolic measures among the three pa groups . although no significant association was observed between pa and bmi ( p = 0.28 ) , increasing pa was significantly associated with decreasing percent body fat ( p < 0.0001 ) , 2-h glucose ( p = 0.001 ) , fasting insulin ( p = 0.0003 ) , and 2-h insulin ( p = 0.0001 ) and increasing -cell function ( p = 0.004 ) . an increasing level of pa was marginally associated with an increasing level of airg ( p = 0.09 ) . the associations between pa and diabetes - related traits appeared to be similar between males and females ( interaction test p > 0.28 for each trait after including sex in the model , details by sex analyses ) ( supplementary tables 1 and 2 ) except ogtt fasting glucose ( p = 0.037 ) . age - adjusted fasting glucose decreased with increasing pa in women ( mean sem for low = 5.1 0.04 mmol / l , moderate = 5.0 0.04 mmol / l , and high = 4.9 0.06 mmol / l ; p = 0.013 ) but not in men ( low = 5.2 0.08 mmol / l , moderate = 5.1 0.05 mmol / l , and high = 5.2 0.06 mmol / l ; p = 0.35 ) . after further adjustment for sex , the association between pa and percent body fat was no longer significant ( p = 0.38 ) ( table 2 ) . the significant associations for 2-h glucose , fasting , and 2-h insulin and di observed in the age - adjusted analyses remained after further adjustment for sex ( table 2 ) . an increasing level of pa was significantly associated with a lower waist - to - hip ratio ( p = 0.012 ) and marginally associated with decreasing fasting glucose ( p = 0.10 ) and increasing si ( p = 0.076 ) after adjustment for age and sex . comparison of metabolic traits across the three pa groups * we assessed the relative contribution of body fat to the observed associations between pa and diabetes - related traits by additionally adjusting for percent body fat , bmi , or waist - to - hip ratio . age- , sex- , and percent body fat adjusted means for ogtt fasting and 2-h glucose and insulin are shown in fig . analogously , adjusted means for si , airg , and di are shown in fig . 2 . associations between pa and 2-h glucose , fasting insulin , 2-h insulin , and di remained significant after further adjusting for percent body fat , and the regression coefficients were only slightly reduced ( < 15% change ) . by contrast , adjustment for body fat reduced the association between pa and si by 26% and increased the association with airg by 27% , although these associations remained insignificant ( fig . results were similar when models were adjusted for bmi or waist - to - hip ratio instead of percent body fat ( data not shown ) . including waist - to - hip ratio in addition to percent body fat or bmi in the model adjustment did not change the conclusion ( data not shown ) . age- , sex- , and percent body fat adjusted means and 95% cis for ogtt fasting and 2-h glucose and insulin by the three pa groups : low , moderate , and high . p values were from the trend association between pa groups and each of the metabolic traits . age- , sex- , and percent body fat adjusted means and 95% cis for insulin sensitivity ( si ) , acute insulin secretion ( airg ) , and -cell compensation for insulin resistance ( di ) by the three pa groups : low , moderate , and high . p values were from the trend association between pa groups and each of the metabolic traits . because higher pa level was typically accompanied by higher caloric intake , we further adjusted for caloric intake . caloric intake did not significantly alter the age- and sex - adjusted associations between pa and any diabetes - related traits . pa was not significantly associated with bmi ( p = 0.66 ) or percent body fat ( p = 0.54 ) after further adjustment for caloric intake . further adjustment for caloric intake had little impact on the age- , sex- , and percent body fat adjusted associations between pa and fasting glucose , 2-h glucose , fasting insulin , 2-h insulin , airg , si , and di ; the additional adjustment resulted in < 10% change in the corresponding regression coefficients , with adjusted p values of 0.098 , 0.017 , 0.0003 , 0.016 , 0.21 , 0.16 , and 0.019 , respectively . in this study , we observed that an increasing level of self - reported pa was significantly associated with decreasing levels of fasting , 2-h insulin , and 2-h glucose and an increasing level of -cell function assessed as the di . these associations were not modified significantly by adjustment for percent body fat , bmi , waist - to - hip ratio , or caloric intake . these findings suggest that more pa , even in a free - living environment , is beneficial to -cell function and glucose regulation . the impact of exercise on glucose and insulin metabolism has been evaluated by several studies . animal studies have demonstrated that exercise increases glucose uptake by stimulating glut4 translocation in muscle cells and increasing glucose uptake by the liver ( 5 ) . additionally , an exercise training study among type 2 diabetic patients revealed that exercise enhances the whole - body glucose disposal ( 6 ) . although the association between pa and insulin sensitivity did not make the statistical significance based on a p value < 0.05 cut point in this cohort , there was a trend that subjects with higher pa levels had better insulin sensitivity ( p = 0.076 after adjustment for age and sex ) . this attenuated relationship was consistent with the lack of association between pa and bmi or percent body fat and could be due to the fact that no exercise training and interventions were applied in this cohort . thus , our results are consistent with the findings from exercise trainings and support the concept that more pa may contribute to lower ogtt glucose and insulin levels and , to a lesser significant extent , better insulin sensitivity . the most novel finding in this study was the significant association between pa and -cell function . of note , > 75% of the participants in this cohort were overweight or obese ( 25th percentile of bmi was 25.5 kg / m ) . two previous studies evaluated short - term exercise training and changes in -cell function before and after training . one of the studies included 12 subjects > 60 years of age ( 11 ) . the other study included overweight adults and demonstrated that both moderate and vigorous exercise training improved insulin sensitivity and -cell function , although the improvement of -cell function was not statistically significant for vigorous activity ( 12 ) . although the biological mechanisms of the impact of pa on -cell function have not been clarified , there has been evidence that exercises expanded -cell mass by stimulating its proliferations and preventing its apoptosis ( 25,26 ) . in these rat studies , exercises were shown to enhance the expression of insulin receptor substrate-2 , which is crucial for -cell growth and survival . the beneficial effect of exercises on -cell function may also be the improvement of adipose tissue biology such as increasing adiponectin and reducing inflammation ( 27 ) . recently , we showed that declining adiponectin was significantly associated with -cell function deterioration in a longitudinal study independent of weight gain ( 28 ) . the mechanism for this association may promote -cell function and survival by increasing ceramidase activity , decreasing intracellular ceramide levels , and increasing antiapoptotic metabolite sphingosine-1 phosphate levels ( 29,30 ) . taken together , the results from this report and previous reports suggest that greater amounts of daily living activity might protect -cell function , even in overweight and obese individuals . a cross - sectional study in mexican children suggested that the impact of pa on -cell function could be mediated by body fat ( 31 ) . they found that the higher cardiorespiratory fitness , which reflects chronic pa behavior , was significantly correlated with -cell function as well as insulin resistance . we did not observe a significant association between percent body fat or bmi and self - reported pa groups after adjustment for age and sex . in addition , the adjustment for percent body fat and bmi did not significantly reduce the association between pa and -cell function . the lack of association between pa and bmi or percent body fat in this adult cohort may be due to the fact that our cohort is primarily composed of women with a history of gdm and their family members , the majority of whom are overweight or obese and are presumably at higher than normal risk for diabetes . we did not measure fitness levels , and pa was self - reported and included work - related activities such as moving heavy furniture , loading trucks , and gardening , as well as sports activities such as aerobics . our finding was consistent with the results from several large studies with long - term follow - up , which showed that self - reported pa was associated with a lower incidence of diabetes independent of bmi ( 32 ) . in other previous studies , energy intake was shown to confound the effect of exercise on body weight or insulin resistance ( 33,34 ) . however , in our analysis , the associations between -cell function , glucose , insulin profiles , and pa were not significantly changed by the adjustment for caloric intake . therefore , our results indicated a more direct contribution of pa in preserving -cell function . moreover , our findings are consistent with the results of a recent study in rats , which demonstrated that voluntary exercise was beneficial for sustaining -cell compensation without preventing dyslipidemia or obesity ( 35 ) . one possible mechanism for such an effect would be mitigation of the adverse metabolic effects of obesity . since pancreatic biopsies can not be performed , a surrogate measure would have been the computed axial tomography scan estimate of peritoneal fat or an ultrasound assessment of hepatic fat content . although the questionnaire has been used in previous studies in mexican americans ( 18 ) , it is well known that self - reported pa tends to be overreported on questionnaires ( 36,37 ) . dhhs recommendation for pa instead of using the minutes of pa as a continuous variable to reduce the impact of measurement errors . as evidence of potential overreporting in this cohort , 57% of participants reported meeting or exceeding the dhhs pa guideline , as compared with 36% for mexican americans in a national report of participation in aerobic activity ( 38 ) . however , we also note that our questionnaire included work - related pas , which might explain the higher than expected percentage . second , we did not measure physical fitness , which is more objective and a better predictor than self - reported pa for many health outcomes , such as diabetes and cardiovascular diseases ( 39,40 ) . third , the cross - sectional and observational design of our study precludes us from examining the dynamic impact of pa on the change in metabolic traits . the strength of the current study is the unique sample , which includes a large cohort of mexican americans with detailed ogtt- and ivgtt - based measures of glucose tolerance , insulin sensitivity , and -cell function . unlike other studies that mostly examined the short - term effect of exercise training / intervention on insulin secretion and insulin resistance with small sample sizes , we described the association with pa in a free - living environment to provide a more realistic model than the impact of a specific , short - term exercise intervention . we showed that increasing pa is associated with better -cell function in a population without overt diabetes . our results suggest that an effect on diabetes prevention by lifestyle change ( 1,2 ) may be mediated by the improvement of -cell function . our findings have implications for a real - world approach to the delay , prevention , and/or early treatment of type 2 diabetes . in conclusion , our study indicates that a greater level of pa might play a role in improving glucose tolerance and protecting -cell function in mexican americans who are at high risk of developing diabetes . the beneficial impact of pa on -cell function does not depend on bmi , percent body fat , and energy intakes . our findings have important public health implications to prevent / slow down the deterioration of -cell function that leads to type 2 diabetes .

objectiveto examine the association between self - reported physical activity ( pa ) and diabetes - related quantitative traits.research design and methodsthe observational cohort was 1,152 mexican american adults with dual - energy x - ray absorptiometry , oral and intravenous glucose tolerance tests , and self - reported dietary and pa questionnaires . pa was categorized into three mutually exclusive groups according to the u.s . department of health and human services pa guidelines for americans : low ( vigorous < 75 min / week and moderate < 150 min / week ) , moderate ( vigorous 75 min / week or moderate 150 min / week ) , and high ( vigorous 75 min / week and moderate 150 min / week ) . trends in pa groups were tested for association with metabolic traits in a cross - sectional analysis.resultsthe participants mean age was 35 years ( range , 1866 years ) , mean bmi was 29.6 kg / m2 , and 73% were female . among them , 501 ( 43% ) , 448 ( 39% ) , and 203 ( 18% ) were classified as having low , moderate , and high pa , respectively . after adjustment for age , a higher pa was significantly associated with lower 2-h glucose , fasting insulin , and 2-h insulin and greater -cell function ( p = 0.001 , 0.0003 , 0.0001 , and 0.004 , respectively ) . the association did not differ significantly by sex . results were similar after further adjustment for age , sex , bmi , or percent body fat.conclusionsan increasing level of pa is associated with a better glucose and insulin profile and enhanced -cell function that is not explained by differences in bmi or percent body fat . our results suggest that pa can be beneficial to -cell function and glucose regulation independent of obesity .

RESEARCH DESIGN AND METHODS Study participants Testing procedures and assays PA and dietary assessment Data analysis RESULTS CONCLUSIONS Supplementary Material

betagene participants are mexican - american adults ( both parents and three or more grandparents are mexican or of mexican descent ) who are 1 ) women who had gestational diabetes mellitus ( gdm ) within the previous 5 years , 2 ) siblings or cousins of those with a history of gdm , or 3 ) women with normal glucose levels during pregnancy in the past 5 years . women without previous gdm were frequency - matched to gdm cases by age , bmi , and parity . research data were collected in two separate visits to the general clinical research center at the university of southern california . the first visit consisted of a physical examination , dietary and pa questionnaires , a 2-h , 75-g oral glucose tolerance test ( ogtt ) , and fasting blood for lipid measurements ( 15 ) . participants with fasting glucose < 7.0 mmol / l were invited for a second visit , which consisted of a dual - energy x - ray absorptiometry scan for body composition and an insulin - modified intravenous glucose tolerance test ( ivgtt ) for measurement of insulin sensitivity and -cell function ( 16 ) . insulin was measured by a two - site immunoenzymometric assay ( tosoh biosciences , san francisco , ca ) that has < 0.1% cross - reactivity with proinsulin and intermediate split products . the amount and intensity of pa was assessed by questionnaires developed in the hawaii - los angeles multiethnic cohort study ( 17,18 ) . this questionnaire is comprised of a list of structured questions describing various types of activity ( sitting , strenuous sports , vigorous work , and moderate activities including sports and work ) during the past year . the u.s . department of health and human services ( dhhs ) recommends at least 75 min / week of vigorous or 150 min / week of moderate activity for americans ( 19 ) . individuals were categorized into three mutually exclusive groups according to how their reported pa matched these dhhs guidelines : low , < 75 min / week of vigorous and < 150 min / week of moderate activity ; moderate , either 75 min / week of vigorous or 150 min / week of moderate activity ; high , reported both 75 min / week of vigorous and 150 min / week of moderate activity ( 20 ) . insulin response to oral glucose ( 30-min insulin ) was computed as the 30-min ogtt insulin concentration minus the fasting insulin concentration . total and incremental ( above basal ) areas under ogtt glucose and insulin curves were calculated by trapezoid method . insulin sensitivity ( si ) , glucose effectiveness ( sg ) , and the incremental insulin response to glucose ( airg ) during the first 10 min of the ivgtt were determined using the millenium version of the bergman minimal model ( 22 ) . the disposition index ( di ) , a measure of -cell function , was computed as the product of airg and si . characteristics of the study cohort were presented by means , medians , and interquartile ranges . fasting insulin , postchallenge insulin , insulin response , si , di , sg , and total calories were log transformed to approximate normal distribution prior to analysis . the relationship between diabetes - related metabolic measures and pa was assessed by testing trend association between the metabolic measures and levels of pa in categories ( low , moderate , and high ) using generalized estimating equations to account for correlations among related individuals within families . if no significant interaction was detected , analyses were conducted using the entire sample with adjustment for age and sex . the impact of body composition on the association was assessed through covariate adjustment for bmi or percent body fat and/or waist - to - hip ratio . betagene participants are mexican - american adults ( both parents and three or more grandparents are mexican or of mexican descent ) who are 1 ) women who had gestational diabetes mellitus ( gdm ) within the previous 5 years , 2 ) siblings or cousins of those with a history of gdm , or 3 ) women with normal glucose levels during pregnancy in the past 5 years . women documented with and without previous gdm were identified from the los angeles county / university of southern california medical center , kaiser permanente southern california s delivery population , and obstetrical / gynecological clinics at local southern california hospitals . women without previous gdm were frequency - matched to gdm cases by age , bmi , and parity . research data were collected in two separate visits to the general clinical research center at the university of southern california . the first visit consisted of a physical examination , dietary and pa questionnaires , a 2-h , 75-g oral glucose tolerance test ( ogtt ) , and fasting blood for lipid measurements ( 15 ) . mmol / l were invited for a second visit , which consisted of a dual - energy x - ray absorptiometry scan for body composition and an insulin - modified intravenous glucose tolerance test ( ivgtt ) for measurement of insulin sensitivity and -cell function ( 16 ) . insulin was measured by a two - site immunoenzymometric assay ( tosoh biosciences , san francisco , ca ) that has < the amount and intensity of pa was assessed by questionnaires developed in the hawaii - los angeles multiethnic cohort study ( 17,18 ) . this questionnaire is comprised of a list of structured questions describing various types of activity ( sitting , strenuous sports , vigorous work , and moderate activities including sports and work ) during the past year . the u.s . department of health and human services ( dhhs ) recommends at least 75 min / week of vigorous or 150 min / week of moderate activity for americans ( 19 ) . individuals were categorized into three mutually exclusive groups according to how their reported pa matched these dhhs guidelines : low , < 75 min / week of vigorous and < 150 min / week of moderate activity ; moderate , either 75 min / week of vigorous or 150 min / week of moderate activity ; high , reported both 75 min / week of vigorous and 150 min / week of moderate activity ( 20 ) . insulin response to oral glucose ( 30-min insulin ) was computed as the 30-min ogtt insulin concentration minus the fasting insulin concentration . total and incremental ( above basal ) areas under ogtt glucose and insulin curves were calculated by trapezoid method . insulin sensitivity ( si ) , glucose effectiveness ( sg ) , and the incremental insulin response to glucose ( airg ) during the first 10 min of the ivgtt the disposition index ( di ) , a measure of -cell function , was computed as the product of airg and si . characteristics of the study cohort were presented by means , medians , and interquartile ranges . fasting insulin , postchallenge insulin , insulin response , si , di , sg , and total calories were log transformed to approximate normal distribution prior to analysis . the relationship between diabetes - related metabolic measures and pa was assessed by testing trend association between the metabolic measures and levels of pa in categories ( low , moderate , and high ) using generalized estimating equations to account for correlations among related individuals within families . if no significant interaction was detected , analyses were conducted using the entire sample with adjustment for age and sex . the impact of body composition on the association was assessed through covariate adjustment for bmi or percent body fat and/or waist - to - hip ratio . a total of 1,250 participants were recruited into the betagene study with completed ivgtts ; of these , 1,152 completed ogtts and pa questionnaires . the mean age was 34.7 years ( range , 17.965.6 years ) , mean bmi was 29.6 kg / m , mean percent body fat was 34.7% , and 72.3% of the cohort were female . normal glucose tolerance was present in 707 ( 61.4% ) , impaired glucose tolerance in 361 ( 31.3% ) , and diabetes by 2-h glucose 11.1 mmol / l ( 24 ) in 84 ( 7.3% ) participants , respectively . among females , the median durations for vigorous and moderate activity were both 45 min / week , considerably below the dhhs recommendation of 150 min / week of moderate activity or 75 min / week of vigorous activity for chronic disease prevention ( 19 ) . cohort characteristics ( n = 1,152 ) of the study participants , 501 ( 43% ) were classified as low in pa , 448 ( 39% ) as moderate , and 203 ( 18% ) as high . the higher activity groups were significantly younger ( mean age = 35.3 , 34.2 , 34.0 years in the low , moderate , and high groups , respectively ; p = 0.012 for trend ) . the distribution of females versus males in the three pa groups was significantly different ( p < 0.001 ) and showed a pattern that indicated lower pa among females : low = 52 vs. 22% for females vs. males , moderate = 33 vs. 55% , and high = 16 vs. 23% , respectively . although no significant association was observed between pa and bmi ( p = 0.28 ) , increasing pa was significantly associated with decreasing percent body fat ( p < 0.0001 ) , 2-h glucose ( p = 0.001 ) , fasting insulin ( p = 0.0003 ) , and 2-h insulin ( p = 0.0001 ) and increasing -cell function ( p = 0.004 ) . an increasing level of pa was marginally associated with an increasing level of airg ( p = 0.09 ) . the associations between pa and diabetes - related traits appeared to be similar between males and females ( interaction test p > 0.28 for each trait after including sex in the model , details by sex analyses ) ( supplementary tables 1 and 2 ) except ogtt fasting glucose ( p = 0.037 ) . age - adjusted fasting glucose decreased with increasing pa in women ( mean sem for low = 5.1 0.04 mmol / l , moderate = 5.0 0.04 mmol / l , and high = 4.9 0.06 mmol / l ; p = 0.013 ) but not in men ( low = 5.2 0.08 mmol / l , moderate = 5.1 0.05 mmol / l , and high = 5.2 0.06 mmol / l ; p = 0.35 ) . after further adjustment for sex , the association between pa and percent body fat was no longer significant ( p = 0.38 ) ( table 2 ) . the significant associations for 2-h glucose , fasting , and 2-h insulin and di observed in the age - adjusted analyses remained after further adjustment for sex ( table 2 ) . an increasing level of pa was significantly associated with a lower waist - to - hip ratio ( p = 0.012 ) and marginally associated with decreasing fasting glucose ( p = 0.10 ) and increasing si ( p = 0.076 ) after adjustment for age and sex . comparison of metabolic traits across the three pa groups * we assessed the relative contribution of body fat to the observed associations between pa and diabetes - related traits by additionally adjusting for percent body fat , bmi , or waist - to - hip ratio . age- , sex- , and percent body fat adjusted means for ogtt fasting and 2-h glucose and insulin are shown in fig . associations between pa and 2-h glucose , fasting insulin , 2-h insulin , and di remained significant after further adjusting for percent body fat , and the regression coefficients were only slightly reduced ( < 15% change ) . by contrast , adjustment for body fat reduced the association between pa and si by 26% and increased the association with airg by 27% , although these associations remained insignificant ( fig . results were similar when models were adjusted for bmi or waist - to - hip ratio instead of percent body fat ( data not shown ) . including waist - to - hip ratio in addition to percent body fat or bmi in the model adjustment did not change the conclusion ( data not shown ) . age- , sex- , and percent body fat adjusted means and 95% cis for ogtt fasting and 2-h glucose and insulin by the three pa groups : low , moderate , and high . p values were from the trend association between pa groups and each of the metabolic traits . age- , sex- , and percent body fat adjusted means and 95% cis for insulin sensitivity ( si ) , acute insulin secretion ( airg ) , and -cell compensation for insulin resistance ( di ) by the three pa groups : low , moderate , and high . p values were from the trend association between pa groups and each of the metabolic traits . caloric intake did not significantly alter the age- and sex - adjusted associations between pa and any diabetes - related traits . pa was not significantly associated with bmi ( p = 0.66 ) or percent body fat ( p = 0.54 ) after further adjustment for caloric intake . further adjustment for caloric intake had little impact on the age- , sex- , and percent body fat adjusted associations between pa and fasting glucose , 2-h glucose , fasting insulin , 2-h insulin , airg , si , and di ; the additional adjustment resulted in < 10% change in the corresponding regression coefficients , with adjusted p values of 0.098 , 0.017 , 0.0003 , 0.016 , 0.21 , 0.16 , and 0.019 , respectively . in this study , we observed that an increasing level of self - reported pa was significantly associated with decreasing levels of fasting , 2-h insulin , and 2-h glucose and an increasing level of -cell function assessed as the di . these associations were not modified significantly by adjustment for percent body fat , bmi , waist - to - hip ratio , or caloric intake . these findings suggest that more pa , even in a free - living environment , is beneficial to -cell function and glucose regulation . the impact of exercise on glucose and insulin metabolism has been evaluated by several studies . although the association between pa and insulin sensitivity did not make the statistical significance based on a p value < 0.05 cut point in this cohort , there was a trend that subjects with higher pa levels had better insulin sensitivity ( p = 0.076 after adjustment for age and sex ) . this attenuated relationship was consistent with the lack of association between pa and bmi or percent body fat and could be due to the fact that no exercise training and interventions were applied in this cohort . thus , our results are consistent with the findings from exercise trainings and support the concept that more pa may contribute to lower ogtt glucose and insulin levels and , to a lesser significant extent , better insulin sensitivity . the most novel finding in this study was the significant association between pa and -cell function . of note , > 75% of the participants in this cohort were overweight or obese ( 25th percentile of bmi was 25.5 kg / m ) . two previous studies evaluated short - term exercise training and changes in -cell function before and after training . the other study included overweight adults and demonstrated that both moderate and vigorous exercise training improved insulin sensitivity and -cell function , although the improvement of -cell function was not statistically significant for vigorous activity ( 12 ) . although the biological mechanisms of the impact of pa on -cell function have not been clarified , there has been evidence that exercises expanded -cell mass by stimulating its proliferations and preventing its apoptosis ( 25,26 ) . recently , we showed that declining adiponectin was significantly associated with -cell function deterioration in a longitudinal study independent of weight gain ( 28 ) . the mechanism for this association may promote -cell function and survival by increasing ceramidase activity , decreasing intracellular ceramide levels , and increasing antiapoptotic metabolite sphingosine-1 phosphate levels ( 29,30 ) . taken together , the results from this report and previous reports suggest that greater amounts of daily living activity might protect -cell function , even in overweight and obese individuals . a cross - sectional study in mexican children suggested that the impact of pa on -cell function could be mediated by body fat ( 31 ) . they found that the higher cardiorespiratory fitness , which reflects chronic pa behavior , was significantly correlated with -cell function as well as insulin resistance . we did not observe a significant association between percent body fat or bmi and self - reported pa groups after adjustment for age and sex . in addition , the adjustment for percent body fat and bmi did not significantly reduce the association between pa and -cell function . the lack of association between pa and bmi or percent body fat in this adult cohort may be due to the fact that our cohort is primarily composed of women with a history of gdm and their family members , the majority of whom are overweight or obese and are presumably at higher than normal risk for diabetes . we did not measure fitness levels , and pa was self - reported and included work - related activities such as moving heavy furniture , loading trucks , and gardening , as well as sports activities such as aerobics . our finding was consistent with the results from several large studies with long - term follow - up , which showed that self - reported pa was associated with a lower incidence of diabetes independent of bmi ( 32 ) . however , in our analysis , the associations between -cell function , glucose , insulin profiles , and pa were not significantly changed by the adjustment for caloric intake . therefore , our results indicated a more direct contribution of pa in preserving -cell function . since pancreatic biopsies can not be performed , a surrogate measure would have been the computed axial tomography scan estimate of peritoneal fat or an ultrasound assessment of hepatic fat content . although the questionnaire has been used in previous studies in mexican americans ( 18 ) , it is well known that self - reported pa tends to be overreported on questionnaires ( 36,37 ) . dhhs recommendation for pa instead of using the minutes of pa as a continuous variable to reduce the impact of measurement errors . as evidence of potential overreporting in this cohort , 57% of participants reported meeting or exceeding the dhhs pa guideline , as compared with 36% for mexican americans in a national report of participation in aerobic activity ( 38 ) . second , we did not measure physical fitness , which is more objective and a better predictor than self - reported pa for many health outcomes , such as diabetes and cardiovascular diseases ( 39,40 ) . third , the cross - sectional and observational design of our study precludes us from examining the dynamic impact of pa on the change in metabolic traits . the strength of the current study is the unique sample , which includes a large cohort of mexican americans with detailed ogtt- and ivgtt - based measures of glucose tolerance , insulin sensitivity , and -cell function . unlike other studies that mostly examined the short - term effect of exercise training / intervention on insulin secretion and insulin resistance with small sample sizes , we described the association with pa in a free - living environment to provide a more realistic model than the impact of a specific , short - term exercise intervention . we showed that increasing pa is associated with better -cell function in a population without overt diabetes . our results suggest that an effect on diabetes prevention by lifestyle change ( 1,2 ) may be mediated by the improvement of -cell function . in conclusion , our study indicates that a greater level of pa might play a role in improving glucose tolerance and protecting -cell function in mexican americans who are at high risk of developing diabetes . the beneficial impact of pa on -cell function does not depend on bmi , percent body fat , and energy intakes . our findings have important public health implications to prevent / slow down the deterioration of -cell function that leads to type 2 diabetes .

many tumors show an initial response to targeted therapies before genetic resistance emerges ; however , little is known about how tumor cells might tolerate therapy before genetic resistance dominates . we show how braf - mutant melanoma cells rapidly become tolerant to plx4720 in areas of high stroma . we demonstrate that plx4720 has an effect on the tumor stroma , leading to enhanced matrix remodeling . we propose that this safe haven enhances the population of cancer cells from which genetic resistance emerges . this work highlights the need to consider the effects of targeted therapies on the tumor microenvironment . since the discovery of oncogenes that encoded protein kinases , it has been hoped that inhibition of the relevant kinases would be an effective chemotherapeutic strategy ( shawver et al . , 2002 ) . this aspiration has become a clinical reality with the development of inhibitors against abl tyrosine kinase ( druker et al . , 2001 , 2006 ) , egfr family kinases ( maemondo et al . , 2010 ; mok et al . , 2009 ; sordella et al . , 2004 ) , and braf ( chapman et al . , 2011 ; flaherty et al . , 2010 ; sosman et al . , however , agents targeting either egfr or braf typically show good efficacy in tumors with matching oncogenic mutations for a number of months before genetically resistant cells dominate the tumor and the therapy fails ( kobayashi et al . poulikakos et al . , 2011 ; poulikakos and rosen , 2011 ; villanueva et al . , 2011 ) . in the case of egfr - mutant lung tumors , it has been shown that resistant cells may be present even before treatment and that these are at a strong selective advantage during therapy ( inukai et al . , 2006 ; maheswaran et al . , 2008 ; rosell et al . , 2011 ; turke et al . , 2010 ) . however , the situation in braf - mutant melanoma treated with braf inhibitors is less clear . there is significant variability in the magnitude of initial response to braf inhibition ( chapman et al . , 2011 ; sosman et al . , 2012 ) and genetically resistant sub - clones have not been detected prior to treatment in tumors that show modest responses . it has been proposed that non - cell autonomous mechanisms involving hgf production by the tumor stroma may drive resistance ( straussman et al . , 2012 ; wilson et al . , however , it is not clear how selective pressure would act on the genetically stable stroma to promote the emergence of resistant disease . establishing the chronology of biochemical responses to targeted therapy and biological changes elicited within the context of complex tumor microenvironments remains challenging . the activity of erk / mapk can be monitored in live tissue using a biosensor construct containing two fluorophores , a long flexible linker , an erk / map kinase binding site , an optimal substrate site for the kinase , and a phospho - threonine binding domain ( harvey et al . , 2008 ; komatsu et al . , 2011 when the substrate site is phosphorylated , it engages in an intra - molecular interaction with the phospho - threonine binding domain , leading to an overall change in the conformation of the molecule and a change in fluorescence resonance energy transfer ( fret ) between the two fluorophores ( komatsu et al . , this system enables the biochemical response to braf inhibition to be monitored with single cell resolution in vivo . genetically engineered syngeneic hosts additionally provide the ability to depict the tumor stroma ( muzumdar et al . , 2007 ) . these technologies can be combined with intravital imaging windows to longitudinally track both the biochemical response to braf inhibition and the distribution of the tumor stroma ( janssen et al . , 2013 ) . to study responses to braf inhibition in a syngeneic tumor microenvironment , we tested the response of braf and nras mutant c57/bl6 mouse melanoma cell lines to the braf inhibitor plx4720 . two different braf mutant lines , 5555 and 4434 , were sensitive to plx4720 whereas , as expected , the nras mutant cells ( c790 ) were refractory to plx4720 in vitro ( figure 1a ) . we next tested the response of these cells to plx4720 when growing as tumors in syngeneic mice . to our surprise , both braf - mutant melanoma cell lines were refractory to plx4720 ( figure 1b ) . this unexpected result suggested to us that these cells might represent a model to probe non - cell autonomous mechanisms of resistance of plx4720 . furthermore , they may represent the small subset of braf - mutant melanoma that exhibit only a small response to vemurafenib . to understand the lack of response of 5555 and 4434 cells to plx4720 in vivo , we reasoned that it would be important to monitor the braf signaling with single cell resolution . we engineered 5555 and 4434 cells to express an erk / map kinase biosensor located in the nucleus called ekarev - nls ( figure s1a ) . the ekarev biosensor faithfully monitored changes in erk / map kinase signaling in response to tpa and either braf or mek inhibitors ( figures s1b s1e ) . in contrast , paradoxical activation of erk / mapk activity is observed in nras mutant cells treated with plx4720 ( figures s1c and s1f ) . we further confirmed that changes in fret signal are absolutely dependent upon the phospho - acceptor site in the biosensor ( figures s1 g and s1h ) . we next generated tumors using 5555-ekarev - nls cells ; in some cases , these were adjacent to titanium imaging windows that allow longitudinal imaging of the same tumor ( janssen et al . , 2013 ) . intravital imaging revealed considerable heterogeneity in ekarev fret signal ( figures 1c1f , s1i , and s1j ) . these data are supported by pperk immunohistochemical analysis ( figure s1k ) . to gain insight into the lack of effect of plx4720 on tumor growth , we performed longitudinal imaging of tumors before and during plx4720 treatment . the results showed that the high ekarev fret signal was reduced 4 hr after administration of plx4720 ( figures 1c and 1d ) . these data suggest that plx4720 can access the tumor and achieve the reduction in erk / mapk activity expected based on the in vitro analysis in figures s1c s1e . however , within 1 day , high levels of ekarev fret signal returned even though plx4720 was administered daily ( figure 1c ) . analysis of the distribution of ekarev signal after 3 days of plx4720 treatment suggested that only a small proportion of 5555 cells show a stable reduction in erk / mapk activity ( figures 1e and 1f ) . these cells were typically located in regions with low levels of host cells , which were demonstrated based on of their expression of the mtomato transgene . to more robustly test this observation , we segmented images of plx4720 treated tumors into regions with high or low levels of mtomato signal ( figure s1l ) . figure 1f shows that regions with low levels of stromal cells had significantly lower levels ekarev fret signal ( similar data were obtained in 4434 tumors ; figure s1 m ) . these data demonstrate braf mutant 5555 and 4434 tumors show a short - lived biochemical response to plx4720 . further , the rapid re - activation of erk / mapk and adaptation of these tumors to plx4720 is correlated with stromal density . we sought to establish a culture system that re - capitulated the plx4720 tolerance that we observed in vivo . the behavior of pure spheroids of melanoma cells was compared with that of equivalent sized tumor pieces when embedded in a collagen matrix . figures 2a and 2b show that pure melanoma spheroids were highly sensitive to plx4720 , with the appearance of many nuclear fragments indicating cell death . in contrast , melanoma explants from either subcutaneous tumors or experimentally established lung metastases were unresponsive to plx4720 ( figures 2a and 2b ) . even in the presence of drug , melanoma cells retained healthy nuclear architecture and invasive capability . thus , the spheroid model recapitulates the stroma - dependent plx4720 tolerance observed in vivo . it also formally excludes any problems relating to drug access that might confound interpretation of the in vivo data . we next sought to identify the stromal cell type that might be responsible for the adaptive behavior of 5555 and 4434 melanoma . immunohistochemical staining of 5555 and 4434 tumors revealed low number of infiltrating lymphocytes and neutrophils , and there was no clear relationship between the location of blood vessels and pperk signals ( figure s2a ) . however , both macrophages and stromal fibroblasts were abundant in both dmso- and plx4720-treated tumors ( figure s2a ) . we therefore explored whether melanoma - associated fibroblasts ( mafs ) or macrophages might be sufficient to confer drug tolerance on melanoma cells . two isolates of mafs were established from patients ( designated maf1 and maf2 , figures s2b and s2c ) and their effect on the response of 5555 and 4434 cells to plx4720 was tested . figures 2a , 2b , and s2d show that co - culture of either maf1 or maf2 with melanoma cells conferred tolerance of plx4720 and invasive behavior . this effect was significantly dependent on close proximity of the two cell types because maf conditioned media had only partial ability to reduce plx4720-induced cell death ( figures 2a , 2b , s2e , and s2f ) . furthermore , we excluded a role for egfr and c - met in mediating maf - dependent plx4720 tolerance ( figures s2 g and s2h ) . co - culture with macrophages was unable to confer drug tolerance on melanoma cells ( figure s2i ) . fret imaging with ekarev - nls shows heterogeneous erk activity in 5555 explants and 5555/maf co - culture spheroids ( figure s2j , and similar results with 4434 cells in figure s2k ) . there was markedly reduced signal / noise of the ekar biosensor in the explant / spheroid center ; therefore , we excluded these regions from our subsequent analyses . we performed time - lapse imaging of spheroid co - cultures before and after the addition of plx4720 . figures 2c2e and movie s1 reveal a marked decrease in ekarev signal 30 min after the addition of the drug . however , within 12 hr , the ekarev signal had returned to the level prior to drug addition . in pure melanoma spheroids , the ekarev signal was stably decreased until cells began to die ( the variable fret signal in apoptotic cultures can not reliably be interpreted ) . to exclude the possibility of drug metabolism or drug degradation , we re - added plx4720 after 12 hr and monitored the response . strikingly , co - cultures that had been exposed to plx4720 for 12 hr were completely refractory to the addition of more plx4720 , while importantly , they remained sensitive to the addition of the mek inhibitor pd184352 ( figures 2f and 2 g ) . these data demonstrate that co - cultures of melanoma cells and mafs switch from braf - dependent erk signaling to braf - independent erk signaling in just 12 hr . the adaptation is too quick to be genetic and unlike the relief of negative feedback described , the mechanism that we observe can not be mediated by melanoma cells alone ( lito et al . , 2012 ) . we therefore hypothesized that plx4720 might be having an effect on the tumor stroma . to test this , we engineered mafs to contain the ekarev - nls biosensor and also express mcherry to enable them to be distinguished from melanoma cells . spheroid co - cultures of 5555 and mafs were imaged before and during the response to plx4720 . strikingly , ekarev fret signal increased in maf shortly after the addition of plx4720 ( figures 3a3c and s3a and movie s2 ) . as expected , ekarev signal in the melanoma cells decreased immediately after the addition of the drug . erk activation in mafs by plx4720 was confirmed by western blotting ( figure s3b ) . a key feature of fibroblastic stroma is its ability to remodel the extracellular matrix ( kalluri and zeisberg , 2006 ) . this can be assayed using collagen gel contraction assays and imaging of collagen fibers by second harmonic generation ( shg ) ( calvo et al . , 2013 ) . plx4720 enhanced the matrix remodeling capability of both maf1 and maf2 and increased phosphorylation of the key regulator of actomyosin contractility , mlc2/myl9 ( figures 3d and 3e ) . plx4720 also induced dynamic protrusions in mafs ( figure 3f and movie s3 ) and promoted the formation of dense collagen fibrils ( figure 3 g ) . in contrast to mafs , plx4720 did not promote significant gel contraction by normal fibroblasts ( figure s3d ) . this indicates that some prior level of activation is likely to be required for plx4720 to modulate fibroblast function . to obtain a global perspective on how plx4720 affects mafs this revealed coordinated upregulation of the expression of many extracellular matrix ( ecm ) molecules in plx4720-treated mafs ( figures s3e and s3f ; table s1 ) . the upregulation of thrombospondin-1 ( thbs1 ) and tenascin - c ( tnc ) were confirmed using quantitative immunofluorescence ( figure 3h ) . in contrast , the expression of soluble growth factors previously implicated in resistance to braf inhibitors was largely unchanged ( figure s3e ) . interestingly , we noticed that plx4720 increased expression of platelet - derived growth factor receptor ( pdgfr ) in mafs ( figure s3e ) , and previous work has shown that stromal fibroblasts are dependent on pdgfr signaling ( erez et al . , 2010 ; kalluri and zeisberg , 2006 ; pietras et al . , 2008 ) . we hypothesized that plx4720 might activate mafs by enhancing the activity of pdgfr- and ras - dependent signaling through the paradoxical activation of craf ( hatzivassiliou et al . therefore , we investigated the effect of inhibiting pdgfr on both basal and plx - induced matrix remodeling by mafs . figure s3 g shows that both basal and plx4720-induced maf activity were greatly reduced by imatinib and sunitinib , two kinase inhibitors that target pdgfr in common . the data above show that plx4720 elicits changes in matrix production and remodeling by mafs . to test if the ecm was sufficient to modulate the response of melanoma cells to plx4720 , we varied both matrix composition and matrix stiffness . figure s4a shows that fibronectin ( fn ) consistently reduces the effect of plx4720 on both 5555 and 4434 melanoma cells . other matrix components , including thbs1/2 and tnc , have more variable effects between the two cell lines . polyacrylamide gels ranging from 0.2 kpa ( similar to adipose tissue ) to 12 kpa ( similar to stiff tumors or muscle ) were coated with either collagen i , fn , or a combination of fn , thbs1/2 , and tnc . melanoma cells on low stiffness collagen i showed high levels of cell death following braf inhibition ( figures 4a and 4b ) . however , this was greatly abrogated if cells were cultured on fn matrices with either 3 kpa or 12 kpa stiffness , with the most impressive cell survival on 12 kpa fn- , thbs1/2- , and tnc , containing matrices ( figures 4a and 4b ) . these data demonstrate that an appropriate matrix composition and stiffness can render braf - mutant melanoma cells insensitive to plx4720 . increasing the stiffness of fibronectin matrices lead to the re - organization of integrin 1 into focal adhesions and elevated pfak levels ( figures 4c and s4b ) . furthermore , treatment of co - cultures with plx4720 led to the relocation of active integrin 1 into fibrillar adhesions and increased pfak signals ( figures 4d and s4c ) . to determine if the changes in integrin organization and fak signaling are relevant for the adaptation of melanoma - maf co - cultures to plx4720 , we investigated the effect of combined plx4720 treatment and experimental perturbation of integrin 1 and fak . combination of plx4720 with either integrin 1 or fak depletion led to prevention of erk / mapk re - activation and synergistic induction of cell death ( figures 4e4 g , s4d , and s4e ) . we next investigated whether combining braf inhibition with pharmacological targeting of signaling downstream of integrin 1 would be effective . multiple fak inhibitors ( pf573228 , pf562271 , faki14 ) prevented the re - activation of erk / mapk signaling in melanoma / maf co - cultured spheroids treated with plx4720 ( figures 5a , 5c , s5a , and s5b and movie s4 , see also figure 2e ; all quantification is collated in figure s5 g , and similar results with 4434 cells in figures s5h and s5i ) . treatment of plx4720-naive cells with fak inhibitors alone did not lead to reduced erk activity ( figures 5a , 5c , s5a , and s5b ) . as expected , combined braf and mek inhibition lead to a stable reduction in erk activity ( figures 5c and s5c ) . in accordance with the results of ekarev fret imaging , combination of plx4720 with fak inhibition led to synergistic induction of cell death in the co - cultured spheroids ( figure 5d ) . because fak activity is often linked to src function , we tested the effect of two src inhibitors in combination with plx4720 . both dasatinib and pp2 effectively prevented the re - activation of erk signaling following plx4720 treatment ( figures 5b , 5c , and s5d and movie s4 ) . furthermore , the combination of plx4720 and dasatinib led to significantly increased melanoma cell death ( figure 5d ) . neither fak nor src inhibition reduced the viability of melanoma mono - cultures ( figure s5j ) . these data demonstrate the critical role of adhesion - mediated signaling via integrin 1 , fak , and src in the adaptive re - activation of erk / mapk following braf inhibition in tumor / stroma co - cultures . both basal and plx4720-enhanced maf activity can be reduced by targeting pdgfr ( figure s3 we tested this by monitor ekarev signals following the combination of plx4720 and imatinib ( autofluorescence of sunitinib prevented us from testing its effect in this assay ) . this combination reduced the re - activation of erk , while imatinib alone had no effect ( figures s5e and s5f ) . these data support our hypothesis that pdgfr signaling in the stroma contributes to erk re - activation in melanoma cells . we next tested whether braf and fak inhibition would synergize to control melanoma growth in vivo . 5555 melanoma tumors were allowed to reach 48 mm before mice began receiving daily doses of plx4720 , pf562271 , plx4720 and pf562271 , or vehicle . figure 5e shows that only the combination of braf and fak inhibitors led to effective control of tumor size ; either inhibitor alone had only a modest effect . plx4720-treated tumors had increased aligned fibrous ecm and fn , and this was associated with fak - dependent elongation of melanoma cells ( figure 5f ) . these data indicate that combined braf and fak inhibition is a promising strategy for improving management of braf mutant melanoma . the data described above demonstrate that mafs provide a mechanism for mouse braf - mutant melanoma cell lines refractory to plx4720 in vivo . however , the majority of human braf - mutant melanoma respond well to braf inhibition before the emergence of resistant disease over many months ( chapman et al . , 2011 ) . we were interested whether our findings regarding the role of the stroma in providing drug tolerance were relevant to the survival of melanoma cells in between the initial administration of braf inhibitors and the ultimate emergence of genetically resistant cells . therefore , we examined erk / mapk activity and stromal changes in two models of human melanoma that exhibit a clear response to braf inhibition in vivo . figures 6a and 6b show that a375 and wm266.4 human melanoma cells are sensitive to plx4720 both in vitro and in vivo . however , tumors did not disappear and typically 24 mm of residual disease remained during plx4720 treatment . intravital imaging of the ekarev biosensor revealed that the residual disease after 1114 days of plx4720 treatment exhibited similar levels of erk / mapk signaling to the pre - treatment tumors ( figures 6c and s6a ) . based on our analysis of 5555 and 4434 models , we predict the following : there should be changes in the stroma of plx4720-treated a375 and wm266.4 tumors , the melanoma cells should not be intrinsically resistant to plx4720 at this stage , but that they should use fak- and src - dependent signaling to sustain erk / mapk activity and survival . both a375 and wm266.4 exhibited clear changes in collagen shg when treated with plx4720 ( figure 6c ) . immunohistochemical staining , and gomori s trichrome staining confirmed that the residual disease was rich in fibroblastic stroma and had higher levels of fibrillar collagen , fn and tnc ( figures 6d and s6a ) . furthermore , conformation specific antibodies revealed increased active integrin 1 levels in plx - treated tumors ( figure s6b ) . to formally confirm that the melanoma cells were dependent on extrinsic signals for their tolerance of plx4720 , we re - isolated a375 and wm266.4 cells from the residual disease that persisted following plx4720 treatment . figure s6c shows that these cells are just as sensitive to plx4720 as parental cells when cultured in isolation without a supportive tumor microenvironment . these data demonstrate the persistence of residual disease is due to cell extrinsic factors that re - activate erk signaling by a braf - independent mechanism . to further test our hypothesis , we investigated whether the erk signaling in residual disease was dependent on fak and src function . combination of either pf573228 or dasatinib with plx4720 led to more prolonged erk inhibition in tumor explants ( figure s6d ) . finally , we tested whether combining braf and fak inhibition would have a synergistic effect on a375 tumor burden . established a375 tumors ( 57 mm ) were treated with plx4720 , pf562271 , plx4720 , and pf562271 , or vehicle for up to 30 days . we observed that combined braf and fak inhibition led to significantly reduced burden compared to either treatment alone ( figure 6e ) ; indeed , no biolumiscence signal could be detected in one mouse . co - targeting braf and fak also triggered extensive neutrophil and macrophage infiltration ( figure s6e ) . more surprisingly , although plx4720 controls total melanoma volume in this tumor model , it has a minimal effect on ki-67 positivity , whereas plx4720 + pf562271 leads to a clear reduction in ki-67 positive melanoma cells ( figures 6f and 6 g ) . these data suggest that plx4720-treated tumors continue to proliferate , albeit balanced by cell death . this would enable the evolution of resistant clones even in a plx4720-treated tumor that is not increasing in size . we next investigated changes in the tumor stroma and the effect of fak inhibition in a patient - derived xenograft ( pdx ) model . figure 7a shows that this grows well in control nod.cg-prkdc il2rg / szj ( nsg ) mice , but the growth of this pdx was controlled by plx4720 , although significant disease remained . in agreement with our analyses of a375 and wm266.4 tumors , histological analysis of the residual disease in plx4720-treated mice revealed increased fibrous ecm , higher numbers of sma - positive cells , and active erk / mapk signaling ( figure 7b ) . we next explored whether this pdx model would eventually fail plx4720 therapy , and whether fak inhibition might reduce this failure . after roughly 50 days of plx4720 treatment , the pdx tumors began to grow , indicating therapy failure ( figure 7c ) . fak inhibition alone had no effect on tumor growth , but when combined with plx4720 lead to significantly increased tumor control both at early time points and , more importantly , after 4 months when plx4720-treated tumors were failing therapy ( figure 7c ) . no adverse effects , such as weight loss , were observed in mice treated with plx4720 and pf562271 for 4 months ( figure s7a ) . the data above demonstrate that braf inhibition can directly affect the fibroblastic stroma leading to remodeled ecm and adhesion - dependent signaling to erk that negates the effect of braf inhibition in the melanoma cells . we find histologic features that are compatible with our experimental models in patient samples prior to vemurafenib treatment and following disease progression on vemurafenib ( table s2 ) : these include changes in the stroma and melanoma cell morphology following vemurafenib treatment . figure s7b shows increased fibrous ecm and sma - positive mafs were visible in patients 1 and 4 after failure of braf inhibition . in patient 4 , there was an increase in elongated melanoma cells . these analyses confirm that braf inhibition can modulate the fibrous ecm and fibroblastic stroma in melanoma patients . taken together with the experimental work , we show how an effect of braf inhibition on the stroma generates a drug - tolerant microenvironment that supports residual disease before the emergence of genetic cell intrinsic drug resistance mechanisms ( figure 7d ) . a common feature of kinase - targeted therapies is a period of response followed by the emergence of resistant disease . in melanoma , different genetic resistance mechanisms can develop in the same patient ( shi et al . , 2014 ; van allen et al . , 2014 ) , often at multiple metastatic sites ( shi et al . , 2014 ) . these data are not easily reconciled with a model of a pre - existing genetically resistant sub - clone ; thus , resistant melanoma cells may arise after treatment with vemurafenib has begun . how significant numbers of braf - mutant cells survive therapy prior to the emergence of genetic mutations or epigenetic changes that enable resistance is not well understood , and here we describe a mechanism enabling the survival of large numbers of braf - mutant melanoma cells following plx4720 treatment . this mechanism has two key features : first , the fibroblastic stroma is paradoxically activated by braf inhibition ; and second , adhesion - dependent signals from the altered tumor microenvironment lead to braf - independent erk / map kinase activity . braf inhibitors can promote ras - driven signaling through craf ( hatzivassiliou et al . , 2010 ; heidorn et al . , 2010 ; nazarian et al . , 2010 ; , 2010 ; villanueva et al . , 2010 ) ; however , it was not clear if this mechanism may act on ras - signaling in the stroma and how this may affect therapeutic efficacy . we find that plx4720 increases erk / mapk signaling , elevates matrix production , and increases mlc phosphorylation and matrix remodeling , which is likely due to enhancing rtk - ras signaling . these compounds probably act through their effects on pdgfr , which is upregulated by plx4720 and has previously been implicated in the function of carcinoma - associated fibroblasts ( erez et al . , 2010 ; kalluri and zeisberg , 2006 ; pietras et al . , 2008 ) dabrafenib is likely to activate the fibroblastic stroma in a similar manner to plx4720 ( gibney and zager , 2013 ; huang et al . , 2013 ) . interestingly , we found that plx4720 did not enhance the activity of normal fibroblasts ; this may be because they do not have elevated rtk signaling . the ability of mafs to confer plx4720 tolerance depends on integrin 1 and fak in the melanoma cells . furthermore , fibronectin - rich matrices of the appropriate stiffness are sufficient to abrogate the effects of braf inhibition . we propose that certain ecm environments provide safe havens for melanoma cells to tolerate braf targeted therapy . although extrinsic signals from the tumor microenvironment can support four different braf - mutant melanoma cell lines in the presence of plx4720 , the magnitude of this protective effect varied therefore , the magnitude of the effect of plx4720 on the stroma would be variable . alternatively , melanoma cells may differ in their ability to activate adhesion - dependent fak and src to erk / map kinase . although we find no role for hgf in our analyses , the ability of c - met to drive rac activation and elongated modes of cell migration could be a point of commonality with the work of strausmann et al . ( straussman et al . , 2012 ; watson et al . , 2014 ) . furthermore , the relief of negative inhibition that can be triggered by braf inhibition may also sensitize melanoma cells to integrin / fak - mediated erk activation ( lito et al . there are currently moves to use combined blockade of braf and mek to improve outcomes in melanoma ( flaherty et al . although this approach extends lifespan , it is rarely curative ( larkin et al . , both strategies are similarly effective at inducing melanoma cell death . in the long term , combined braf and fak blockade improves tumor control but rarely eliminates tumors entirely , and some pdx tumors resume very slow growth after 23 months . our results also indicate that braf and src inhibition will be effective and recent results using a combined raf and src inhibitor support this ( girotti et al . , 2015 ) . furthermore , because many rtks require signals from cell adhesions to function ( butcher et al . , 2009 ; paszek et al . , 2005 ; 2014 ) , fak targeting may attenuate divergent signaling from cell adhesion receptors and rtks to cell survival and proliferation machinery , and this may provide some benefit over simply targeting erk signaling . to conclude , multiple mechanisms of cell - intrinsic resistance to melanoma have been documented . in patients , cell - intrinsic resistance the mechanisms that sustain melanoma cells in the period between initial response and disease progression on therapy are unclear . we describe how paradoxical activation of the fibroblastic stroma enables matrix - derived signaling via integrin 1 and fak to promote erk activation and cell survival . we propose that by reducing the numbers of residual melanoma cells , there would less material from which truly resistant cells could arise . this should lead to longer periods of progression - free survival and possibly even cure . braf - mutant ( 5555 and 4434 ) and nras - mutant ( c790 ) mouse melanoma cell lines were established from c57bl/6_braf + /lsl - brafv600e ; tyr::creert2+/o ( dhomen et al . , 2009 ) and c57bl/6_nras + /lsl - nrasg12d ; tyr::cre - a ( pedersen et al . , 2013 b16f10 mouse melanoma cell lines , and a375 and wm266.4 human melanoma cell lines were a gift from prof . melanoma cells were maintained in dmem ( invitrogen ) with 10% fetal bovine serum/1% penstrep ( gibco ) . the fret biosensors for erk / mapk ( ekarev - nls / nes ) was described previously ( komatsu et al . , 2011 ) and were introduced into the cells with piggybac transposon system ( wellcome trust sanger institute , hinxton , uk ) . animal experiments were done in accordance with uk regulations under project license ppl/80/2368 . for melanoma allograft model , 2 10 5555 or 4434 cells were suspended into 200 l pbs and subcutaneously injected in the flanks of c57bl/6 mice or c57bl/6_rosa26_mtmg mice . after 1014 days when the cells form palpable tumors , the mice were dosed daily by oral gavage with vehicle ( 4% dmso ) , plx4720 ( 25 mg / kg ) , pf562271 ( 50 mg / kg ) , or the combination . the tumor size was monitored daily by calculating length width ( mm ) . for experimental lung metastasis , 2 10 5555 cells in 200 l pbs were injected from the tail vein and , after 4 weeks , the mice were killed and the lung tumors are examined further . for human melanoma xenograft model , 2 10 a375 or wm266.4 cells in 200 l pbs were subcutaneously injected in the flanks of nude mice . after about 2 weeks when palpable tumors formed , we started further experiments . for skin - flap tumor imaging , the mouse was anesthetized and the tumor was surgically exposed and imaged through a coverslip with a zeiss lsm780 inverted microscope as described elsewhere ( giampieri et al . , 2009 ) . for bioluminescence imaging , nude mice bearing a375 tumors stably expressing firefly luciferase were anesthetized , intraperitoneally injected with d - luciferin ( 150 mg / kg , perkinelmer ) , and imaged under ivis spectrum ( perkinelmer ) . tumor samples were collected under the manchester cancer research centre ( mcrc ) biobank ethics application no . 07/h1003/161 + 5 with full informed consent from the patients . the work presented in this manuscript was approved by mcrc biobank access committee application 13_rima_01 . necrotic parts of the tumors were removed and 5 5 5 mm pieces were implanted subcutaneously in the right flanks of 5- to 6-week - old female il-2 nsg mice . when the pdxs reached 1500 mm volume , they were excised , and viable tissue was dissected into 5 mm cubes and transplanted into additional mice using the same procedure . genomic and histological analyses had confirmed that the tumors at each point were derived from the starting material . following transplantation , tumors were allowed to grow to 6080 mm , and the mice were randomized before initiation of treatment by daily orogastric gavage of plx4720 ( 45 mg / kg ) , pf562271 ( 50 mg / kg ) , vehicle ( 5% dmso , 95% water ) , or the combination for the indicated days . tumor spheroids cultured in collagen gel on a glass bottom dish / plate were imaged with an lsm780 inverted microscope through 20 objective every 5 min for up to 13 hr . for the dual - emission ratio imaging , the fret biosensor was excited by a chameleon ti : sapphire laser ( coherent inc . ) at 820 nm excitation wavelength . the emission light was separated by beam splitters into 463506 nm for cfp and 515559 nm for fret . details in microscopy , data analysis , and image processing are available in the supplemental experimental procedures .

summaryintravital imaging of braf - mutant melanoma cells containing an erk / mapk biosensor reveals how the tumor microenvironment affects response to braf inhibition by plx4720 . initially , melanoma cells respond to plx4720 , but rapid reactivation of erk / mapk is observed in areas of high stromal density . this is linked to paradoxical activation of melanoma - associated fibroblasts by plx4720 and the promotion of matrix production and remodeling leading to elevated integrin 1/fak / src signaling in melanoma cells . fibronectin - rich matrices with 312 kpa elastic modulus are sufficient to provide plx4720 tolerance . co - inhibition of braf and fak abolished erk reactivation and led to more effective control of braf - mutant melanoma . we propose that paradoxically activated mafs provide a safe haven for melanoma cells to tolerate braf inhibition .

Significance Introduction Results Discussion Experimental Procedures

we show how braf - mutant melanoma cells rapidly become tolerant to plx4720 in areas of high stroma . we demonstrate that plx4720 has an effect on the tumor stroma , leading to enhanced matrix remodeling . we propose that this safe haven enhances the population of cancer cells from which genetic resistance emerges . this work highlights the need to consider the effects of targeted therapies on the tumor microenvironment . , however , agents targeting either egfr or braf typically show good efficacy in tumors with matching oncogenic mutations for a number of months before genetically resistant cells dominate the tumor and the therapy fails ( kobayashi et al . in the case of egfr - mutant lung tumors , it has been shown that resistant cells may be present even before treatment and that these are at a strong selective advantage during therapy ( inukai et al . however , the situation in braf - mutant melanoma treated with braf inhibitors is less clear . there is significant variability in the magnitude of initial response to braf inhibition ( chapman et al . the activity of erk / mapk can be monitored in live tissue using a biosensor construct containing two fluorophores , a long flexible linker , an erk / map kinase binding site , an optimal substrate site for the kinase , and a phospho - threonine binding domain ( harvey et al . , 2011 when the substrate site is phosphorylated , it engages in an intra - molecular interaction with the phospho - threonine binding domain , leading to an overall change in the conformation of the molecule and a change in fluorescence resonance energy transfer ( fret ) between the two fluorophores ( komatsu et al . , this system enables the biochemical response to braf inhibition to be monitored with single cell resolution in vivo . these technologies can be combined with intravital imaging windows to longitudinally track both the biochemical response to braf inhibition and the distribution of the tumor stroma ( janssen et al . to study responses to braf inhibition in a syngeneic tumor microenvironment , we tested the response of braf and nras mutant c57/bl6 mouse melanoma cell lines to the braf inhibitor plx4720 . we next tested the response of these cells to plx4720 when growing as tumors in syngeneic mice . to our surprise , both braf - mutant melanoma cell lines were refractory to plx4720 ( figure 1b ) . furthermore , they may represent the small subset of braf - mutant melanoma that exhibit only a small response to vemurafenib . to understand the lack of response of 5555 and 4434 cells to plx4720 in vivo , we reasoned that it would be important to monitor the braf signaling with single cell resolution . we engineered 5555 and 4434 cells to express an erk / map kinase biosensor located in the nucleus called ekarev - nls ( figure s1a ) . the ekarev biosensor faithfully monitored changes in erk / map kinase signaling in response to tpa and either braf or mek inhibitors ( figures s1b s1e ) . in contrast , paradoxical activation of erk / mapk activity is observed in nras mutant cells treated with plx4720 ( figures s1c and s1f ) . we next generated tumors using 5555-ekarev - nls cells ; in some cases , these were adjacent to titanium imaging windows that allow longitudinal imaging of the same tumor ( janssen et al . to gain insight into the lack of effect of plx4720 on tumor growth , we performed longitudinal imaging of tumors before and during plx4720 treatment . these data suggest that plx4720 can access the tumor and achieve the reduction in erk / mapk activity expected based on the in vitro analysis in figures s1c s1e . analysis of the distribution of ekarev signal after 3 days of plx4720 treatment suggested that only a small proportion of 5555 cells show a stable reduction in erk / mapk activity ( figures 1e and 1f ) . to more robustly test this observation , we segmented images of plx4720 treated tumors into regions with high or low levels of mtomato signal ( figure s1l ) . these data demonstrate braf mutant 5555 and 4434 tumors show a short - lived biochemical response to plx4720 . further , the rapid re - activation of erk / mapk and adaptation of these tumors to plx4720 is correlated with stromal density . we sought to establish a culture system that re - capitulated the plx4720 tolerance that we observed in vivo . the behavior of pure spheroids of melanoma cells was compared with that of equivalent sized tumor pieces when embedded in a collagen matrix . figures 2a and 2b show that pure melanoma spheroids were highly sensitive to plx4720 , with the appearance of many nuclear fragments indicating cell death . in contrast , melanoma explants from either subcutaneous tumors or experimentally established lung metastases were unresponsive to plx4720 ( figures 2a and 2b ) . even in the presence of drug , melanoma cells retained healthy nuclear architecture and invasive capability . thus , the spheroid model recapitulates the stroma - dependent plx4720 tolerance observed in vivo . we therefore explored whether melanoma - associated fibroblasts ( mafs ) or macrophages might be sufficient to confer drug tolerance on melanoma cells . two isolates of mafs were established from patients ( designated maf1 and maf2 , figures s2b and s2c ) and their effect on the response of 5555 and 4434 cells to plx4720 was tested . figures 2a , 2b , and s2d show that co - culture of either maf1 or maf2 with melanoma cells conferred tolerance of plx4720 and invasive behavior . furthermore , we excluded a role for egfr and c - met in mediating maf - dependent plx4720 tolerance ( figures s2 g and s2h ) . co - culture with macrophages was unable to confer drug tolerance on melanoma cells ( figure s2i ) . we performed time - lapse imaging of spheroid co - cultures before and after the addition of plx4720 . strikingly , co - cultures that had been exposed to plx4720 for 12 hr were completely refractory to the addition of more plx4720 , while importantly , they remained sensitive to the addition of the mek inhibitor pd184352 ( figures 2f and 2 g ) . these data demonstrate that co - cultures of melanoma cells and mafs switch from braf - dependent erk signaling to braf - independent erk signaling in just 12 hr . spheroid co - cultures of 5555 and mafs were imaged before and during the response to plx4720 . we hypothesized that plx4720 might activate mafs by enhancing the activity of pdgfr- and ras - dependent signaling through the paradoxical activation of craf ( hatzivassiliou et al . the data above show that plx4720 elicits changes in matrix production and remodeling by mafs . to test if the ecm was sufficient to modulate the response of melanoma cells to plx4720 , we varied both matrix composition and matrix stiffness . melanoma cells on low stiffness collagen i showed high levels of cell death following braf inhibition ( figures 4a and 4b ) . these data demonstrate that an appropriate matrix composition and stiffness can render braf - mutant melanoma cells insensitive to plx4720 . furthermore , treatment of co - cultures with plx4720 led to the relocation of active integrin 1 into fibrillar adhesions and increased pfak signals ( figures 4d and s4c ) . to determine if the changes in integrin organization and fak signaling are relevant for the adaptation of melanoma - maf co - cultures to plx4720 , we investigated the effect of combined plx4720 treatment and experimental perturbation of integrin 1 and fak . combination of plx4720 with either integrin 1 or fak depletion led to prevention of erk / mapk re - activation and synergistic induction of cell death ( figures 4e4 g , s4d , and s4e ) . we next investigated whether combining braf inhibition with pharmacological targeting of signaling downstream of integrin 1 would be effective . multiple fak inhibitors ( pf573228 , pf562271 , faki14 ) prevented the re - activation of erk / mapk signaling in melanoma / maf co - cultured spheroids treated with plx4720 ( figures 5a , 5c , s5a , and s5b and movie s4 , see also figure 2e ; all quantification is collated in figure s5 g , and similar results with 4434 cells in figures s5h and s5i ) . in accordance with the results of ekarev fret imaging , combination of plx4720 with fak inhibition led to synergistic induction of cell death in the co - cultured spheroids ( figure 5d ) . both dasatinib and pp2 effectively prevented the re - activation of erk signaling following plx4720 treatment ( figures 5b , 5c , and s5d and movie s4 ) . furthermore , the combination of plx4720 and dasatinib led to significantly increased melanoma cell death ( figure 5d ) . these data demonstrate the critical role of adhesion - mediated signaling via integrin 1 , fak , and src in the adaptive re - activation of erk / mapk following braf inhibition in tumor / stroma co - cultures . this combination reduced the re - activation of erk , while imatinib alone had no effect ( figures s5e and s5f ) . these data support our hypothesis that pdgfr signaling in the stroma contributes to erk re - activation in melanoma cells . we next tested whether braf and fak inhibition would synergize to control melanoma growth in vivo . 5555 melanoma tumors were allowed to reach 48 mm before mice began receiving daily doses of plx4720 , pf562271 , plx4720 and pf562271 , or vehicle . figure 5e shows that only the combination of braf and fak inhibitors led to effective control of tumor size ; either inhibitor alone had only a modest effect . plx4720-treated tumors had increased aligned fibrous ecm and fn , and this was associated with fak - dependent elongation of melanoma cells ( figure 5f ) . these data indicate that combined braf and fak inhibition is a promising strategy for improving management of braf mutant melanoma . the data described above demonstrate that mafs provide a mechanism for mouse braf - mutant melanoma cell lines refractory to plx4720 in vivo . however , the majority of human braf - mutant melanoma respond well to braf inhibition before the emergence of resistant disease over many months ( chapman et al . we were interested whether our findings regarding the role of the stroma in providing drug tolerance were relevant to the survival of melanoma cells in between the initial administration of braf inhibitors and the ultimate emergence of genetically resistant cells . therefore , we examined erk / mapk activity and stromal changes in two models of human melanoma that exhibit a clear response to braf inhibition in vivo . figures 6a and 6b show that a375 and wm266.4 human melanoma cells are sensitive to plx4720 both in vitro and in vivo . intravital imaging of the ekarev biosensor revealed that the residual disease after 1114 days of plx4720 treatment exhibited similar levels of erk / mapk signaling to the pre - treatment tumors ( figures 6c and s6a ) . based on our analysis of 5555 and 4434 models , we predict the following : there should be changes in the stroma of plx4720-treated a375 and wm266.4 tumors , the melanoma cells should not be intrinsically resistant to plx4720 at this stage , but that they should use fak- and src - dependent signaling to sustain erk / mapk activity and survival . to formally confirm that the melanoma cells were dependent on extrinsic signals for their tolerance of plx4720 , we re - isolated a375 and wm266.4 cells from the residual disease that persisted following plx4720 treatment . figure s6c shows that these cells are just as sensitive to plx4720 as parental cells when cultured in isolation without a supportive tumor microenvironment . to further test our hypothesis , we investigated whether the erk signaling in residual disease was dependent on fak and src function . combination of either pf573228 or dasatinib with plx4720 led to more prolonged erk inhibition in tumor explants ( figure s6d ) . finally , we tested whether combining braf and fak inhibition would have a synergistic effect on a375 tumor burden . established a375 tumors ( 57 mm ) were treated with plx4720 , pf562271 , plx4720 , and pf562271 , or vehicle for up to 30 days . we observed that combined braf and fak inhibition led to significantly reduced burden compared to either treatment alone ( figure 6e ) ; indeed , no biolumiscence signal could be detected in one mouse . co - targeting braf and fak also triggered extensive neutrophil and macrophage infiltration ( figure s6e ) . we next investigated changes in the tumor stroma and the effect of fak inhibition in a patient - derived xenograft ( pdx ) model . figure 7a shows that this grows well in control nod.cg-prkdc il2rg / szj ( nsg ) mice , but the growth of this pdx was controlled by plx4720 , although significant disease remained . in agreement with our analyses of a375 and wm266.4 tumors , histological analysis of the residual disease in plx4720-treated mice revealed increased fibrous ecm , higher numbers of sma - positive cells , and active erk / mapk signaling ( figure 7b ) . fak inhibition alone had no effect on tumor growth , but when combined with plx4720 lead to significantly increased tumor control both at early time points and , more importantly , after 4 months when plx4720-treated tumors were failing therapy ( figure 7c ) . no adverse effects , such as weight loss , were observed in mice treated with plx4720 and pf562271 for 4 months ( figure s7a ) . the data above demonstrate that braf inhibition can directly affect the fibroblastic stroma leading to remodeled ecm and adhesion - dependent signaling to erk that negates the effect of braf inhibition in the melanoma cells . figure s7b shows increased fibrous ecm and sma - positive mafs were visible in patients 1 and 4 after failure of braf inhibition . these analyses confirm that braf inhibition can modulate the fibrous ecm and fibroblastic stroma in melanoma patients . taken together with the experimental work , we show how an effect of braf inhibition on the stroma generates a drug - tolerant microenvironment that supports residual disease before the emergence of genetic cell intrinsic drug resistance mechanisms ( figure 7d ) . how significant numbers of braf - mutant cells survive therapy prior to the emergence of genetic mutations or epigenetic changes that enable resistance is not well understood , and here we describe a mechanism enabling the survival of large numbers of braf - mutant melanoma cells following plx4720 treatment . this mechanism has two key features : first , the fibroblastic stroma is paradoxically activated by braf inhibition ; and second , adhesion - dependent signals from the altered tumor microenvironment lead to braf - independent erk / map kinase activity . , 2010 ) ; however , it was not clear if this mechanism may act on ras - signaling in the stroma and how this may affect therapeutic efficacy . we find that plx4720 increases erk / mapk signaling , elevates matrix production , and increases mlc phosphorylation and matrix remodeling , which is likely due to enhancing rtk - ras signaling . these compounds probably act through their effects on pdgfr , which is upregulated by plx4720 and has previously been implicated in the function of carcinoma - associated fibroblasts ( erez et al . the ability of mafs to confer plx4720 tolerance depends on integrin 1 and fak in the melanoma cells . furthermore , fibronectin - rich matrices of the appropriate stiffness are sufficient to abrogate the effects of braf inhibition . we propose that certain ecm environments provide safe havens for melanoma cells to tolerate braf targeted therapy . although extrinsic signals from the tumor microenvironment can support four different braf - mutant melanoma cell lines in the presence of plx4720 , the magnitude of this protective effect varied therefore , the magnitude of the effect of plx4720 on the stroma would be variable . alternatively , melanoma cells may differ in their ability to activate adhesion - dependent fak and src to erk / map kinase . furthermore , the relief of negative inhibition that can be triggered by braf inhibition may also sensitize melanoma cells to integrin / fak - mediated erk activation ( lito et al . there are currently moves to use combined blockade of braf and mek to improve outcomes in melanoma ( flaherty et al . in the long term , combined braf and fak blockade improves tumor control but rarely eliminates tumors entirely , and some pdx tumors resume very slow growth after 23 months . we describe how paradoxical activation of the fibroblastic stroma enables matrix - derived signaling via integrin 1 and fak to promote erk activation and cell survival . we propose that by reducing the numbers of residual melanoma cells , there would less material from which truly resistant cells could arise . braf - mutant ( 5555 and 4434 ) and nras - mutant ( c790 ) mouse melanoma cell lines were established from c57bl/6_braf + /lsl - brafv600e ; tyr::creert2+/o ( dhomen et al . the fret biosensors for erk / mapk ( ekarev - nls / nes ) was described previously ( komatsu et al . for skin - flap tumor imaging , the mouse was anesthetized and the tumor was surgically exposed and imaged through a coverslip with a zeiss lsm780 inverted microscope as described elsewhere ( giampieri et al . following transplantation , tumors were allowed to grow to 6080 mm , and the mice were randomized before initiation of treatment by daily orogastric gavage of plx4720 ( 45 mg / kg ) , pf562271 ( 50 mg / kg ) , vehicle ( 5% dmso , 95% water ) , or the combination for the indicated days .

all experiments were conducted in accordance with the national institutes of health guidelines for the care and use of experimental animals , and all procedures involving mice were preapproved by the committee on use and care of animals at the university of michigan . generation of the pitx2 , pitx2 , ubc - creer , and tfap2b strains has been described previously . mice were mated to generate timed pregnancies , and the morning that a plug was identified was designated embryonic day 0.5 ( e0.5 ) . the relevant crosses were ubc - creer ; pitx2 pitx2 ; r26r / r26r , and tfap2b tfap2b . when indicated , a single intraperitoneal injection of tamoxifen ( sigma - aldrich corp . , st . louis , mo , usa ) suspended in corn oil at a dose of 100 mg per gram body weight was administered to the pregnant dam at noon on the day noted . the resulting embryos were genotyped as appropriate using pcr - based methods . timed pregnant dams from mating ubc - creer ; pitx2 pitx2 ; r26r / r26r were injected with tamoxifen at e10.5 , and embryos were collected at e12.5 , e13.5 , or 16.5 . embryos were also collected at e12.5 from timed pregnant dams following the mating tfap2b tfap2b . corneal tissue was micro dissected from frozen sections using a leica lmd7000 workstation ( buffalo grove , il , usa ) . corneal tissue from the pair of eyes from each embryo was collected and considered as one sample . total rna was isolated from each sample using the rneasy micro kit ( qiagen , hilden , germany ) and used to generate cdna by the ovation pico wta system v2 method ( nugen , san carlos , ca , usa ) . relative expression of tfap2b , vegfa , foxc1 , lmx1b , mmp2 , mmp9 , and flt1 were measured in replicate samples ( n = 3 - 6/genotype ) using taqman gene expression assays ( life sciences technologies , carlsbad , ca , usa ) . amplification and labeling of rna was performed using 200 pg of total rna for each sample and the targetamp 2-round aminoallyl - arna amplification kit 1.0 ( epicentre , madison , wi , usa ) . hybridization of alexa555 labeled rna to microarrays ( mouse gene expression v.2 , 8x60k ) was performed per the protocol of agilent technologies ( santa clara , ca , usa ) . microarrays were scanned with an agilent dual - laser scanner , and images were analyzed using agilent technologies ' feature extraction software version 11.0.1.1 . expression values were quantile normalized for each array . for each pair of conditions compared , genes were first filtered to select those having detectable expression in at least 75% of the samples for either condition . a moderated t - test and the benjamini and hochberg multiple test correction minimal criteria were 2-fold change in expression and a false discovery rate of 5% . embryos generated for histology were fixed in 4% paraformaldehyde diluted in pbs , washed in pbs , dehydrated through graded alcohols , and processed into paraplast plus ( mccormick scientific , st . louis , mo , usa ) for paraffin sectioning . mounted paraffin sections for morphologic analysis primary antibodies against pitx2 ( gift from tord hjalt ) , ap-2 ( abnova , walnut , ca , usa ) , cytokeratin 4 ( ck4 , abcam , cambridge , ca , usa ) , cytokeratin 12 ( ck12 , gift from winston kao ) , endomucin ( affymetrix ebioscience , san diego , ca , usa ) , zo-1 ( invitrogen , carlsbad , ca , usa ) , and lyve-1 ( abcam ) were used . digoxigenin - labeled riboprobes against keratocan ( gift from winston kao ) and dkk1 ( gift from christoff niehrs ) were generated from donated plasmid templates and used to stain paraffin sections as previously described . all experiments were conducted in accordance with the national institutes of health guidelines for the care and use of experimental animals , and all procedures involving mice were preapproved by the committee on use and care of animals at the university of michigan . generation of the pitx2 , pitx2 , ubc - creer , and tfap2b strains has been described previously . mice were mated to generate timed pregnancies , and the morning that a plug was identified was designated embryonic day 0.5 ( e0.5 ) . the relevant crosses were ubc - creer ; pitx2 pitx2 ; r26r / r26r , and tfap2b tfap2b . when indicated , a single intraperitoneal injection of tamoxifen ( sigma - aldrich corp . , st . louis , mo , usa ) suspended in corn oil at a dose of 100 mg per gram body weight was administered to the pregnant dam at noon on the day noted . timed pregnant dams from mating ubc - creer ; pitx2 pitx2 ; r26r / r26r were injected with tamoxifen at e10.5 , and embryos were collected at e12.5 , e13.5 , or 16.5 . embryos were also collected at e12.5 from timed pregnant dams following the mating tfap2b tfap2b . corneal tissue was micro dissected from frozen sections using a leica lmd7000 workstation ( buffalo grove , il , usa ) . corneal tissue from the pair of eyes from each embryo was collected and considered as one sample . total rna was isolated from each sample using the rneasy micro kit ( qiagen , hilden , germany ) and used to generate cdna by the ovation pico wta system v2 method ( nugen , san carlos , ca , usa ) . relative expression of tfap2b , vegfa , foxc1 , lmx1b , mmp2 , mmp9 , and flt1 were measured in replicate samples ( n = 3 - 6/genotype ) using taqman gene expression assays ( life sciences technologies , carlsbad , ca , usa ) . amplification and labeling of rna was performed using 200 pg of total rna for each sample and the targetamp 2-round aminoallyl - arna amplification kit 1.0 ( epicentre , madison , wi , usa ) . hybridization of alexa555 labeled rna to microarrays ( mouse gene expression v.2 , 8x60k ) was performed per the protocol of agilent technologies ( santa clara , ca , usa ) . microarrays were scanned with an agilent dual - laser scanner , and images were analyzed using agilent technologies ' feature extraction software version 11.0.1.1 . expression values were quantile normalized for each array . for each pair of conditions compared , genes were first filtered to select those having detectable expression in at least 75% of the samples for either condition . a moderated t - test and the benjamini and hochberg multiple test correction minimal criteria were 2-fold change in expression and a false discovery rate of 5% . embryos generated for histology were fixed in 4% paraformaldehyde diluted in pbs , washed in pbs , dehydrated through graded alcohols , and processed into paraplast plus ( mccormick scientific , st . louis , mo , usa ) for paraffin sectioning . mounted paraffin sections for morphologic analysis were dewaxed , rehydrated , and stained with hematoxylin and eosin . paraffin sections were immunostained as previously described . primary antibodies against pitx2 ( gift from tord hjalt ) , ap-2 ( abnova , walnut , ca , usa ) , cytokeratin 4 ( ck4 , abcam , cambridge , ca , usa ) , cytokeratin 12 ( ck12 , gift from winston kao ) , endomucin ( affymetrix ebioscience , san diego , ca , usa ) , zo-1 ( invitrogen , carlsbad , ca , usa ) , and lyve-1 ( abcam ) were used . digoxigenin - labeled riboprobes against keratocan ( gift from winston kao ) and dkk1 ( gift from christoff niehrs ) were generated from donated plasmid templates and used to stain paraffin sections as previously described . we combined laser microdissection and microarray analysis using e12.5 embryos to identify genes regulated by pitx2 during corneal development . because anterior segment morphogenesis and early corneal development are blocked prior to initiation of corneal development in both global and neural crest - specific pitx2-null embryos , we employed a temporal knockout strategy described previously to selectively ablate pitx2 at the onset of corneal development . briefly , we crossed mice carrying our conditional pitx2 allele with mice carrying the ubc - creer transgene , which ubiquitously expresses a cre fusion protein that is activated by tamoxifen . females carrying prospective mutants ( ubc - creer : pitx2 ) and controls ( pitx2 ) were injected with tamoxifen at e10.5 to ablate pitx2 in the prospective mutant ( pitx2-tko ) embryos . our initial comparison of the resulting expression profiles identified 1917 genes whose expression was predicted to be different in wild - type versus pitx2-tko corneas . we chose tfap2b as an attractive candidate for further analysis because it encodes ap-2 , a member of the ap-2 family of transcription factors that are widely required for normal eye development , and it is expressed at the appropriate time in neural crest mesenchyme of the developing cornea . furthermore , the loss of ap-2 transcription factors is associated with pathologic angiogenesis , a prominent phenotype in developing corneas of pitx2-deficient embryos . we used quantitative rt - pcr to confirm that tfap2b expression in pitx2-tko corneas taken from e12.5 and e16.5 eyes is significantly reduced when compared with corneas taken from wild - type pitx2 control embryos ( fig . immunohistochemistry confirmed that , in control mice at e12.5 , ap-2 protein has a restricted pattern of expression in mesenchymal cells that will ultimately contribute to the cornea and subsequently the limbus and structures within the iridocorneal angle . notably , ap-2 is not present in the primordia to other ocular structures that derive from the mesenchyme such as the sclera ( fig . ap-2 is also expressed in the surface ectoderm of the presumptive eyelid conjunctiva of control mice at this timepoint ( fig . 1b ) . in pitx2-tko embryos , ap-2 is present in surface ectoderm of the eyelid conjunctiva , but in contrast to control embryos , ap-2 expression is absent in mesenchyme of the presumptive cornea and limbus as well as the presumptive corneal ectoderm ( fig . collectively , these results identify tfap2b as a genetic target of pitx2 in the neural crest of the developing cornea and potentially additional anterior segment structures that differentiate from neural crest later during eye development . ( a ) quantitative rt - pcr assays were used to compare relative tfap2b expression levels in corneas isolated from e12.5 control and pitx2-tko mutant corneas by laser microdissection . ( b , c ) immunohistochemistry was used to detect ap-2 protein ( green ) in e12.5 control and pitx2-tko mutant eyes . expression is specifically lost from the corneal mesenchyme in pitx2-tko mutant eyes ; expression is identical in eyelid ectoderm of eyes from each genotype . key : cm , corneal mesenchyme ; el , eyelid , eyelid ectoderm ; l , lens . to determine if the ap-2 transcription factor is essential for corneal development , we examined eye morphogenesis in wild type , tfap2b , and tfap2b embryos . the initial steps in corneal morphogenesis , including the migration of periocular mesenchyme into the space between the newly formed lens vesicle and the overlying presumptive corneal ectoderm , appear to occur normally in the absence of ap-2 ( data not shown ) . however , notable differences are apparent in the corneas of tfap2b mice during later gestation . by e16.5 in control eyes , the corneal endothelium is readily visible as a single monolayer overlying the anterior lens epithelium . in contrast , the monolayer of endothelium is not apparent in the corneas of tfap2b eyes ( figs . patent blood vessels containing red blood cells are readily visible extending into the central cornea of all tfap2b eyes examined ( fig . 2c ' ) . interestingly , blood vessels containing red blood cells are also present in a subset of corneas ( 2/8 ) from tfap2b eyes ( fig . these data suggested that ap-2 is an important effector downstream of pitx2 during corneal development . differentiated corneal ectoderm and stroma do not require ap-2. ( a c ) evaluation of eyes from e18.5 embryos of the indicated genotypes following staining with h&e revealed disorganized lamination within the stroma layer and the presence of blood vessels containing red blood cells in all homozygous eyes examined . ( d f ) immunohistochemistry was used to detect ck12 , a specific marker of differentiated corneal ectoderm in e18.5 embryos of the indicated genotypes . ( g i ) in situ hybridization was used to detect keratocan , a specific marker of differentiated corneal stroma in e18.5 embryos . however , in contrast to the uniform staining pattern found in eye of wild - type and heterozygous individuals , the staining pattern in homozygous - mutant individuals defines two layers based on differential expression levels . en , endothelium ; ep , epithelium ; l , len ; s , stroma . pitx2 is required for differentiation of all three major cell lineages during corneal development . to definitively determine whether the loss of the ap-2 transcription factor is a contributing mechanism underlying these defects in pitx2-tko mice in wild - type mice , the intermediate filament protein ck12 is a specific marker of the differentiated corneal ectoderm ( fig . 2d ) , and the related protein ck4 is expressed in the adjacent conjunctival ectoderm ( supplemental fig . ck12 expression in the corneas is comparable with wild - type eyes , suggesting that the corneal ectoderm is specified normally in these mice ( figs . collectively , these data indicate that ap-2 function in the presumptive corneal neural crest is not required for correct specification of the overlying corneal ectoderm during embryogenesis . stromal keratocytes are marked by the uniform expression of keratocan by late gestation , and we observed an analogous expression pattern in tfap2b heterozygous eyes as well ( figs . in contrast , keratocan expression in the corneas of tfap2b homozygous mutants defines two distinct layers based on apparent signal intensity . expression is high within the posterior stroma adjacent to the lens and appears to match expression in wild - type eyes , whereas expression is notably reduced in the anterior stroma located adjacent to the corneal ectoderm ( fig . this location corresponds to the cells that show the greatest degree of disorganization in histologic sections ( fig . 2c ' ) . collectively , these data suggest that ap-2 function is likely not required for the differentiation of stromal keratocytes . finally , we sought to confirm the absence of a corneal endothelium in tfap2b eyes by examining markers specific for this layer . expression of the tight junction protein zo-1 specifically marks the corneal endothelium in the eyes of wild - type and tfap2b heterozygous mice ( figs . in contrast , zo-1 expression in the corneas of tfap2b mice is either discontinuous or absent ( fig . we also examined expression of the dkk1 , encoding an inhibitor of canonical wnt signaling . dkk1 expression labels the corneal endothelium in wild - type and heterozygous tfpap2b mice but is missing from the cornea in tfap2b eyes ( figs . f ) . collectively , these data suggest that ap-2 is required for normal differentiation of the corneal endothelium and that this layer is likely absent or undifferentiated in tfap2b mice ( a c ) immunohistochemistry was used to detect zo-1 ( green ) , a specific marker of differentiated corneal endothelium in e16.5 embryos of the indicated genotypes . ( d f ) in situ hybridization was used to detect dkk1 , a second specific marker of differentiated corneal endothelium in the same embryos . the appearance of patent blood vessels extending to the central cornea of tfap2b mutant mice is reminiscent of pitx2-tko eyes , where both angiogenic and lymphangiogenic privilege is lost in corneas following temporal ablation of pitx2 . to further classify the blood vessels present in the corneas of heterozygous and homozygous tfap2b mutant corneas , we stained sections for the presence of endomucin , a specific marker of blood vessel endothelium , and lyve-1 , a specific marker of lymphatic vessels endothelium . endomucin staining was absent from the corneas of all wild - type eyes , as expected ( fig . endomucin - positive blood vessels were present in a subset ( 2/8 eyes scored ) of tfap2b corneas as well as in the corneas of all tfap2b embryos examined ( figs . 4b , 4c ) . strikingly , although present in the eyelids of tfap2b embryos as a positive control , lyve-1 staining was absent from the corneas of all tfap2b mutant embryos , irrespective of genotype ( figs . collectively , these data indicate that ap-2 is an essential effector downstream of pitx2 that is required to establish angiogenic but not lymphangiogenic privilege during corneal development . ( a f ) immunohistochemistry revealed that all vessels found in e16.5 eyes of homozygous and heterozygous individuals stained positively for a specific marker of blood vessel endothelium ( endomucin ) ( green ) but negative for a specific marker of lymphatic vessel endothelium ( lyve-1 ) ( green ) . note : although corneas of all genotypes were negative for a specific marker of lymphatic vessel endothelium ( lyve1 ) , positive staining of lymphatic vessels ( upper right [ b ] ) in the eyelids of tfap2b eyes provides a positive control for this marker . alterations in the expression of several genes have previously been associated with phenotypes observed in tfap2b - null mice . therefore , we used quantitative rt - pcr to analyze the expression of these genes in rna samples isolated from developing e12.5 wild - type and mutant corneas to gain further insight into the role of ap-2. the homeodomain gene , lmx1b and the forkhead gene , foxc1 , each encode transcription factors that are expressed in neural crest during corneal development and are required for normal differentiation of the endothelium layer . we found that lmx1b expression is not significantly altered in the developing corneas of tfap2b when compared with wild - type littermates ( fig . in contrast , foxc1 expression is significantly reduced in the absence of ap-2. these results suggest that the normal expression of foxc1 , but not lmx1b , is dependent on ap-2 and that reduced foxc1 levels may contribute to altered development of the corneal endothelium in tfap2b mutant eyes . quantitative rt - pcr was used to assess relative expression of the indicated genes in total rna from corneal tissue isolated from e14.5 wild - type control and tfap2b embryos ( n = 3 - 4 embryos / genotype ) by laser microdissection . n.s . , not significant . vegfa encodes the signaling molecule vegf , a potent inducer of angiogenesis , including in the developing cornea . we assessed vegfa expression but found no significant difference between wild - type and tfap2b mutant corneas . to prevent abnormal angiogenesis , the levels of biologically available vegf protein are posttranscriptionally regulated at several levels , including sequestration by the extracellular matrix or soluble inhibitory proteins that prevent binding to and subsequent activation of cognate receptors . mmp2 and mmp9 encode matrix metalloproteinases that promote the release of biologically active vegf from the extracellular matrix . interestingly , ap-2 is required to activate mmp2 expression in the cornea , whereas both mmp2 and mmp9 are derepressed in foxc1-mutant mice , leading to vegf-dependent loss of angiogenic privilege in the cornea . mmp2 expression is significantly reduced in the absence of ap-2 , whereas mmp9 expression could not be reliably detected in the corneas of either wild - type or tfap2b - null embryos . these data suggest that activation of mmp2 or mmp9 is unlikely to contribute to a loss of angiogenic privilege in the corneas of tfap2b - null mice . finally , we assessed the expression of the secreted isoform of vegf receptor 1 ( sflt1 ) , which prevents the binding of vegf to the membrane - bound form of the receptor and is essential for maintaining angiogenic privilege in the adult cornea . we found that the expression of sflt1 is not reduced in developing tfap2b - null corneas , making it unlikely that the loss of this protein accounts for the loss of angiogenic privilege in these eyes . we combined laser microdissection and microarray analysis using e12.5 embryos to identify genes regulated by pitx2 during corneal development . because anterior segment morphogenesis and early corneal development are blocked prior to initiation of corneal development in both global and neural crest - specific pitx2-null embryos , we employed a temporal knockout strategy described previously to selectively ablate pitx2 at the onset of corneal development . briefly , we crossed mice carrying our conditional pitx2 allele with mice carrying the ubc - creer transgene , which ubiquitously expresses a cre fusion protein that is activated by tamoxifen . females carrying prospective mutants ( ubc - creer : pitx2 ) and controls ( pitx2 ) were injected with tamoxifen at e10.5 to ablate pitx2 in the prospective mutant ( pitx2-tko ) embryos . our initial comparison of the resulting expression profiles identified 1917 genes whose expression was predicted to be different in wild - type versus pitx2-tko corneas . we chose tfap2b as an attractive candidate for further analysis because it encodes ap-2 , a member of the ap-2 family of transcription factors that are widely required for normal eye development , and it is expressed at the appropriate time in neural crest mesenchyme of the developing cornea . furthermore , the loss of ap-2 transcription factors is associated with pathologic angiogenesis , a prominent phenotype in developing corneas of pitx2-deficient embryos . we used quantitative rt - pcr to confirm that tfap2b expression in pitx2-tko corneas taken from e12.5 and e16.5 eyes is significantly reduced when compared with corneas taken from wild - type pitx2 control embryos ( fig . immunohistochemistry confirmed that , in control mice at e12.5 , ap-2 protein has a restricted pattern of expression in mesenchymal cells that will ultimately contribute to the cornea and subsequently the limbus and structures within the iridocorneal angle . notably , ap-2 is not present in the primordia to other ocular structures that derive from the mesenchyme such as the sclera ( fig . ap-2 is also expressed in the surface ectoderm of the presumptive eyelid conjunctiva of control mice at this timepoint ( fig . 1b ) . in pitx2-tko embryos , ap-2 is present in surface ectoderm of the eyelid conjunctiva , but in contrast to control embryos , ap-2 expression is absent in mesenchyme of the presumptive cornea and limbus as well as the presumptive corneal ectoderm ( fig . collectively , these results identify tfap2b as a genetic target of pitx2 in the neural crest of the developing cornea and potentially additional anterior segment structures that differentiate from neural crest later during eye development . ( a ) quantitative rt - pcr assays were used to compare relative tfap2b expression levels in corneas isolated from e12.5 control and pitx2-tko mutant corneas by laser microdissection . ( b , c ) immunohistochemistry was used to detect ap-2 protein ( green ) in e12.5 control and pitx2-tko mutant eyes . expression is specifically lost from the corneal mesenchyme in pitx2-tko mutant eyes ; expression is identical in eyelid ectoderm of eyes from each genotype . key : cm , corneal mesenchyme ; el , eyelid , eyelid ectoderm ; l , lens . to determine if the ap-2 transcription factor is essential for corneal development , we examined eye morphogenesis in wild type , tfap2b , and tfap2b embryos . the initial steps in corneal morphogenesis , including the migration of periocular mesenchyme into the space between the newly formed lens vesicle and the overlying presumptive corneal ectoderm , appear to occur normally in the absence of ap-2 ( data not shown ) . however , notable differences are apparent in the corneas of tfap2b mice during later gestation . by e16.5 in control eyes , the corneal endothelium is readily visible as a single monolayer overlying the anterior lens epithelium . in contrast , the monolayer of endothelium is not apparent in the corneas of tfap2b eyes ( figs . patent blood vessels containing red blood cells are readily visible extending into the central cornea of all tfap2b eyes examined ( fig . 2c ' ) . interestingly , blood vessels containing red blood cells are also present in a subset of corneas ( 2/8 ) from tfap2b eyes ( fig . 2b ' ) . these data suggested that ap-2 is an important effector downstream of pitx2 during corneal development . differentiated corneal ectoderm and stroma do not require ap-2. ( a c ) evaluation of eyes from e18.5 embryos of the indicated genotypes following staining with h&e revealed disorganized lamination within the stroma layer and the presence of blood vessels containing red blood cells in all homozygous eyes examined . ( d f ) immunohistochemistry was used to detect ck12 , a specific marker of differentiated corneal ectoderm in e18.5 embryos of the indicated genotypes . ( g i ) in situ hybridization was used to detect keratocan , a specific marker of differentiated corneal stroma in e18.5 embryos . however , in contrast to the uniform staining pattern found in eye of wild - type and heterozygous individuals , the staining pattern in homozygous - mutant individuals defines two layers based on differential expression levels . en , endothelium ; ep , epithelium ; l , len ; s , stroma . pitx2 is required for differentiation of all three major cell lineages during corneal development . to definitively determine whether the loss of the ap-2 transcription factor is a contributing mechanism underlying these defects in pitx2-tko mice , we assessed the expression of lineage - specific protein markers in tfap2b mice . in wild - type mice , the intermediate filament protein ck12 is a specific marker of the differentiated corneal ectoderm ( fig . 2d ) , and the related protein ck4 is expressed in the adjacent conjunctival ectoderm ( supplemental fig . ck12 expression in the corneas is comparable with wild - type eyes , suggesting that the corneal ectoderm is specified normally in these mice ( figs . collectively , these data indicate that ap-2 function in the presumptive corneal neural crest is not required for correct specification of the overlying corneal ectoderm during embryogenesis . stromal keratocytes are marked by the uniform expression of keratocan by late gestation , and we observed an analogous expression pattern in tfap2b heterozygous eyes as well ( figs . in contrast , keratocan expression in the corneas of tfap2b homozygous mutants defines two distinct layers based on apparent signal intensity . expression is high within the posterior stroma adjacent to the lens and appears to match expression in wild - type eyes , whereas expression is notably reduced in the anterior stroma located adjacent to the corneal ectoderm ( fig . this location corresponds to the cells that show the greatest degree of disorganization in histologic sections ( fig . 2c ' ) . collectively , these data suggest that ap-2 function is likely not required for the differentiation of stromal keratocytes . finally , we sought to confirm the absence of a corneal endothelium in tfap2b eyes by examining markers specific for this layer . expression of the tight junction protein zo-1 specifically marks the corneal endothelium in the eyes of wild - type and tfap2b heterozygous mice ( figs . in contrast , zo-1 expression in the corneas of tfap2b mice is either discontinuous or absent ( fig . we also examined expression of the dkk1 , encoding an inhibitor of canonical wnt signaling . dkk1 expression labels the corneal endothelium in wild - type and heterozygous tfpap2b mice but is missing from the cornea in tfap2b eyes ( figs . f ) . collectively , these data suggest that ap-2 is required for normal differentiation of the corneal endothelium and that this layer is likely absent or undifferentiated in tfap2b mice ( a c ) immunohistochemistry was used to detect zo-1 ( green ) , a specific marker of differentiated corneal endothelium in e16.5 embryos of the indicated genotypes . ( d f ) in situ hybridization was used to detect dkk1 , a second specific marker of differentiated corneal endothelium in the same embryos . the appearance of patent blood vessels extending to the central cornea of tfap2b mutant mice is reminiscent of pitx2-tko eyes , where both angiogenic and lymphangiogenic privilege is lost in corneas following temporal ablation of pitx2 . to further classify the blood vessels present in the corneas of heterozygous and homozygous tfap2b mutant corneas , we stained sections for the presence of endomucin , a specific marker of blood vessel endothelium , and lyve-1 , a specific marker of lymphatic vessels endothelium . endomucin staining was absent from the corneas of all wild - type eyes , as expected ( fig . endomucin - positive blood vessels were present in a subset ( 2/8 eyes scored ) of tfap2b corneas as well as in the corneas of all tfap2b embryos examined ( figs . 4b , 4c ) . strikingly , although present in the eyelids of tfap2b embryos as a positive control , lyve-1 staining was absent from the corneas of all tfap2b mutant embryos , irrespective of genotype ( figs . collectively , these data indicate that ap-2 is an essential effector downstream of pitx2 that is required to establish angiogenic but not lymphangiogenic privilege during corneal development . ( a f ) immunohistochemistry revealed that all vessels found in e16.5 eyes of homozygous and heterozygous individuals stained positively for a specific marker of blood vessel endothelium ( endomucin ) ( green ) but negative for a specific marker of lymphatic vessel endothelium ( lyve-1 ) ( green ) . note : although corneas of all genotypes were negative for a specific marker of lymphatic vessel endothelium ( lyve1 ) , positive staining of lymphatic vessels ( upper right [ b ] ) in the eyelids of tfap2b eyes provides a positive control for this marker . alterations in the expression of several genes have previously been associated with phenotypes observed in tfap2b - null mice . therefore , we used quantitative rt - pcr to analyze the expression of these genes in rna samples isolated from developing e12.5 wild - type and mutant corneas to gain further insight into the role of ap-2. the homeodomain gene , lmx1b and the forkhead gene , foxc1 , each encode transcription factors that are expressed in neural crest during corneal development and are required for normal differentiation of the endothelium layer . we found that lmx1b expression is not significantly altered in the developing corneas of tfap2b when compared with wild - type littermates ( fig . in contrast , foxc1 expression is significantly reduced in the absence of ap-2. these results suggest that the normal expression of foxc1 , but not lmx1b , is dependent on ap-2 and that reduced foxc1 levels may contribute to altered development of the corneal endothelium in tfap2b mutant eyes . quantitative rt - pcr was used to assess relative expression of the indicated genes in total rna from corneal tissue isolated from e14.5 wild - type control and tfap2b embryos ( n = 3 - 4 embryos / genotype ) by laser microdissection . n.s . , not significant . vegfa encodes the signaling molecule vegf , a potent inducer of angiogenesis , including in the developing cornea . we assessed vegfa expression but found no significant difference between wild - type and tfap2b mutant corneas . to prevent abnormal angiogenesis , the levels of biologically available vegf protein are posttranscriptionally regulated at several levels , including sequestration by the extracellular matrix or soluble inhibitory proteins that prevent binding to and subsequent activation of cognate receptors . mmp2 and mmp9 encode matrix metalloproteinases that promote the release of biologically active vegf from the extracellular matrix . interestingly , ap-2 is required to activate mmp2 expression in the cornea , whereas both mmp2 and mmp9 are derepressed in foxc1-mutant mice , leading to vegf-dependent loss of angiogenic privilege in the cornea . mmp2 expression is significantly reduced in the absence of ap-2 , whereas mmp9 expression could not be reliably detected in the corneas of either wild - type or tfap2b - null embryos . these data suggest that activation of mmp2 or mmp9 is unlikely to contribute to a loss of angiogenic privilege in the corneas of tfap2b - null mice . finally , we assessed the expression of the secreted isoform of vegf receptor 1 ( sflt1 ) , which prevents the binding of vegf to the membrane - bound form of the receptor and is essential for maintaining angiogenic privilege in the adult cornea . we found that the expression of sflt1 is not reduced in developing tfap2b - null corneas , making it unlikely that the loss of this protein accounts for the loss of angiogenic privilege in these eyes . the correct development of the cornea is essential for normal vision , but the genes critical for differentiation of the three major cell lineages and establishment of an avascular environment during corneal development remain largely unknown . a detailed understanding of the molecular mechanisms regulating these processes could in the future contribute to more effective treatments of corneal diseases . recently , we demonstrated that the homeodomain transcription factor pitx2 is essential for multiple developmental events in the cornea , but the essential downstream effectors of pitx2 function remained to be identified . in this report , we examined gene expression changes in the cornea caused by a loss of pitx2 and determined there was a dramatic reduction in tfap2b transcripts . we subsequently demonstrated that tfap2b , encoding the ap-2 transcription factor , functions downstream of pitx2 and is required for correct differentiation of corneal endothelium and for establishing angiogenic privilege . the endothelium is unique among mature corneal cell lineages because cells lost to disease or trauma normally are not replaced . loss of the barrier function provided by the endothelium leads to swelling and opacity within the adjacent stroma , which leads to vision loss . transplantation is the only currently available therapy with the potential to replace lost endothelium cells . however , because sources of endothelium for engraftment are extremely limited , there is considerable interest in the development of stem cell based therapies . a detailed understanding of the genes and mechanisms required for embryonic development of the endothelium is likely to aid in the design of strategies to program stem cells to differentiate into this lineage . specification of the endothelium from the neural crest located between the newly formed lens vesicle and the overlying surface ectoderm requires inductive signaling from the adjacent anterior lens epithelium and the anterior rim of the optic cup . the complete composition of the signal remains to be determined but it includes retinoic acid . a key molecular response to retinoic acid signaling within the neural crest is the induction of pitx2 expression , which is subsequently required for the differentiation of all three major corneal cell lineages . previously , we have shown that pitx2-dependent expression of dkk2 , and the resulting suppression of canonical wnt signaling within the adjacent surface ectoderm , is essential for the differentiation of the corneal epithelium . in contrast , the differentiation of cell lineages from the neural crest is relatively unaffected by loss of dkk2 . in the present report , we establish that pitx2 is required for the expression of tfap2b throughout the corneal neural crest and that ap-2 is required for specification of the corneal endothelium . two additional transcription factors that are required for specification of the corneal endothelium have been identified . retinoic acid signaling also induces expression of the gene encoding the forkhead transcription factor foxc1 within the neural crest . mutations in human foxc1 are a second cause of axenfeld - rieger syndrome , suggesting that pitx2 and foxc1 likely coregulate common molecular pathways during anterior segment development . like pitx2 , foxc1 is also required for normal specification of the corneal endothelium as well as the stroma . although a dependence on retinoic acid signaling has not been reported , the homeobox gene lmx1b encodes a fourth transcription factor that is expressed within the neural crest and is required for specification of the endothelium . our data suggest that full expression of foxc1 , but not lmx1b , depends on ap-2 and that foxc1 may act downstream effector of ap-2 required for normal development of the corneal endothelium . ultimately , specification of the corneal endothelium must depend on the unique combinatorial expression of transcription factors in this lineage . although these four genes are each individually required for differentiation of the corneal endothelium , they are also coexpressed in the ocular neural crest in cells not fated to form the endothelium , and similar to pitx2 , foxc1 and lmx1b are required for specification of additional lineages during eye development . these observations provide compelling evidence that our understanding of the transcription factor code required for specification of the corneal endothelium remains incomplete and that additional components remain to be identified . completely elucidating additional upstream transcription factors essential for tfap2b expression , as well as cofactors that function together with ap-2 , will provide important opportunities to discover additional components of the code . in addition , the identification of the cascade of molecular events located downstream of ap-2 will help uncover additional components of the regulatory cascade required for differentiation of the corneal endothelium . in this context , at present it is not clear if pitx2 regulates the expression of tfap2b via direct or indirect mechanisms and will require the identification and characterization of the tfap2b cis - acting sequences responsible for expression in the developing cornea . therefore , further studies will be required to identify the regulatory interactions between these four transcriptional regulators of corneal endothelium development . the genes and mechanisms required for establishing angiogenic and lymphangiogenic privilege during development also remain largely unknown despite the importance of these properties for normal vision and the likelihood that elucidation of these pathways could have a positive impact on the development of new therapeutic strategies . foxc1 is required for the establishment of both properties through a mechanism that includes the repression of genes encoding matrix metalloproteinases 2 and 9 , which enhance the generation of available bioactive forms of vegf . recently , we showed that pitx2 is also essential for both properties but downstream effectors were not identified . we now extend the knowledge of this pathway by establishing the gene encoding ap-2 as a required pitx2-dependent mediator of angiogenic but not lymphangiogenic privilege during corneal development . previously , overexpression of the gene encoding ap-2 had been shown to correlate with angiogenesis and poor prognosis in human lung adenocarcinomas through mechanisms that include modulation of vegf / pdgf signaling . these observations imply that the effects of ap-2 on blood vessel growth versus nongrowth are context dependent . this property is consistent with the established functions of the highly related family member ap-2 , which has been demonstrated to promote or suppress angiogenesis depending on context . the activator and repressor functions of ap-2 transcription factors are generally dictated by the identity of essential interacting partners , suggesting that the differences in angiogenic responses are likely to result from the complement of cofactors available in each context . the need to understand ap-2 mediated suppression of angiogenesis during corneal development highlights the importance of identifying the complement of available cofactors during this process . our data demonstrate that ap-2 is required for the establishment of angiogenic privilege during corneal development , but the underlying mechanism remains unknown . in the cornea , as elsewhere , the growth or nongrowth of new blood vessels is ultimately the net result of a tightly regulated balance between the local availability of proangiogenic and antiangiogenic factors . the vegfa promoter is a direct target of both ap-2 and ap-2 in certain tissues , with the net effect dependent on context . furthermore , the overexpression of ap-2 correlates with increased levels of hif-1 , a potent modulator of the vegfa promoter , in certain contexts . ap-2 suppresses the expression of mmp2 and mmp9 in some tissues but activates mmp2 expression in the cornea . interestingly , although the expression levels of vegfa , mmp2 , and mmp9 are all elevated in corneas from pitx2-tko mice , our data suggest that enhanced transcription of these genes does not account for a loss of angiogenic privilege in developing tfap2b corneas . therefore , additional experiments are required to identify the factor(s ) required downstream of ap-2 during the establishment of angiogenic privilege in the cornea . the endothelium is unique among mature corneal cell lineages because cells lost to disease or trauma normally are not replaced . loss of the barrier function provided by the endothelium leads to swelling and opacity within the adjacent stroma , which leads to vision loss . transplantation is the only currently available therapy with the potential to replace lost endothelium cells . however , because sources of endothelium for engraftment are extremely limited , there is considerable interest in the development of stem cell based therapies . a detailed understanding of the genes and mechanisms required for embryonic development of the endothelium is likely to aid in the design of strategies to program stem cells to differentiate into this lineage . specification of the endothelium from the neural crest located between the newly formed lens vesicle and the overlying surface ectoderm requires inductive signaling from the adjacent anterior lens epithelium and the anterior rim of the optic cup . the complete composition of the signal remains to be determined but it includes retinoic acid . a key molecular response to retinoic acid signaling within the neural crest is the induction of pitx2 expression , which is subsequently required for the differentiation of all three major corneal cell lineages . previously , we have shown that pitx2-dependent expression of dkk2 , and the resulting suppression of canonical wnt signaling within the adjacent surface ectoderm , is essential for the differentiation of the corneal epithelium . in contrast , the differentiation of cell lineages from the neural crest is relatively unaffected by loss of dkk2 . in the present report , we establish that pitx2 is required for the expression of tfap2b throughout the corneal neural crest and that ap-2 is required for specification of the corneal endothelium . two additional transcription factors that are required for specification of the corneal endothelium have been identified . retinoic acid signaling also induces expression of the gene encoding the forkhead transcription factor foxc1 within the neural crest . mutations in human foxc1 are a second cause of axenfeld - rieger syndrome , suggesting that pitx2 and foxc1 likely coregulate common molecular pathways during anterior segment development . like pitx2 , foxc1 is also required for normal specification of the corneal endothelium as well as the stroma . although a dependence on retinoic acid signaling has not been reported , the homeobox gene lmx1b encodes a fourth transcription factor that is expressed within the neural crest and is required for specification of the endothelium . our data suggest that full expression of foxc1 , but not lmx1b , depends on ap-2 and that foxc1 may act downstream effector of ap-2 required for normal development of the corneal endothelium . ultimately , specification of the corneal endothelium must depend on the unique combinatorial expression of transcription factors in this lineage . although these four genes are each individually required for differentiation of the corneal endothelium , they are also coexpressed in the ocular neural crest in cells not fated to form the endothelium , and similar to pitx2 , foxc1 and lmx1b are required for specification of additional lineages during eye development . these observations provide compelling evidence that our understanding of the transcription factor code required for specification of the corneal endothelium remains incomplete and that additional components remain to be identified . completely elucidating additional upstream transcription factors essential for tfap2b expression , as well as cofactors that function together with ap-2 , will provide important opportunities to discover additional components of the code . in addition , the identification of the cascade of molecular events located downstream of ap-2 will help uncover additional components of the regulatory cascade required for differentiation of the corneal endothelium . in this context , at present it is not clear if pitx2 regulates the expression of tfap2b via direct or indirect mechanisms and will require the identification and characterization of the tfap2b cis - acting sequences responsible for expression in the developing cornea . therefore , further studies will be required to identify the regulatory interactions between these four transcriptional regulators of corneal endothelium development . the genes and mechanisms required for establishing angiogenic and lymphangiogenic privilege during development also remain largely unknown despite the importance of these properties for normal vision and the likelihood that elucidation of these pathways could have a positive impact on the development of new therapeutic strategies . foxc1 is required for the establishment of both properties through a mechanism that includes the repression of genes encoding matrix metalloproteinases 2 and 9 , which enhance the generation of available bioactive forms of vegf . recently , we showed that pitx2 is also essential for both properties but downstream effectors were not identified . we now extend the knowledge of this pathway by establishing the gene encoding ap-2 as a required pitx2-dependent mediator of angiogenic but not lymphangiogenic privilege during corneal development . previously , overexpression of the gene encoding ap-2 had been shown to correlate with angiogenesis and poor prognosis in human lung adenocarcinomas through mechanisms that include modulation of vegf / pdgf signaling . these observations imply that the effects of ap-2 on blood vessel growth versus nongrowth are context dependent . this property is consistent with the established functions of the highly related family member ap-2 , which has been demonstrated to promote or suppress angiogenesis depending on context . the activator and repressor functions of ap-2 transcription factors are generally dictated by the identity of essential interacting partners , suggesting that the differences in angiogenic responses are likely to result from the complement of cofactors available in each context . the need to understand ap-2 mediated suppression of angiogenesis during corneal development highlights the importance of identifying the complement of available cofactors during this process . our data demonstrate that ap-2 is required for the establishment of angiogenic privilege during corneal development , but the underlying mechanism remains unknown . in the cornea , as elsewhere , the growth or nongrowth of new blood vessels is ultimately the net result of a tightly regulated balance between the local availability of proangiogenic and antiangiogenic factors . the vegfa promoter is a direct target of both ap-2 and ap-2 in certain tissues , with the net effect dependent on context . furthermore , the overexpression of ap-2 correlates with increased levels of hif-1 , a potent modulator of the vegfa promoter , in certain contexts . ap-2 suppresses the expression of mmp2 and mmp9 in some tissues but activates mmp2 expression in the cornea . interestingly , although the expression levels of vegfa , mmp2 , and mmp9 are all elevated in corneas from pitx2-tko mice , our data suggest that enhanced transcription of these genes does not account for a loss of angiogenic privilege in developing tfap2b corneas . therefore , additional experiments are required to identify the factor(s ) required downstream of ap-2 during the establishment of angiogenic privilege in the cornea . our current results identify ap-2 as an important downstream effect of pitx2 in corneas during embryogenesis but a number of critical questions remain regarding the roles of both proteins in developing and mature ocular anterior segment structures . for pitx2 , although we have identified ap-2 as an important factor that accounts at least in part for the requirement for pitx2 in differentiation of the endothelium and establishment of angiogenic privilege , other factors must be required for pitx2-dependent processes such as differentiation of the stroma and for lymphangiogenic privilege . for ap-2 , persistent expression throughout corneal development and in the mature cornea suggests additional potential roles at later timepoints . both pitx2 and ap-2 are present at high levels in neural - crest derived structures within in the iridocorneal angle , highlighting the possibility that these factors may also serve important functions in the development and/or maintenance of these structures as well . conditional alleles for both genes together with suitable cre transgenic mouse lines will be essential for future experiments addressing the tissue - specific and temporal requirements of pitx2 and tfap2b in anterior segment development .

purposethe homeodomain transcription factor , pitx2 , is at the apex of a genetic pathway required for corneal development , but the critical effector genes regulated by the pitx2 remain unknown . the purpose of this study was to discover and validate pitx2-dependent mechanisms required for specifying cell lineages and establishing angiogenic privilege within the developing cornea.methodsmicroarrays were used to compare gene expression in corneas isolated from temporal pitx2 knockout embryos and control littermates . quantitative rt - pcr and immunohistochemistry was used to further validate tfap2b expression differences in pitx2 knockout versus control corneas . in situ hybridization and protein immunohistochemistry were used to assay eyes of a tfap2b allelic series of embryos to identify differentiated cellular lineages in the cornea , blood vessel endothelium , or lymphatic vessel endothelium.resultswe show that pitx2 is required for the expression of tfap2b , encoding the ap-2 transcription factor , in the neural crest during corneal development . markers of differentiated corneal epithelium and stroma are expressed in the absence of ap-2. in contrast , markers of differentiated corneal endothelium are not expressed in the absence of ap-2. endomucin+ blood vessels are present throughout the developing corneal stroma in the absence of ap-2 , whereas lyve1 + lymphatic vessels are not found.conclusionsthe ap-2 transcription factor is an important effector of pitx2 function during corneal development , required for differentiation of corneal endothelium and establishment of angiogenic privilege . unlike pitx2 , ap-2 is not required for the early expression of available lineage specific markers for the corneal epithelium and stroma during embryogenesis , nor establishment of lymphangiogenic privilege . therefore , additional pitx2-dependent factors likely regulate these latter processes during embryonic development . these results extend our understanding of the genetic mechanisms regulating cornea development .

Materials and Methods Mouse Strains and Animal Husbandry Laser Micro Dissection and Quantitative RT-PCR Microarray Analysis Embryo Processing and Histochemistry Immunostaining and RNA In Situ Hybridization Results Tfap2b Expression During Corneal Development Requires PITX2 AP-2 Is Required for Normal Development of Corneal Cell Lineages From the Neural Crest AP-2 Is Required for Establishment of Angiogenic but Not Lymphangiogenic Privilege During Corneal Development Loss of AP-2 Alters Expression of a Subset of Genes Required for Normal Corneal Development and Establishment of Angiogenic Privilege Discussion AP-2 Contributes to a Transcriptional Code Required for Specification of Corneal Endothelium AP-2 Is Required for Establishment of Angiogenic Privilege During Corneal Development Summary Supplementary Material

we combined laser microdissection and microarray analysis using e12.5 embryos to identify genes regulated by pitx2 during corneal development . we chose tfap2b as an attractive candidate for further analysis because it encodes ap-2 , a member of the ap-2 family of transcription factors that are widely required for normal eye development , and it is expressed at the appropriate time in neural crest mesenchyme of the developing cornea . we used quantitative rt - pcr to confirm that tfap2b expression in pitx2-tko corneas taken from e12.5 and e16.5 eyes is significantly reduced when compared with corneas taken from wild - type pitx2 control embryos ( fig . collectively , these results identify tfap2b as a genetic target of pitx2 in the neural crest of the developing cornea and potentially additional anterior segment structures that differentiate from neural crest later during eye development . ( a ) quantitative rt - pcr assays were used to compare relative tfap2b expression levels in corneas isolated from e12.5 control and pitx2-tko mutant corneas by laser microdissection . to determine if the ap-2 transcription factor is essential for corneal development , we examined eye morphogenesis in wild type , tfap2b , and tfap2b embryos . in contrast , the monolayer of endothelium is not apparent in the corneas of tfap2b eyes ( figs . these data suggested that ap-2 is an important effector downstream of pitx2 during corneal development . ( d f ) immunohistochemistry was used to detect ck12 , a specific marker of differentiated corneal ectoderm in e18.5 embryos of the indicated genotypes . ( g i ) in situ hybridization was used to detect keratocan , a specific marker of differentiated corneal stroma in e18.5 embryos . pitx2 is required for differentiation of all three major cell lineages during corneal development . to definitively determine whether the loss of the ap-2 transcription factor is a contributing mechanism underlying these defects in pitx2-tko mice in wild - type mice , the intermediate filament protein ck12 is a specific marker of the differentiated corneal ectoderm ( fig . collectively , these data indicate that ap-2 function in the presumptive corneal neural crest is not required for correct specification of the overlying corneal ectoderm during embryogenesis . in contrast , keratocan expression in the corneas of tfap2b homozygous mutants defines two distinct layers based on apparent signal intensity . expression of the tight junction protein zo-1 specifically marks the corneal endothelium in the eyes of wild - type and tfap2b heterozygous mice ( figs . in contrast , zo-1 expression in the corneas of tfap2b mice is either discontinuous or absent ( fig . collectively , these data suggest that ap-2 is required for normal differentiation of the corneal endothelium and that this layer is likely absent or undifferentiated in tfap2b mice ( a c ) immunohistochemistry was used to detect zo-1 ( green ) , a specific marker of differentiated corneal endothelium in e16.5 embryos of the indicated genotypes . ( d f ) in situ hybridization was used to detect dkk1 , a second specific marker of differentiated corneal endothelium in the same embryos . to further classify the blood vessels present in the corneas of heterozygous and homozygous tfap2b mutant corneas , we stained sections for the presence of endomucin , a specific marker of blood vessel endothelium , and lyve-1 , a specific marker of lymphatic vessels endothelium . collectively , these data indicate that ap-2 is an essential effector downstream of pitx2 that is required to establish angiogenic but not lymphangiogenic privilege during corneal development . note : although corneas of all genotypes were negative for a specific marker of lymphatic vessel endothelium ( lyve1 ) , positive staining of lymphatic vessels ( upper right [ b ] ) in the eyelids of tfap2b eyes provides a positive control for this marker . therefore , we used quantitative rt - pcr to analyze the expression of these genes in rna samples isolated from developing e12.5 wild - type and mutant corneas to gain further insight into the role of ap-2. the homeodomain gene , lmx1b and the forkhead gene , foxc1 , each encode transcription factors that are expressed in neural crest during corneal development and are required for normal differentiation of the endothelium layer . in contrast , foxc1 expression is significantly reduced in the absence of ap-2. these results suggest that the normal expression of foxc1 , but not lmx1b , is dependent on ap-2 and that reduced foxc1 levels may contribute to altered development of the corneal endothelium in tfap2b mutant eyes . quantitative rt - pcr was used to assess relative expression of the indicated genes in total rna from corneal tissue isolated from e14.5 wild - type control and tfap2b embryos ( n = 3 - 4 embryos / genotype ) by laser microdissection . interestingly , ap-2 is required to activate mmp2 expression in the cornea , whereas both mmp2 and mmp9 are derepressed in foxc1-mutant mice , leading to vegf-dependent loss of angiogenic privilege in the cornea . mmp2 expression is significantly reduced in the absence of ap-2 , whereas mmp9 expression could not be reliably detected in the corneas of either wild - type or tfap2b - null embryos . we found that the expression of sflt1 is not reduced in developing tfap2b - null corneas , making it unlikely that the loss of this protein accounts for the loss of angiogenic privilege in these eyes . we combined laser microdissection and microarray analysis using e12.5 embryos to identify genes regulated by pitx2 during corneal development . we chose tfap2b as an attractive candidate for further analysis because it encodes ap-2 , a member of the ap-2 family of transcription factors that are widely required for normal eye development , and it is expressed at the appropriate time in neural crest mesenchyme of the developing cornea . we used quantitative rt - pcr to confirm that tfap2b expression in pitx2-tko corneas taken from e12.5 and e16.5 eyes is significantly reduced when compared with corneas taken from wild - type pitx2 control embryos ( fig . in pitx2-tko embryos , ap-2 is present in surface ectoderm of the eyelid conjunctiva , but in contrast to control embryos , ap-2 expression is absent in mesenchyme of the presumptive cornea and limbus as well as the presumptive corneal ectoderm ( fig . collectively , these results identify tfap2b as a genetic target of pitx2 in the neural crest of the developing cornea and potentially additional anterior segment structures that differentiate from neural crest later during eye development . ( a ) quantitative rt - pcr assays were used to compare relative tfap2b expression levels in corneas isolated from e12.5 control and pitx2-tko mutant corneas by laser microdissection . to determine if the ap-2 transcription factor is essential for corneal development , we examined eye morphogenesis in wild type , tfap2b , and tfap2b embryos . in contrast , the monolayer of endothelium is not apparent in the corneas of tfap2b eyes ( figs . these data suggested that ap-2 is an important effector downstream of pitx2 during corneal development . ( d f ) immunohistochemistry was used to detect ck12 , a specific marker of differentiated corneal ectoderm in e18.5 embryos of the indicated genotypes . ( g i ) in situ hybridization was used to detect keratocan , a specific marker of differentiated corneal stroma in e18.5 embryos . pitx2 is required for differentiation of all three major cell lineages during corneal development . to definitively determine whether the loss of the ap-2 transcription factor is a contributing mechanism underlying these defects in pitx2-tko mice , we assessed the expression of lineage - specific protein markers in tfap2b mice . collectively , these data indicate that ap-2 function in the presumptive corneal neural crest is not required for correct specification of the overlying corneal ectoderm during embryogenesis . expression of the tight junction protein zo-1 specifically marks the corneal endothelium in the eyes of wild - type and tfap2b heterozygous mice ( figs . collectively , these data suggest that ap-2 is required for normal differentiation of the corneal endothelium and that this layer is likely absent or undifferentiated in tfap2b mice ( a c ) immunohistochemistry was used to detect zo-1 ( green ) , a specific marker of differentiated corneal endothelium in e16.5 embryos of the indicated genotypes . ( d f ) in situ hybridization was used to detect dkk1 , a second specific marker of differentiated corneal endothelium in the same embryos . the appearance of patent blood vessels extending to the central cornea of tfap2b mutant mice is reminiscent of pitx2-tko eyes , where both angiogenic and lymphangiogenic privilege is lost in corneas following temporal ablation of pitx2 . to further classify the blood vessels present in the corneas of heterozygous and homozygous tfap2b mutant corneas , we stained sections for the presence of endomucin , a specific marker of blood vessel endothelium , and lyve-1 , a specific marker of lymphatic vessels endothelium . collectively , these data indicate that ap-2 is an essential effector downstream of pitx2 that is required to establish angiogenic but not lymphangiogenic privilege during corneal development . note : although corneas of all genotypes were negative for a specific marker of lymphatic vessel endothelium ( lyve1 ) , positive staining of lymphatic vessels ( upper right [ b ] ) in the eyelids of tfap2b eyes provides a positive control for this marker . therefore , we used quantitative rt - pcr to analyze the expression of these genes in rna samples isolated from developing e12.5 wild - type and mutant corneas to gain further insight into the role of ap-2. the homeodomain gene , lmx1b and the forkhead gene , foxc1 , each encode transcription factors that are expressed in neural crest during corneal development and are required for normal differentiation of the endothelium layer . in contrast , foxc1 expression is significantly reduced in the absence of ap-2. these results suggest that the normal expression of foxc1 , but not lmx1b , is dependent on ap-2 and that reduced foxc1 levels may contribute to altered development of the corneal endothelium in tfap2b mutant eyes . quantitative rt - pcr was used to assess relative expression of the indicated genes in total rna from corneal tissue isolated from e14.5 wild - type control and tfap2b embryos ( n = 3 - 4 embryos / genotype ) by laser microdissection . interestingly , ap-2 is required to activate mmp2 expression in the cornea , whereas both mmp2 and mmp9 are derepressed in foxc1-mutant mice , leading to vegf-dependent loss of angiogenic privilege in the cornea . mmp2 expression is significantly reduced in the absence of ap-2 , whereas mmp9 expression could not be reliably detected in the corneas of either wild - type or tfap2b - null embryos . we found that the expression of sflt1 is not reduced in developing tfap2b - null corneas , making it unlikely that the loss of this protein accounts for the loss of angiogenic privilege in these eyes . the correct development of the cornea is essential for normal vision , but the genes critical for differentiation of the three major cell lineages and establishment of an avascular environment during corneal development remain largely unknown . a detailed understanding of the molecular mechanisms regulating these processes could in the future contribute to more effective treatments of corneal diseases . recently , we demonstrated that the homeodomain transcription factor pitx2 is essential for multiple developmental events in the cornea , but the essential downstream effectors of pitx2 function remained to be identified . we subsequently demonstrated that tfap2b , encoding the ap-2 transcription factor , functions downstream of pitx2 and is required for correct differentiation of corneal endothelium and for establishing angiogenic privilege . a detailed understanding of the genes and mechanisms required for embryonic development of the endothelium is likely to aid in the design of strategies to program stem cells to differentiate into this lineage . a key molecular response to retinoic acid signaling within the neural crest is the induction of pitx2 expression , which is subsequently required for the differentiation of all three major corneal cell lineages . previously , we have shown that pitx2-dependent expression of dkk2 , and the resulting suppression of canonical wnt signaling within the adjacent surface ectoderm , is essential for the differentiation of the corneal epithelium . in contrast , the differentiation of cell lineages from the neural crest is relatively unaffected by loss of dkk2 . in the present report , we establish that pitx2 is required for the expression of tfap2b throughout the corneal neural crest and that ap-2 is required for specification of the corneal endothelium . retinoic acid signaling also induces expression of the gene encoding the forkhead transcription factor foxc1 within the neural crest . although a dependence on retinoic acid signaling has not been reported , the homeobox gene lmx1b encodes a fourth transcription factor that is expressed within the neural crest and is required for specification of the endothelium . our data suggest that full expression of foxc1 , but not lmx1b , depends on ap-2 and that foxc1 may act downstream effector of ap-2 required for normal development of the corneal endothelium . although these four genes are each individually required for differentiation of the corneal endothelium , they are also coexpressed in the ocular neural crest in cells not fated to form the endothelium , and similar to pitx2 , foxc1 and lmx1b are required for specification of additional lineages during eye development . these observations provide compelling evidence that our understanding of the transcription factor code required for specification of the corneal endothelium remains incomplete and that additional components remain to be identified . in addition , the identification of the cascade of molecular events located downstream of ap-2 will help uncover additional components of the regulatory cascade required for differentiation of the corneal endothelium . in this context , at present it is not clear if pitx2 regulates the expression of tfap2b via direct or indirect mechanisms and will require the identification and characterization of the tfap2b cis - acting sequences responsible for expression in the developing cornea . foxc1 is required for the establishment of both properties through a mechanism that includes the repression of genes encoding matrix metalloproteinases 2 and 9 , which enhance the generation of available bioactive forms of vegf . we now extend the knowledge of this pathway by establishing the gene encoding ap-2 as a required pitx2-dependent mediator of angiogenic but not lymphangiogenic privilege during corneal development . our data demonstrate that ap-2 is required for the establishment of angiogenic privilege during corneal development , but the underlying mechanism remains unknown . therefore , additional experiments are required to identify the factor(s ) required downstream of ap-2 during the establishment of angiogenic privilege in the cornea . a detailed understanding of the genes and mechanisms required for embryonic development of the endothelium is likely to aid in the design of strategies to program stem cells to differentiate into this lineage . a key molecular response to retinoic acid signaling within the neural crest is the induction of pitx2 expression , which is subsequently required for the differentiation of all three major corneal cell lineages . previously , we have shown that pitx2-dependent expression of dkk2 , and the resulting suppression of canonical wnt signaling within the adjacent surface ectoderm , is essential for the differentiation of the corneal epithelium . in contrast , the differentiation of cell lineages from the neural crest is relatively unaffected by loss of dkk2 . in the present report , we establish that pitx2 is required for the expression of tfap2b throughout the corneal neural crest and that ap-2 is required for specification of the corneal endothelium . retinoic acid signaling also induces expression of the gene encoding the forkhead transcription factor foxc1 within the neural crest . although a dependence on retinoic acid signaling has not been reported , the homeobox gene lmx1b encodes a fourth transcription factor that is expressed within the neural crest and is required for specification of the endothelium . our data suggest that full expression of foxc1 , but not lmx1b , depends on ap-2 and that foxc1 may act downstream effector of ap-2 required for normal development of the corneal endothelium . although these four genes are each individually required for differentiation of the corneal endothelium , they are also coexpressed in the ocular neural crest in cells not fated to form the endothelium , and similar to pitx2 , foxc1 and lmx1b are required for specification of additional lineages during eye development . these observations provide compelling evidence that our understanding of the transcription factor code required for specification of the corneal endothelium remains incomplete and that additional components remain to be identified . in addition , the identification of the cascade of molecular events located downstream of ap-2 will help uncover additional components of the regulatory cascade required for differentiation of the corneal endothelium . in this context , at present it is not clear if pitx2 regulates the expression of tfap2b via direct or indirect mechanisms and will require the identification and characterization of the tfap2b cis - acting sequences responsible for expression in the developing cornea . foxc1 is required for the establishment of both properties through a mechanism that includes the repression of genes encoding matrix metalloproteinases 2 and 9 , which enhance the generation of available bioactive forms of vegf . our data demonstrate that ap-2 is required for the establishment of angiogenic privilege during corneal development , but the underlying mechanism remains unknown . therefore , additional experiments are required to identify the factor(s ) required downstream of ap-2 during the establishment of angiogenic privilege in the cornea . for pitx2 , although we have identified ap-2 as an important factor that accounts at least in part for the requirement for pitx2 in differentiation of the endothelium and establishment of angiogenic privilege , other factors must be required for pitx2-dependent processes such as differentiation of the stroma and for lymphangiogenic privilege .

mismatch repair is one of the key dna repair systems in the cell that repair incorrectly formed base pairs and insertion / deletion loops during replication , significantly increasing the stability of the genome [ 15 ] . additional importance comes from the contribution of defects in mmr proteins to cancer development , progression and therapeutic success . in addition to the recognition of replication errors , key mmr proteins msh2/msh6 recognize a number of dna lesions that are irreparable by these proteins , which , in turn , initiate a cell death pathway . the role of mmr proteins in the cell death response to cytotoxic agents has been the subject of much debate [ 6 , 7 ] . whether dna damage is recognized in form of a mismatch at the site of the damage , or dna damage itself is recognized recent data suggest that some types of dna lesions are recognized without the requirements for a mismatch . as a result of their ability to recognize certain dna lesions and their participation in the initiation of cell death , this participation is particularly evident in cells with defects in mmr proteins which reduce the sensitivity to chemotherapeutic agents sufficiently to prove to be a problem for the treatment of tumors [ 1 , 6 , 912 ] . the exact mechanism of the cytotoxic response initiated by msh2/msh6 interactions with dna damage remains under investigation and speculation . recent data suggest that the response pathway depends on the nature of the lesion rather than the unification of different signals into a common pathway . at least two hypotheses have been put forward to address the mechanism behind the mmr protein - dependent cell death : the futile cycles of repair hypothesis is based on the formation of a mismatch opposite the dna damage , followed by attempted repair events that retain the damage and the formation of dna strand breaks as factual initiators of cell death [ 1 , 4 , 14 ] . the second hypothesis suggests a direct involvement of mmr proteins in the initiation of the cell death event . we and others have identified separation - of - function mutants that demonstrate that repair - defective mutants of mmr proteins retain the ability to induce cell death , suggesting that repair is not required for cytotoxic response to all types of dna damage [ 8 , 1517 ] , but also suggesting that specific mmr proteins form recognition complexes for both mismatch repair , and at least some types of dna damage . computational modeling predicted the formation of a death conformation with distinctly different features to the repair conformation of muts and its homologs ( msh ) . this model of a muts death conformation was validated in extensive mutational analyses on eukaryotic proteins . although the mechanistic details of this pathway are largely unknown , targeting the apparent recognition complex for cell death , that is , the death conformation , has shown initial successes . hence , we aim to exploit this pathway via small molecule binding to the death conformation . recently , we have demonstrated that the msh2-dependent cell death response can be induced by small molecules that mimic binding to dna damage and promote the induction of this cell death . the prototype for this induction of msh2-dependent cell death without genotoxic insult is reserpine , a drug previously in clinical use for hypertension . the predicted binding of the molecule to the protein occurs in the dna binding pocket and induces specific conformational changes that allow access to the death conformation of the protein . at present , the direct binding of reserpine , and its derivatives , to the death conformation has been predicted , and the ability of reserpine and rescinnamine to induce msh2-dependent apoptosis has been verified experimentally in this work as well as in an earlier study . unfortunately , it does not seem possible , with present technology , to unequivocally demonstrate physical association between msh2/msh6 and either reserpine or rescinnamine . attempts to use spr and calorimetry ( data not shown ) have lacked the precision to detect these small alkaloids binding to such a large proteins complex . we expect that the technology used to detect such direct binding of small molecules to large protein complexes will be refined over the next few years , and that presently challenging complexes , such as the proposed msh2/msh6/rescinnamine or reserpine complex , will be directly studied . for now , given the circumstantial evidence reported herein and in we consider the hypothesis that reserpine binds to msh2/msh6 as predicted to be a reasonable starting point for further studies . here , we determine the parameters for small molecules to initiate the mmr protein - dependent cell - death response , where the cell - death response is measured via two distinct standard assays , mts assay and caspase activation , which provide multiple pieces of evidence for the induction of cell - death . based on our models , we have hypothesized that specific structural parameters are required of a small molecule to mimic the interaction of msh2/msh6 with dna damage , show the correct orientation in the protein - binding pocket and initiate msh2-dependent cell death . in a combination of computer - based structural modeling , chemical synthesis , and cell biology , we have identified these distinct requirements , which consist of the size of the molecule , the presence and location of methoxy groups , and the orientation of the molecule within the protein - binding pocket . while the computational modeling assumes that the small molecules bind in a specific pocket and in specific orientations , as predicted , the cellular assays do not require such assumptions . these small - molecule structural parameters will provide the foundation for any subsequent searches for new small molecule compounds that initiate the specific msh2-dependent cell death targeted in this approach . autodock 3.0 was used to perform 3d docking into structural models generated from the molecular dynamics simulations . docking grids from vdw and electrostatic interactions were generated using standard autodock charges , vdw , and electrostatic parameters on cubic grids , 18.75 on a side . the grids were centered on the midpoint of the line connecting the two guanine nitrogens that are crosslinked in the platinated simulation , but the grids were generated in the absence of dna and the platinum adduct . the mol2 files for the small molecules were generated using marvin and assigned charges and rotatable bonds using autodock , which was also used to perform the preparatory work for the grid docking . the ligands were docked using default autodock lamarckian genetic algorithm parameters except the number of runs was increased to 256 . the structure with the lowest estimated inhibition constant , ki , as calculated by autodock was used as the docked pose . the structures used for the docking are the centroids from the final equilibrated clusters of two molecular dynamics simulations . the clusters were obtained by k - means clustering with a 1 radius cutoff on the alpha - carbon positions of structures in the molecular dynamics trajectories . the simulation protocol is largely the same as described elsewhere , differing only in the use of a constant pressure algorithm , and differing simulation lengths , and is briefly summarized here . the x - ray structure of escherichia coli muts in complex with dna was the initial starting point ; hydrogen atoms were added using the building facility of charmm and tip3p water molecules and counterions were added using the solvate package of the visual molecular dynamics ( vmd ) package . the charmm27 force field was used for the entire complex with additional parameters added based on preexisting cisplatin parameters . the platinum crosslinks two adjacent guanines . the simulation was performed in namd using standard parameters : a 2.0 fs timestep using shake on all bonds to hydrogen atoms , a 12 cutoff , particle mesh ewald with a 128 grid points on a side , berendsen 's constant pressure algorithm with a target pressure of 1.01325 bar , a compressibility of 45.7 mbar , a relaxation time of 1 ps , and a pressure frequency of 40 fs , and a coordinate save frequency of 200 fs ; all as implemented in namd . the simulation protocol consisted of 250 ps of thermal equilibration to 300 k , followed by a 10 ns production simulation 15 . the partition coefficients were calculated using extended group contribution approach . in this approach , a chemical structure is automatically decomposed into fragments and atom types , and the contributions of different fragments and atom types are summed together , along with correction terms to account for interactions between different fragments . all other reserpine analogs were generated in a similar fashion by condensation of methyl reserpate with the commercially available acid chloride . all new compounds were purified by flash chromatography and characterized by proton and carbon nmr spectroscopy and mass spectrometry ( see supplementary material for specific compounds ) . hec59 cells ( msh2 deficient ) and the paired cell line with chromosome 2 transfer hec59 ( 2 ) have been extensively characterized previously . the cells were grown in standard growth media of dmem - f12 + 10% fbs . cells were plated in 96 well plates at an appropriate concentration in 100 l media and incubated overnight . media was replaced with fresh media containing drug and allowed to incubate for 24 hours at indicated concentrations . one solution reagent ( celltiter 96(r ) aqueous one solution ) was added to existing media ( 20 l / well ) and allowed to incubate 3 - 4 hrs . dose - dependent response to nineteen increasing concentrations of the respective compound was determined . od measurements were used to determine cell viability at each of the concentrations as percentage of control and analyzed for ic50 values using graphpad prism 4 . reserpine ( figure 1(a ) ) , a small molecule identified through computational modeling has been predicted to bind muts homologs mimicking dna damage . we have used lc - ms total ion chromatography to provide evidence for the binding of reserpine to yeast msh2/msh6 ( see figure 1 in supplementary material available online at doi:10.4061/2011/162018 ) . data analysis was performed for m / z 609 , which is characteristic of reserpine alone . the chromatogram of pure reserpine standard showed one prominent peak that eluted at 1.62 minutes with a mass / charge of 609.325 ( suppl . this peak was fragmented by the later ms sectors and two fragments397.08 and 194.82 were reproducibly observed . when the reserpine was mixed in a one - to - one ratio with the protein sample , the peak very reproducibly eluted with a longer retention time ( 1.88 minutes ) relative to pure reserpine . the fact that we detected the peak at 609 could lead to several conclusions : the detected reserpine may represent never bound or excess unbound reserpine . if the presence of protein simply blocked sites of interaction on the stationary phase , it would be surprising to see this as a highly reproducible effect . an alternate explanation would be that the detected reserpine indeed had protein bound , but that the bound reserpine had been liberated ( unbound ) during the electrospray stage of the analysis . since no excess reserpine was present in the mix , we would not expect two peaks ( unbound , excess , and bound reserpine ) . taken together , our data indicate that reserpine was bound to protein through the lc column , and freed during the electrospray stage . the weakness of interaction may not be surprising , given that the compound is much smaller than the normal dna substrate , and hence provides much fewer opportunities for interactions . this does provide indirect evidence that reserpine does bind , albeit , potentially weakly to the protein complex , which suggests that modification of reserpine may be fruitful to improve targeting of the msh2-dependent pathway . cell viability assays show that even though reserpine decreases viability of both msh2-deficient ( ic50 93 m ) and msh2-proficient ( ic50 61 m ) cells after 24-hr treatment , it preferentially kills msh2-proficient cells ( figure 1(b ) ) . the 1.5-fold difference in cell viability between proficient and deficient cells is reminiscent of the activity of cisplatin , though reserpine activity is observed considerably earlier . rescinnamine ( figure 1(a ) ) , a derivative of reserpine that adds additional length via the substitution of the trimethoxybenzoyl group with a trimethoxycinnamoyl group , likewise requires functional msh2 for full reduction of cell viability ( msh2-deficient : ic50 115 m ; msh2-proficient : ic50 38 m ) ( figure 1(b ) ) . the increase in length , without alteration of functional groups , doubled msh2-dependent cytotoxicity to a 3-fold difference in cell viability between proficient and deficient cells . we next determined if the decrease in mitochondrial integrity , as determined by the mts assay for cell viability is indicative of the induction of an apoptotic response pathway . cell extracts of treated cells were analyzed for the specific activation of caspase-3 , a proapoptotic protein commonly used as an indicator for apoptotic cell death . the treatment of msh2-deficient cells with reserpine ( 85 m ) , rescinnamine ( 60 m ) or the control compound staurosporine for 24 hours induces caspase-3 cleavage equally and only weakly above the untreated background control ( figure 1(c ) ) . drug concentrations were chosen based on the ic50 in these cells , which is reflective of the different toxicity of the compounds . the difference in the efficiency in inducing cell death response between reserpine and rescinnamine is apparent , as rescinnamine induces stronger caspase-3 activation than reserpine ( figure 1(c ) ) . msh2 forms a functional heterodimer with msh6 in its ability to recognize dna lesions and initiate response pathways . we next determined if msh6 is also required in the initiation of proper responses to small molecules , such as reserpine and rescinnamine . msh6-deficient and msh6-deficient cells were treated with reserpine ( 85 m ) and rescinnamine ( 60 m ) for 24 hours . the detection of cleaved caspase-3 in extracts from both cell lines revealed activation primarily in the msh6-proficient cells , consistent with a functional requirement of the protein for cytotoxic response ( figure 1(d ) ) . cisplatin is a chemotherapeutic that primarily forms ( 1,2)-intrastrand crosslinks between adjacent guanines in dna . this compound was previously used as a model agent to characterize the msh2/msh6-dependent induction of a protein death conformation and cytotoxic response . since reserpine was predicted to likewise stabilize the msh2/msh6 death conformation , and rescinnamine showed an improved activity over reserpine , we asked whether a combination of rescinnamine with cisplatin would improve the activity of either drug alone . msh2-deficient and msh2-proficient cells were treated with sublethal doses of cisplatin ( 10 m ) and increasing concentrations of rescinnamine ( 025 m ) . cell viability was determined after 24 , 48 , and 72 hours by mts assay ( figure 2 ) . twenty - four hours after treatment ( figure 2(a ) ) , a rapid decrease in cell viability was observed that led to the complete elimination of viable cells after 48 hours at much lower concentration of either cisplatin or rescinnamine alone ( figure 1(b ) ) . overall cell viability was reminiscent of the treatment of msh2-proficient cells with rescinnamine alone , and increased tolerance to the drug was eliminated by the concomitant treatment with cisplatin ( figure 2(a ) ) . increased exposure to the combination treatment eradicated cell viability entirely ( figures 2(b ) and 2(c ) ) , which was previously not observed for much higher concentrations of either drug alone [ 13 , 18 ] . full eradication of cells was observed after 48 or 72 hour treatment with 20 m cisplatin and 15 m rescinnamine . the addition of cisplatin resulted in a 7.3-fold decrease in ic50 value for rescinnamine after 24 hours , and a 9.7-fold decrease after 48 hours in msh2-deficient cells . the same levels of rescinnamine reduced cell viability in msh2-proficient cells ( table 1 ) . an additional 24 hours of treatment for a total of 72 hours did not significantly alter the ic50 any further . these findings are confirmed by immunoblotting ; upon treatment with 24 hours of rescinnamine ( 60 m ) and cisplatin ( 10 m ) comparable levels of caspase-3 cleavage in msh2-deficient and msh2-proficient cells are observed ( figure 2(d ) ) . at 48 and 72 hours , all cells treated with the combination treatment were eliminated and no lysates could be collected and assessed for caspase-3 cleavage . next , we wanted to identify the specific parameters that are required for the msh2-dependent induction of cell death , and when altered , reduce or abolish this function . modeling of reserpine and rescinnamine into the muts structure ( figure 3 ) predicted functional groups and parts of the molecules with favorable ( red ) or unfavorable ( blue ) interactions with the protein . differences in the predicted favorable and unfavorable interactions of reserpine and rescinnamine , respectively , represent the difference in their activity . the methoxy group on ring 1 of reserpine is predicted to be nonessential for interactions , while , in rescinnamine , it is proposed to be important for interactions . as for ring 6 , all three methoxy groups in reserpine are predicted to have favorable interactions , but only two of them are highly favorable for interactions between rescinnamine and muts / msh proteins . in reserpine , the carbon of ring 5 that connects to carbon chain leading to ring 6 is pertinent for interactions . these computational predictions were subsequently experimentally analyzed to determine the validity of the prediction . derivatives of reserpine and rescinnamine were purchased or synthesized ( if not commercially available ) ( see table 1 ) . through structural predictions , the compounds were subdivided into four distinct modes of binding to muts / msh proteins : reserpine - like conformation , flipped conformation , mismatch - like conformation , and nonspecific conformation ( see figure 5 ) . death conformation of msh2/msh6 [ 8 , 18 ] , predicting a similar interaction with the dna - binding pocket of muts ( figure 3(a ) ) . likewise , rescinnamine is predicted to show similar interactions ( figure 3(b ) ) . the molecules are stabilized by hydrogen bonds between their methoxy groups and at least three amino acids ( g38 , r58 , r108 of muts ) . these residues were previously identified as being important for the interaction with cisplatinated dna . the phenylalanine ( f36 ) shown to be indispensable for mismatch repair is far removed from the small molecules and shows no significant binding activity [ 8 , 22 ] . this lack of significance is reminiscent of its failure to exhibit a significant effect on the binding to cisplatinated dna or , when mutated , on the cytotoxic response to cisplatin . previously , it was suggested that an acquired or preexisting bend in the ligand for muts homologous proteins is required for robust binding to their substrates . the existence of such a bend may be a prerequisite for small molecules to specifically bind to muts homologous proteins and induce a specific response . however , the predicted orientation of the bend in the small molecules appears to be inverted compared to dna , which may suggest that the actual presence of a bend is not a requirement for cytotoxic response . among all the compounds tested , reserpine and rescinnamine are the only molecules predicted to preferentially bind in this specific conformation and exhibit these specific interactions with the protein binding pocket . to determine the functional requirements of a small molecule to induce msh2-dependent cell death , we constructed a number of analogs based on the reserpine structure . we hypothesized , based on the structural predictions , that the methoxy groups on ring 6 will have significant impact . we constructed several compounds that either lack all or some of the methoxy groups , resulting in seven different molecules : benzoyl , 3-methoxybenzoyl , 4-methoxybenzoyl , 3,4-dimethoxybenozyl , 3,5-dimethoxybenzoyl , methylendioxybenzoyl , and cinnamoyl resperine ( figure 4(a ) ) . interestingly , the structural predictions suggest a preferred altered binding orientation of these molecules , even for those lacking only one methoxy group ( 3,4-dimethoxy benzoyl ; 3,5-dimethoxy benzoyl ) . this altered binding mode would flip the molecule and have it oriented the opposite way to the reserpine / rescinnamine . the binding of each one of the five molecules in this orientation is predicted to be strong , with predicted ki values of 1532 m . experimental determination of the cell death demonstrates the ability of these compounds to induce cell death , however in an msh2-independent manner ( figure 6(a ) , table 1 ) . the 3,4-dimethoxy benzoyl compound is an exception and the only molecule that initiates a significant msh2-dependent cell death response . the ic50 values for this compound result in 85 m in msh2-deficient cells versus 66 m in msh2-proficient cells , with a 1.3-fold difference , reminiscent of the response observed with reserpine ( table 1 ) . when forced by computational modeling into the correct orientation , the predicted binding constant is 93 m , which would substantiate the cell biology data and suggest a poorer binding to muts than reserpine . the limitations of the resolution of structural modeling make it impossible to distinguish the binding affinity of this compound from the one of other methoxy compounds in the correct orientation . since ring 6 is attached to the remainder of the protein by a rotatable bond , the 3,4-dimethoxy benzoyl compound is indistinguishable from the 4,5-dimethoxy molecule . whether the presence of two methoxy groups on this ring is sufficient for the induction of msh2-dependent cell death , or this rotation and the resulting presence of methoxy groups in all three positions enable the appropriate response is unknown . this compound is predicted to bind to muts with a ki of 19 m ; however , the predicted binding is also found to be in a flipped conformation . the cell death response is opposite to the damage response generally observed in dependence of mismatch repair proteins . msh2-deficient cells show a higher sensitivity to the compound than proficient ones ( ic50 of 36 m in msh2-deficient ones , versus 51 m in msh2-proficient cells , table 1 ) , reversing the resistance phenotype seen with cisplatin and reserpine / rescinnamine . cinnamoyl reserpine is a rescinnamine derivative that lacks the methoxy groups on ring 6 , similar to the reserpine analog benzoyl . again , similar to the corresponding reserpine analog , the predicted binding of cinnamoyl reserpine to muts is predicted to occur in the flipped conformation with a binding constant of 14 m . the compound induces cell death that is independent of functional msh2 ( ic50 value of 150 m in msh2-deficient cells versus 130 m in msh2-proficient cells ; figure 6(a ) , table 1 ) . together , these results suggest that the absence of essential methoxy groups on ring 6 that appear to position the molecules in the correct conformation triggers a different binding mode ( figure 5(b ) ) that does not result in msh2-dependent cell death . removal of these suggested , essential functional groups eliminates the msh2-dependent cell death response . the methoxy group on ring 1 was predicted to show unfavorable interactions with the muts protein for reserpine , but not rescinnamine ( figure 3 ) . a clinically relevant compound exists that has the general structure of reserpine , but lacks this methoxy group ( deserpidine , figure 4(b ) ) . though eliminating a suggestively unfavorable interaction , the predicted binding constant is roughly 2-fold worse than that for reserpine ( 130 m ) , which suggests that even an unfavorable interaction may be required to correctly orient the molecule in the binding pocket . the predicted binding mode for deserpidine is reminiscent of the binding of mismatched dna to the protein . the molecule shows a stronger bending than other compounds and altered orientation that results in the molecule wrapping around the phenylalanine involved in coordinating mismatch binding ( f36 ) ( figure 5(c ) ) . the predicted specificity for the death conformation is reduced for this molecule , which would further confirm the predicted the cell biology of this compound revealed cell death induction with no significant specificity for the presence of msh2 ( ic50 values of 56 m in msh2-deficient versus 49 m in msh2-proficient cells ; figure 6(b ) , table 1 ) , confirming the structural predictions . this result suggests that binding in the mismatched dna mode eliminates msh2-dependent cytotoxic response . though binding is still observed in or close to the dna - binding pocket , the general orientation of these compounds is altered when compared to the binding of reserpine / rescinnamine ( figure 5(d ) ) . this compound is predicted to bind in a distance to the actual binding pocket with a ki of 101 m . the molecule only weakly induces cell death in either msh2-proficient and msh2-deficient cells with no preference for either cell line . syrosingopine is a commercially available reserpine analog that has found application as an antihypertensive drug . its predicted binding to the mismatch repair protein is the worst among the tested compounds , with a predicted ki of 1310 m . this is largely due to the fact that its substitution on position 4 of ring 6 increases its length considerably , which interferes with the binding to the protein ( figures 4(c ) and 5(d ) ) . the binding of the molecule is hence highly distorted when compared to the interaction of reserpine / rescinnamine with the protein ( compare figures 5(a ) and 5(d ) ) . evodiamine , a compound described as aiding in diet - induced obesity , only contains ring systems 1 - 5 and lacks all methoxy groups seen in reserpine / rescinnamine . this compound shows no specific binding in the computational docking experiments , and hence does not result in a predictable binding constant . in the cell system , this molecule shows no significant cytotoxic effect , in either the presence or absence of msh2 ( figure 6(a ) ) . this is presumably due to the fact that evodiamine can not establish any of the required interactions with the mmr protein . cells were treated with selected reserpine derivatives to determine their effect on cell viability and caspase-3 cleavage . msh2-proficient and msh2-deficient cells were treated with increasing concentrations ( 0250 m ) of 3-methoxy reserpine , 3,4-dimethoxybenzoyl reserpine , 3,5-dimethoxybenzoyl reserpine , evodiamine , and deserpidine for 24 hours after which time their viability was assessed via mts assay ( figure 6(a ) and data not shown ) . upon treatment with 3,5-dimethoxybenzoyl , no msh2-dependent cell death was observed ( data not shown ) . upon treatment with 3,4-dimethoxybenzoyl reserpine , however , a 1.3-fold difference between msh2-proficient and msh2-deficient cells was observed , reminiscent of the increase in resistance to cisplatin in msh2-deficient cells . this result indicates the importance of two methoxy groups on ring 6 of the small molecule . this cellular result provides further indirect evidence that the computational model , which is one of direct binding to a specific protein conformation , is valid . since rescinnamine exhibits the activity of an ace inhibitor , we determined if the observed effect on cell viability is due to this activity . in comparison , another , structurally different ace inhibitor , lisinopril , was added and cell viability determined . no effect on cell viability and caspase-3 activation was observed in either msh2-proficient and msh2-deficient cells ( figure 6(b ) ) , suggesting a different function for rescinnamine in msh2-dependent cell death . mmr is one of the primary dna repair pathways that maintain genome stability within every cell . its significance is displayed by its contribution to carcinogenesis in cells deficient for a key mmr protein . based on the increasing number of additional protein interactions mmr proteins undergo , they may participate in numerous other cellular functions that remain to be understood . among these , their participation in the response to cytotoxic agents and the initiation of cell death is of importance , particularly since defects in mmr proteins increase resistance to certain chemotherapeutic agents and lead to the clonal selection of mmr - deficient cells . we have previously described the response of mmr proteins to the chemotherapeutic agent cisplatin and predicted a this death conformation can be accessed by a small molecule , reserpine that induces dna damage - independent cell death . our multiple cellular assays have provided indirect evidence that this model , which required direct binding of reserpine , is indeed correct . however , we do look forward to the day when direct binding can be observed with either refinements of current technologies , or with brand new technologies . we have here determined the particular parameters that allow reserpine and its analogs to access the predicted death conformation these parameters , although predicted based on our model , were verified by cellular assays , which provided indirect evidence for the appropriateness of our model . our experimental data indicates that the length of the molecule and specific functional groups are required for the induction of cell death . in our model , these parameters are critical to maintain the correct orientation of the small molecules within the msh2/msh6-binding pocket . small changes in this structure of reserpine abrogate the ability to induce msh2-dependent cell death ; which would not be surprising if our model of direct binding were correct . these results suggest that the development of an improved molecule for the induction of cell death that is based on reserpine will need to be closely designed upon the original structure of the molecule . since rescinnamine proved to be the best compound , future designs would do well to be based on this structure . since cinnamate , a derivative of rescinnamine without the trimethoxy groups on ring system 6 , abrogates the msh2-dependent cell death response , these groups are required for the observed activity . our data demonstrate the combining virtual screening with conformational modeling is a novel , valid approach to identify compounds that target highly specific structures and pathways . we show that , in at least the case , molecular dynamics simulations provide molecular conformations that are sufficiently accurate as to allow for targeting of specific conformations associated with specific molecular functions . in particular , we have shown that cell death can be induced by small molecules that have been screened against models for a specific death conformation of the msh2/msh6 complexes . we have demonstrated the effects of these small molecules , reserpine - analogs , via two distinct cell assays ; mts assay and caspase - activation , which are standard and commonly used tests for cell viability and apoptotic cell death . furthermore , the combinatorial treatment of reserpine and cisplatin resulted in rapid , msh2-independent cell death , suggesting that the combination treatment overloads the msh2-dependent system , and the cell induces death via a different pathway . this observation suggests that future mechanistic studies , well beyond the scope of this paper as few of the actors involved are known , would be fruitful in determining the precise signaling pathways induced in the msh2-dependent and msh2-independent cell - death pathways . a more detailed mechanistic study of the msh2-dependent pathway would be useful in potentially providing another protein target . understanding the mechanism of the msh2-dependent pathway may provide treatment options for mmr - deficient tumors , which are found in many patients with colorectal cancers . taken together , our data determine the parameters required of reserpine analogs to induce mmr - dependent cell death that can be utilized to further develop chemicals targeting this particular cell death pathway .

mismatch repair proteins modulate the cytotoxicity of several chemotherapeutic agents . we have recently proposed a death conformation of the muts homologous proteins that is distinguishable from their repair conformation . this conformation can be induced by a small molecule , reserpine , leading to dna - independent cell death . we investigated the parameters for a small reserpine - like molecule that are required to interact with msh2/msh6 to induce msh2/msh6-dependent cytotoxic response . a multidisciplinary approach involving structural modeling , chemical synthesis , and cell biology analyzed reserpine analogs and modifications . we demonstrate that the parameters controlling the induction of msh2/msh6-dependent cytotoxicity for reserpine - analogous molecules reside in the specific requirements for methoxy groups , the size of the molecule , and the orientation of molecules within the protein - binding pocket . reserpine analog rescinnamine showed improved msh2-dependent cytotoxicity . these results have important implications for the identification of compounds that require functional mmr proteins to exhibit their full cytotoxicity , which will avoid resistance in mmr - deficient cells .

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

mismatch repair is one of the key dna repair systems in the cell that repair incorrectly formed base pairs and insertion / deletion loops during replication , significantly increasing the stability of the genome [ 15 ] . additional importance comes from the contribution of defects in mmr proteins to cancer development , progression and therapeutic success . in addition to the recognition of replication errors , key mmr proteins msh2/msh6 recognize a number of dna lesions that are irreparable by these proteins , which , in turn , initiate a cell death pathway . the role of mmr proteins in the cell death response to cytotoxic agents has been the subject of much debate [ 6 , 7 ] . whether dna damage is recognized in form of a mismatch at the site of the damage , or dna damage itself is recognized recent data suggest that some types of dna lesions are recognized without the requirements for a mismatch . as a result of their ability to recognize certain dna lesions and their participation in the initiation of cell death , this participation is particularly evident in cells with defects in mmr proteins which reduce the sensitivity to chemotherapeutic agents sufficiently to prove to be a problem for the treatment of tumors [ 1 , 6 , 912 ] . the exact mechanism of the cytotoxic response initiated by msh2/msh6 interactions with dna damage remains under investigation and speculation . recent data suggest that the response pathway depends on the nature of the lesion rather than the unification of different signals into a common pathway . at least two hypotheses have been put forward to address the mechanism behind the mmr protein - dependent cell death : the futile cycles of repair hypothesis is based on the formation of a mismatch opposite the dna damage , followed by attempted repair events that retain the damage and the formation of dna strand breaks as factual initiators of cell death [ 1 , 4 , 14 ] . the second hypothesis suggests a direct involvement of mmr proteins in the initiation of the cell death event . we and others have identified separation - of - function mutants that demonstrate that repair - defective mutants of mmr proteins retain the ability to induce cell death , suggesting that repair is not required for cytotoxic response to all types of dna damage [ 8 , 1517 ] , but also suggesting that specific mmr proteins form recognition complexes for both mismatch repair , and at least some types of dna damage . computational modeling predicted the formation of a death conformation with distinctly different features to the repair conformation of muts and its homologs ( msh ) . although the mechanistic details of this pathway are largely unknown , targeting the apparent recognition complex for cell death , that is , the death conformation , has shown initial successes . hence , we aim to exploit this pathway via small molecule binding to the death conformation . recently , we have demonstrated that the msh2-dependent cell death response can be induced by small molecules that mimic binding to dna damage and promote the induction of this cell death . the prototype for this induction of msh2-dependent cell death without genotoxic insult is reserpine , a drug previously in clinical use for hypertension . the predicted binding of the molecule to the protein occurs in the dna binding pocket and induces specific conformational changes that allow access to the death conformation of the protein . at present , the direct binding of reserpine , and its derivatives , to the death conformation has been predicted , and the ability of reserpine and rescinnamine to induce msh2-dependent apoptosis has been verified experimentally in this work as well as in an earlier study . we expect that the technology used to detect such direct binding of small molecules to large protein complexes will be refined over the next few years , and that presently challenging complexes , such as the proposed msh2/msh6/rescinnamine or reserpine complex , will be directly studied . here , we determine the parameters for small molecules to initiate the mmr protein - dependent cell - death response , where the cell - death response is measured via two distinct standard assays , mts assay and caspase activation , which provide multiple pieces of evidence for the induction of cell - death . based on our models , we have hypothesized that specific structural parameters are required of a small molecule to mimic the interaction of msh2/msh6 with dna damage , show the correct orientation in the protein - binding pocket and initiate msh2-dependent cell death . in a combination of computer - based structural modeling , chemical synthesis , and cell biology , we have identified these distinct requirements , which consist of the size of the molecule , the presence and location of methoxy groups , and the orientation of the molecule within the protein - binding pocket . while the computational modeling assumes that the small molecules bind in a specific pocket and in specific orientations , as predicted , the cellular assays do not require such assumptions . these small - molecule structural parameters will provide the foundation for any subsequent searches for new small molecule compounds that initiate the specific msh2-dependent cell death targeted in this approach . the grids were centered on the midpoint of the line connecting the two guanine nitrogens that are crosslinked in the platinated simulation , but the grids were generated in the absence of dna and the platinum adduct . the simulation protocol is largely the same as described elsewhere , differing only in the use of a constant pressure algorithm , and differing simulation lengths , and is briefly summarized here . in this approach , a chemical structure is automatically decomposed into fragments and atom types , and the contributions of different fragments and atom types are summed together , along with correction terms to account for interactions between different fragments . reserpine ( figure 1(a ) ) , a small molecule identified through computational modeling has been predicted to bind muts homologs mimicking dna damage . we have used lc - ms total ion chromatography to provide evidence for the binding of reserpine to yeast msh2/msh6 ( see figure 1 in supplementary material available online at doi:10.4061/2011/162018 ) . when the reserpine was mixed in a one - to - one ratio with the protein sample , the peak very reproducibly eluted with a longer retention time ( 1.88 minutes ) relative to pure reserpine . an alternate explanation would be that the detected reserpine indeed had protein bound , but that the bound reserpine had been liberated ( unbound ) during the electrospray stage of the analysis . since no excess reserpine was present in the mix , we would not expect two peaks ( unbound , excess , and bound reserpine ) . this does provide indirect evidence that reserpine does bind , albeit , potentially weakly to the protein complex , which suggests that modification of reserpine may be fruitful to improve targeting of the msh2-dependent pathway . the 1.5-fold difference in cell viability between proficient and deficient cells is reminiscent of the activity of cisplatin , though reserpine activity is observed considerably earlier . the increase in length , without alteration of functional groups , doubled msh2-dependent cytotoxicity to a 3-fold difference in cell viability between proficient and deficient cells . we next determined if the decrease in mitochondrial integrity , as determined by the mts assay for cell viability is indicative of the induction of an apoptotic response pathway . cell extracts of treated cells were analyzed for the specific activation of caspase-3 , a proapoptotic protein commonly used as an indicator for apoptotic cell death . drug concentrations were chosen based on the ic50 in these cells , which is reflective of the different toxicity of the compounds . the detection of cleaved caspase-3 in extracts from both cell lines revealed activation primarily in the msh6-proficient cells , consistent with a functional requirement of the protein for cytotoxic response ( figure 1(d ) ) . this compound was previously used as a model agent to characterize the msh2/msh6-dependent induction of a protein death conformation and cytotoxic response . since reserpine was predicted to likewise stabilize the msh2/msh6 death conformation , and rescinnamine showed an improved activity over reserpine , we asked whether a combination of rescinnamine with cisplatin would improve the activity of either drug alone . overall cell viability was reminiscent of the treatment of msh2-proficient cells with rescinnamine alone , and increased tolerance to the drug was eliminated by the concomitant treatment with cisplatin ( figure 2(a ) ) . next , we wanted to identify the specific parameters that are required for the msh2-dependent induction of cell death , and when altered , reduce or abolish this function . modeling of reserpine and rescinnamine into the muts structure ( figure 3 ) predicted functional groups and parts of the molecules with favorable ( red ) or unfavorable ( blue ) interactions with the protein . in reserpine , the carbon of ring 5 that connects to carbon chain leading to ring 6 is pertinent for interactions . through structural predictions , the compounds were subdivided into four distinct modes of binding to muts / msh proteins : reserpine - like conformation , flipped conformation , mismatch - like conformation , and nonspecific conformation ( see figure 5 ) . death conformation of msh2/msh6 [ 8 , 18 ] , predicting a similar interaction with the dna - binding pocket of muts ( figure 3(a ) ) . this lack of significance is reminiscent of its failure to exhibit a significant effect on the binding to cisplatinated dna or , when mutated , on the cytotoxic response to cisplatin . previously , it was suggested that an acquired or preexisting bend in the ligand for muts homologous proteins is required for robust binding to their substrates . the existence of such a bend may be a prerequisite for small molecules to specifically bind to muts homologous proteins and induce a specific response . however , the predicted orientation of the bend in the small molecules appears to be inverted compared to dna , which may suggest that the actual presence of a bend is not a requirement for cytotoxic response . among all the compounds tested , reserpine and rescinnamine are the only molecules predicted to preferentially bind in this specific conformation and exhibit these specific interactions with the protein binding pocket . to determine the functional requirements of a small molecule to induce msh2-dependent cell death , we constructed a number of analogs based on the reserpine structure . we hypothesized , based on the structural predictions , that the methoxy groups on ring 6 will have significant impact . we constructed several compounds that either lack all or some of the methoxy groups , resulting in seven different molecules : benzoyl , 3-methoxybenzoyl , 4-methoxybenzoyl , 3,4-dimethoxybenozyl , 3,5-dimethoxybenzoyl , methylendioxybenzoyl , and cinnamoyl resperine ( figure 4(a ) ) . interestingly , the structural predictions suggest a preferred altered binding orientation of these molecules , even for those lacking only one methoxy group ( 3,4-dimethoxy benzoyl ; 3,5-dimethoxy benzoyl ) . experimental determination of the cell death demonstrates the ability of these compounds to induce cell death , however in an msh2-independent manner ( figure 6(a ) , table 1 ) . the 3,4-dimethoxy benzoyl compound is an exception and the only molecule that initiates a significant msh2-dependent cell death response . when forced by computational modeling into the correct orientation , the predicted binding constant is 93 m , which would substantiate the cell biology data and suggest a poorer binding to muts than reserpine . the limitations of the resolution of structural modeling make it impossible to distinguish the binding affinity of this compound from the one of other methoxy compounds in the correct orientation . since ring 6 is attached to the remainder of the protein by a rotatable bond , the 3,4-dimethoxy benzoyl compound is indistinguishable from the 4,5-dimethoxy molecule . whether the presence of two methoxy groups on this ring is sufficient for the induction of msh2-dependent cell death , or this rotation and the resulting presence of methoxy groups in all three positions enable the appropriate response is unknown . the cell death response is opposite to the damage response generally observed in dependence of mismatch repair proteins . cinnamoyl reserpine is a rescinnamine derivative that lacks the methoxy groups on ring 6 , similar to the reserpine analog benzoyl . again , similar to the corresponding reserpine analog , the predicted binding of cinnamoyl reserpine to muts is predicted to occur in the flipped conformation with a binding constant of 14 m . the compound induces cell death that is independent of functional msh2 ( ic50 value of 150 m in msh2-deficient cells versus 130 m in msh2-proficient cells ; figure 6(a ) , table 1 ) . together , these results suggest that the absence of essential methoxy groups on ring 6 that appear to position the molecules in the correct conformation triggers a different binding mode ( figure 5(b ) ) that does not result in msh2-dependent cell death . the methoxy group on ring 1 was predicted to show unfavorable interactions with the muts protein for reserpine , but not rescinnamine ( figure 3 ) . though eliminating a suggestively unfavorable interaction , the predicted binding constant is roughly 2-fold worse than that for reserpine ( 130 m ) , which suggests that even an unfavorable interaction may be required to correctly orient the molecule in the binding pocket . the predicted binding mode for deserpidine is reminiscent of the binding of mismatched dna to the protein . the molecule shows a stronger bending than other compounds and altered orientation that results in the molecule wrapping around the phenylalanine involved in coordinating mismatch binding ( f36 ) ( figure 5(c ) ) . the predicted specificity for the death conformation is reduced for this molecule , which would further confirm the predicted the cell biology of this compound revealed cell death induction with no significant specificity for the presence of msh2 ( ic50 values of 56 m in msh2-deficient versus 49 m in msh2-proficient cells ; figure 6(b ) , table 1 ) , confirming the structural predictions . this result suggests that binding in the mismatched dna mode eliminates msh2-dependent cytotoxic response . though binding is still observed in or close to the dna - binding pocket , the general orientation of these compounds is altered when compared to the binding of reserpine / rescinnamine ( figure 5(d ) ) . the molecule only weakly induces cell death in either msh2-proficient and msh2-deficient cells with no preference for either cell line . this is largely due to the fact that its substitution on position 4 of ring 6 increases its length considerably , which interferes with the binding to the protein ( figures 4(c ) and 5(d ) ) . the binding of the molecule is hence highly distorted when compared to the interaction of reserpine / rescinnamine with the protein ( compare figures 5(a ) and 5(d ) ) . this compound shows no specific binding in the computational docking experiments , and hence does not result in a predictable binding constant . msh2-proficient and msh2-deficient cells were treated with increasing concentrations ( 0250 m ) of 3-methoxy reserpine , 3,4-dimethoxybenzoyl reserpine , 3,5-dimethoxybenzoyl reserpine , evodiamine , and deserpidine for 24 hours after which time their viability was assessed via mts assay ( figure 6(a ) and data not shown ) . upon treatment with 3,4-dimethoxybenzoyl reserpine , however , a 1.3-fold difference between msh2-proficient and msh2-deficient cells was observed , reminiscent of the increase in resistance to cisplatin in msh2-deficient cells . this result indicates the importance of two methoxy groups on ring 6 of the small molecule . this cellular result provides further indirect evidence that the computational model , which is one of direct binding to a specific protein conformation , is valid . among these , their participation in the response to cytotoxic agents and the initiation of cell death is of importance , particularly since defects in mmr proteins increase resistance to certain chemotherapeutic agents and lead to the clonal selection of mmr - deficient cells . we have previously described the response of mmr proteins to the chemotherapeutic agent cisplatin and predicted a this death conformation can be accessed by a small molecule , reserpine that induces dna damage - independent cell death . our multiple cellular assays have provided indirect evidence that this model , which required direct binding of reserpine , is indeed correct . we have here determined the particular parameters that allow reserpine and its analogs to access the predicted death conformation these parameters , although predicted based on our model , were verified by cellular assays , which provided indirect evidence for the appropriateness of our model . our experimental data indicates that the length of the molecule and specific functional groups are required for the induction of cell death . in our model , these parameters are critical to maintain the correct orientation of the small molecules within the msh2/msh6-binding pocket . small changes in this structure of reserpine abrogate the ability to induce msh2-dependent cell death ; which would not be surprising if our model of direct binding were correct . these results suggest that the development of an improved molecule for the induction of cell death that is based on reserpine will need to be closely designed upon the original structure of the molecule . since cinnamate , a derivative of rescinnamine without the trimethoxy groups on ring system 6 , abrogates the msh2-dependent cell death response , these groups are required for the observed activity . in particular , we have shown that cell death can be induced by small molecules that have been screened against models for a specific death conformation of the msh2/msh6 complexes . we have demonstrated the effects of these small molecules , reserpine - analogs , via two distinct cell assays ; mts assay and caspase - activation , which are standard and commonly used tests for cell viability and apoptotic cell death . furthermore , the combinatorial treatment of reserpine and cisplatin resulted in rapid , msh2-independent cell death , suggesting that the combination treatment overloads the msh2-dependent system , and the cell induces death via a different pathway . this observation suggests that future mechanistic studies , well beyond the scope of this paper as few of the actors involved are known , would be fruitful in determining the precise signaling pathways induced in the msh2-dependent and msh2-independent cell - death pathways . understanding the mechanism of the msh2-dependent pathway may provide treatment options for mmr - deficient tumors , which are found in many patients with colorectal cancers . taken together , our data determine the parameters required of reserpine analogs to induce mmr - dependent cell death that can be utilized to further develop chemicals targeting this particular cell death pathway .

biogenesis of most eukaryotic mrnas involves splicing and cleavage and polyadenylation ( 3 end processing ) ( derti et al . , 2012 , tian et al . , 2005 ) . both mechanisms are required to produce functional mrnas and are also important to regulate gene expression by producing alternative mrna isoforms and for efficient transcription termination ( di giammartino et al . , 2011 , the alternative isoforms are produced by alternative splicing or by use of alternative polyadenylation ( apa ) sites . both alternative splicing and apa are regulated by rna - binding proteins ( rbps ) ( di giammartino et al . , 2011 , , 2013 , fu and ares , 2014 , shi , 2012 , witten and ule , 2011 ) . however , few tools are available to study both processes in an integrated manner , and the overlap between their regulatory programs is poorly understood . the regulatory function of many rbps depends on their binding position in respect to the regulated exon ( witten and ule , 2011 ) . such position - dependent regulatory principles have been visualized at high - resolution in the form of rna maps ( ule et al . , 2006 ) , and were exploited to derive codes that can predict tissue - specific splicing patterns ( alipanahi et al . it is clear that the rbp binding on nascent rna can also affect apa in a position - dependent manner ( batra et al . , 2014 , three studies have used crosslinking and immunoprecipitation ( clip ) to define the rna map of apa ( licatalosi et al . , 2008 , masuda et al . , 2015 , batra et al . , 2014 ) , but these studies have plotted the position of full clip reads around the regulated poly(a ) sites , rather than the position of crosslink sites , and have not evaluated the statistical significance of identified enrichments . therefore , the importance in binding position for guiding the repressing or enhancing effects of rbps remains unclear . the nucleotide resolution that is obtained by the truncated cdnas in iclip , and the quantitative nature gained by the analysis of unique molecular identifiers ( umis ) , allowed us to examine the rna maps of apa regulation in much greater detail . our study examines how the tar ( transactive response ) dna - binding protein 43 ( tdp-43 , also referred to as tardbp ) regulates splicing and apa via position - dependent principles . tdp-43 is an rbp involved in several neurodegenerative diseases , including frontotemporal lobar degeneration ( ftld - tdp ) and amyotrophic lateral sclerosis ( als ) ( ratti and buratti , 2016 ) . tdp-43 regulates alternative splicing in a position - dependent manner by binding to intronic dinucleotide of uridine and guanosine ( ug)-rich motifs , such that binding close to the splice sites represses splicing , whereas binding further downstream of the exon enhances splicing ( tollervey et al . , 2011 ) . the binding of tdp-43 is also enriched in 3 utrs , and it can regulate apa of its own transcript ( ratti and buratti , 2016 , tollervey et al . , 2011 ) , indicating a possible role for regulating apa of other transcripts . we identified poly(a ) sites regulated by tdp-43 in hek293 cells by 3 mrna sequencing and defined tdp-43 binding sites with individual nucleotide crosslinking and immunoprecipitation ( iclip ) . to perform 3 end data analysis , we developed expressrna , a modular bioinformatics research platform that manages data and inter - connects recently developed ( expressrna.apa module , rnamotifs2 ) and existing ( star , dexseq ) analysis software . the expressrna.apa module identifies the sites where cleavage and polyadenylation takes place ( poly(a ) sites ) , marks and annotates the differentially regulated poly(a ) sites , and visualizes binding patterns of rbps around these sites . the resulting rna maps defined the binding positions where tdp-43 can enhance or repress multiple types of poly(a ) sites , and we validated its direct action with a minigene reporter . we demonstrated that the role of tdp-43 can be replaced in this minigene by tia1 , another known splicing regulator , if ug - rich motifs are replaced by ua - rich motifs . to identify enriched clusters of diverse regulatory motifs around the regulated exons or poly(a ) sites , we extended and upgraded rna motifs ( cereda et al . , 2014 ) this identified enriched clusters composed of multiple types of motifs around tissue - specific exons or poly(a ) sites , which can indicate a potential for combinatorial regulation of splicing and apa . to study how tdp-43 regulates apa , rna was isolated from six independent tdp-43 knockdown ( kd ) and control hek293 cells . the 3 ends of mrnas were amplified with the quantseq rev 3 mrna sequencing ( mrna - seq ) library prep kit ( lexogen ) , which uses a poly(t ) primer to reverse transcribe the mrnas . the library was sequenced with hiseq , producing 60-nt single - end reads and 10-nt index reads . for data analysis , we developed the expressrna web application and analysis platform ( figures 1 and s1b ) . the first step of expressrna is to process and map the 3 mrna - seq data to the genome , classify the sites where cleavage and polyadenylation takes place ( poly(a ) sites ) , and identify the differentially regulated poly(a ) sites . we first aligned the reads to the hg19 reference genome with star ( dobin et al . , 2013 ) , allowing soft clipping from both 5 and 3 end . of the total 52.96 million reads , 54.83% aligned uniquely to the human genome ( table s1 ) . to construct the database of identified poly(a ) sites , we considered sequence data from all experiments as one dataset . some of the aligned reads were soft - clipped due to the sequencing running into the poly(a ) tail or imperfect primer annealing ( figure s2a).figure 1expressrna research platformthe expressrna platform performs analyses of alternative polyadenylation datasets and can include external alternative splicing datasets . the identified or provided regulated features ( poly(a ) sites , alternative exons ) are then combined with rna protein binding information ( iclip ) . expressrna research platform the expressrna platform performs analyses of alternative polyadenylation datasets and can include external alternative splicing datasets . the identified or provided regulated features ( poly(a ) sites , alternative exons ) since the longest 3 utr isoforms are in some cases not fully annotated , we added 5 kb of the intergenic region downstream of each gene . if two genes are closer than 10 kb , only the region up to the middle was added . we found that alternative positions of cleavage and polyadenylation can be clustered in close proximity of a dominant poly(a ) site , with most variation occurring within 5 nt of the dominant site , indicating that the cleavage position is not always precise and can vary by a few nucleotides ( figure s2b ) . such cleavage positions occur downstream of a single polyadenylation signal ( pas ) , and therefore their variation likely reflects lack of cleavage precision by the cleavage and polyadenylation machinery . for quantification of poly(a ) sites , we therefore summed up the counts of reads that identified cleavage up to 5 nt away from each dominant poly(a ) site ( figure s2c ) . published studies also indicate that auxiliary rna motifs tend to be enriched in the region approximately up to 75 nt upstream ( ure , upstream regulatory elements ) and 50 nt downstream ( dre , downstream regulatory elements ) of each cleavage site ( beaudoing et al . , we wished to focus our study on fully independent cleavage sites that contain their own pas and auxiliary motifs . therefore , we identified the dominant poly(a ) sites based on read count , such that all resulting sites were at least 125 nt apart . quantseq relies on annealing a poly(t ) primer to the poly(a ) tail of mrnas to identify the 3 end of the mrnas . however , as has been shown previously , poly(t ) primers often also anneal to internal a - rich sites in mrnas ( derti et al . , therefore , poly(a ) sites with a - tracts in the vicinity [ 10 .. 10 ] were first filtered out . the poly(a ) sites were further classified into three classes by using the software poly(a)r ( akhtar et al . , 2010 ) , which evaluates the presence of preceding pas . this identified 17,102 pas - strong , 2,599 pas - weak , and 10,513 pas - less sites ( table s4 ) . we examined the nucleotide composition , the efficiency of cleavage , and the level of overlap with two past studies ( derti et al . , 2012 , gruber et al . , 2012 ) , which confirms that the pas - strong and pas - weak sites are the most reliable and efficiently used sites ( figures s2d and s2e s2h ) . classification and broad identification of poly(a ) sites in hek293 cells has been achieved earlier by a comprehensive analysis of 3 end targeted sequencing datasets ( gruber et al . , 2016 ) . in contrast , the aim of our study is not to further characterize newly identified sites , but rather to use a stringent filtering approach to examine the regulatory patterns at the functional poly(a ) sites . we therefore focused only on the pas - strong and pas - weak sites , which identified a total of 19,701 poly(a ) sites , 16,599 sites were annotated to genes ( ensembl v.74 ) . to ensure robust analysis , poly(a ) sites with less than ten read counts in either control or kd experiments we then identified the poly(a ) site in each gene that contained the highest read count , which we consider as the major poly(a ) site . to avoid poorly used sites , we then discarded those poly(a ) sites that have less than 5% of reads compared to the number of reads present at the major poly(a ) site in the same gene . 15,113 sites in 10,069 genes remained after these filtering steps ( figure s2d ) . comparisons of our 15,113 sites to the published poly(a ) database in human tissues ( derti et al . , 2012 ) and hek293 cells ( gruber et al . , 2012 ) demonstrates that the majority of sites overlap at nucleotide precision ( figure 2d ) . allowing for up to a 10-nt spacing , approximately 70% of our sites overlap with the sites in derti et al . ( 2012 ) and 65% with the previously defined sites in hek293 cells from gruber et al . the overlapping sites have the characteristic nucleotide signature of a - rich pas upstream of the cleavage site , which is followed by t - rich sequences , in agreement with past studies ( sheets et al . , 1990 ) ( figure s2f ) . a larger number of poly(a ) sites were identified in hek293 cells by a previous study due to the use of milder filtering criteria ( gruber et al . , 2012 ) , but the sites that are not shared with our study have weaker nucleotide signature compared to the overlapping sites ( figure s2 g ) . however , the sites identified in our study have similar nucleotide signature to the ones that overlap with the published sites , indicating that the stringent filtering criteria applied by our study are sufficient to ensure that most of the identified poly(a ) sites are valid ( figure s2 g ) . moreover , de novo search of the most enriched hexamers in the region where the pas is normally located ( [ 30 .. 18 ] upstream of the poly(a ) site ) found the expected consensus sequences ( beaudoing et al . , 2000 ) ( figure s2h ; supplemental experimental procedures).figure 2mapping reads and evaluating poly(a ) site loci and expression(a ) read alignment to the hg19 reference genome . reads are soft clipped ( red ) due to poly(a ) tail sequence at the 5 end or imperfect primer annealing at the 3 end.(b ) classification of proximal and distal poly(a ) site pairs ( 5 s1 = 5 splice site 1 ) . the same - exon pairs are not limited to the last exons in the gene , 9% ( 201 of 2,202 ) of the same - exon pairs are annotated to a non - terminal exon.(c ) dexseq computes direction ( log2 fold change ) and significance ( adjusted p value ) of poly(a ) sites in control versus tdp-43 kd . the proximal - distal site pair with highest fold change is selected among significantly changed poly(a ) sites ( adjusted p value < 0.05 ) . control distributions are drawn as black lines in all rna maps.(d ) overlap with derti et al . , 2012 , gruber et al . , 2012 , and nam et al . ( 2014 ) poly(a ) sites around the poly(a ) sites defined by the present study . mapping reads and evaluating poly(a ) site loci and expression ( a ) read alignment to the hg19 reference genome . reads are soft clipped ( red ) due to poly(a ) tail sequence at the 5 end or imperfect primer annealing at the 3 end . ( b ) classification of proximal and distal poly(a ) site pairs ( 5 s1 = 5 splice site 1 ) . the same - exon pairs are not limited to the last exons in the gene , 9% ( 201 of 2,202 ) of the same - exon pairs are annotated to a non - terminal exon . ( c ) dexseq computes direction ( log2 fold change ) and significance ( adjusted p value ) of poly(a ) sites in control versus tdp-43 kd . the proximal - distal site pair with highest fold change is selected among significantly changed poly(a ) sites ( adjusted p value < 0.05 ) . , 2012 , gruber et al . , 2012 , and nam et al . ( 2014 ) poly(a ) sites around the poly(a ) sites defined by the present study . we further tested the validity of our detected poly(a ) sites by plotting the coverage of rna sequencing ( rna - seq ) experiments from published studies performed in hek293 cells ( pham et al . , 2016 , trakman et al . , 2016 ) ( figures s2j s2l ) . we computed the read coverage in the region 500 .. 200 around proximal and distal poly(a ) sites , defined by the present study , which were separated into the 85% that are shared with the previous study by nam et al . we find a peak of increased rna - seq average coverage upstream of both types of poly(a ) sites , and a drop to negligible levels of rna - seq coverage downstream of the distal poly(a ) sites ( figure s2j ) . this significant difference in read coverage just upstream and downstream of both the annotated and poly(a ) sites that we have annotated additionally supports the validity of poly(a ) sites uncovered here . for further comparison , we also plotted heatmaps of rna - seq read coverage around the 50 known and poly(a ) sites uncovered by this analysis that contain most quantseq reads by displaying the quantseq 3 end targeted sequencing coverage ( data from our study ) alongside the rna - seq coverage data ( figures s2k and s2l ) . this shows that the rna - seq read distribution at individual poly(a ) sites is similar for known and sites uncovered by this analysis , since most sites contain enrichment of rna - seq reads upstream , and lack the reads downstream of the poly(a ) sites . taken together , this indicates that the poly(a ) sites are accurately identified by the expressrna analysis of quantseq data in hek293 cells . to define the regulatory principles with high fidelity , we focused our analyses on 3,291 genes where we can robustly annotate multiple poly(a ) sites . this represents about 33% of detected genes , which is slightly lower compared to most past studies , which reported approximately 40% of protein coding genes with > 1 poly(a ) site ( ni et al . , 2013 ) . it is much lower than the report of 70% with multiple poly(a ) sites that were detected with the cross - tissue analysis ( derti et al . , 2012 ) , but that is expected given that a single cell type has less rna diversity compared to all the tissues . the lower proportion of genes with multiple poly(a ) sites likely reflects also our stringent requirement for that each poly(a ) site contains at least 5% of the reads that map to each gene . next , we wished to identify those genes with multiple poly(a ) sites where the use of the poly(a ) sites changes upon tdp-43 kd . statistically significant changes in poly(a ) site use between control and kd conditions were identified using dexseq ( anders et al . , 2012 ) if more than two poly(a ) sites were identified in a gene , then the two poly(a ) sites most significantly changed ( adjusted p value < 0.05 ) were considered for further analyses of regulated sites . if only one site had an adjusted p value < 0.05 , then the second site was selected based on highest read count . if no site had p value < 0.05 , then both sites were selected based on highest read count . our approach identified 3,291 genes ( poly(a ) site pairs ) that we then classified based on the position of the two poly(a ) sites in the gene ( figure 2b ) . if both poly(a ) sites are in the same exon , we classified them as same exon ( the most common apa type , also referred as tandem in previous studies ) ( elkon et al . , 2013 ) . if the proximal poly(a ) site is part of a composite exon that contains an internal 5 splice site , and the two poly(a ) sites were generated by alternative 5 splice site use , we classified them as a composite exon . finally , if the proximal poly(a ) site is in an exon that is fully skipped when the distal poly(a ) site is used , we classified them as skipped exon ( figure 2b ) . the final filtered poly(a ) data included 3,291 poly(a ) site pairs , of which 2,202 belonged to the same - exon , 662 to the skipped - exon , and 427 to the composite - exon class . to identify changes in apa between control and tdp-43 kd , we examined the changes in relative read counts at the proximal and distal poly(a ) sites in each gene , which is referred to as fold change ( reported by dexseq ) . the fold change is used to determine the direction of change as repressed ( fold change < 0 ) or enhanced ( fold change > 0 ) . for genes with no significantly regulated poly(a ) sites , two sites with highest read counts across both control and kd conditions were selected and classified into control enhanced and control repressed ( figure 2c ) . to identify tdp-43 binding sites in hek293 cells , we performed iclip of tdp-43 in two replicate experiments , which together generated 12,622,661 uniquely mapped iclip cdnas . we report the number of iclip cdnas around each regulated poly(a ) site ( table s2 ) . we then identified 415,238 significant crosslink clusters , 9.4% of which map to annotated 3 utrs ( figure s2i ) . we visualized the positions of these crosslink clusters around the same - exon class of regulated poly(a ) sites , including both the proximal ( figures 3a3c ) and the distal poly(a ) sites ( figure 3d ) . we also examined the ug - rich binding motifs ( see defining tdp-43 binding positions with iclip ) , which are known to bind tdp-43 . reassuringly , crosslink clusters and enriched ug - rich motifs are abundant at similar positions ( figures 3a and s3a s3d ) , further indicating that these two independent approaches correctly define the binding sites of tdp-43.figure 3rna maps of tdp-43 around proximal and distal same - exon poly(a ) sites with iclip clusters(a ) proximal poly(a ) site rna map of tdp-43 iclip at regulated genes . tdp-43 is bound around repressed poly(a ) sites and to a lesser extent ( sparsely ) further upstream and downstream of enhanced poly(a ) sites.(b ) top 20 iclip mrna targets contributing to proximal poly(a ) site repression.(c ) top 20 iclip mrna targets contributing to proximal poly(a ) site enhancement.(d ) distal site rna map of tdp-43 binding around regulated genes . the level of binding is lower compared to the proximal sites ( table s6 ) . rna maps of tdp-43 around proximal and distal same - exon poly(a ) sites with iclip clusters ( a ) proximal poly(a ) site rna map of tdp-43 iclip at regulated genes . tdp-43 is bound around repressed poly(a ) sites and to a lesser extent ( sparsely ) further upstream and downstream of enhanced poly(a ) sites . ( b ) top 20 iclip mrna targets contributing to proximal poly(a ) site repression . ( c ) the level of binding is lower compared to the proximal sites ( table s6 ) . to further assess the specificity of the rna map , we redrew it by using just the 13,034 poly(a ) sites that overlap with the ones identified also with the 3p - seq method in the previous study ( figure 2d ) . the resulting same - exon proximal rna map is almost identical to the map that used all the sites ( figures 3a and s3e ) , which demonstrates that the map is robust and largely unaffected by the identification of additional poly(a ) sites . moreover , we compared iclip binding of tdp-43 , tial1 , and stau1 around the same - exon proximal poly(a ) sites regulated by tdp-43 ( figure s3f ) . we chose tia1 and stau1 as controls , since both of these rbps also have enriched crosslinking to 3 utrs . we plotted the enrichment of crosslink clusters for each protein by comparing regulated versus control poly(a ) sites , which demonstrated much stronger and position - specific enrichment for tdp-43 compared to the other control rbps . since tdp-43 is a known regulator of splicing , we also examined whether use of the poly(a ) sites might be indirectly affected due to co - regulation ( binding ) at nearby splice sites . this is possible in the case of composite - exon and skipped - exon poly(a ) sites , which contain at least one splice between the regulated poly(a ) sites ( s1 and s2 , as marked in figure 2b ) . we found position - dependent tdp-43 crosslinking at the regulated composite - exon and skipped - exon poly(a ) sites ( figures s4a s4d ) and much less at splice sites flanking these poly(a ) sites ( figures s5a s5f ; table s6 ) . this indicates that tdp-43 rarely regulates apa indirectly via splicing but rather directly regulates both types of competing poly(a ) sites . to demonstrate in a simple way that tdp-43 binds at different positions to repress or enhance the poly(a ) sites , we defined the 40-nt window around each type of regulated poly(a ) sites that had the strongest enrichment of crosslink clusters compared to controls ( repressed / enhanced versus control genes ) ( table s6 ) . to determine the number of genes that contain a specific class of alternative poly(a ) site that is directly regulated by tdp-43 , we counted the number of iclip crosslink clusters at this 40-nt window , which allowed us to calculate the bound regulated genes score ( brg ) ( see experimental procedures and table s6 ) . this shows that tdp-43 most often binds next to the proximal same - exon poly(a ) sites ( 97 .. 57 relative to proximal poly(a ) site , brg = 33 , p value 1e-6 , figure 3 ) , while enriched binding is also seen further downstream of the enhanced sites ( 72 .. 112 relative to proximal poly(a ) site , brg = 19 , p value 3e-5 , figure 3 ) . this pattern is reminiscent of the rna maps of splicing regulation , where tdp-43 binds directly upstream or within the exon to repress and further downstream of the exon to enhance splicing ( tollervey et al . , 2011 ) . to understand whether the rna map of apa regulation by tdp-43 can be discovered without any knowledge of its binding specificity so that it could assess regulated poly(a ) sites , in addition to alternative exons . moreover , while the original rnamotifs could only identify clusters of highly similar motifs around alternative exons , the version ( named rnamotifs2 ) can identify clusters of diverse motifs enriched around groups of exons or poly(a ) sites . this can assess the potential for multiple rbps to combinatorially regulate pre - mrna processing . we applied rnamotifs2 to analysis of the same - exon class of poly(a ) sites regulated by tdp-43 . the enrichment of the detected significant motif clusters ( fisher s exact test , p < 0.01 ) is plotted in blue at the repressed and in red at enhanced poly(a ) sites ( figure 4 ) . ug - rich motif clusters were enriched mainly around the proximal regulated sites at similar positions as the iclip crosslink sites ( figures 3a and s3a ; table s6 ) . interestingly , the distal sites mainly had enrichment of u - rich and ya - rich motifs ( figure 4b ) , indicating a potential for regulation of competing poly(a ) sites by different rbps.figure 4motif analysis around tdp-43-regulated same - exon poly(a ) sitesup to two of the most significant motif clusters are shown for each search region ( r1 , r2 , r3).(a ) motif analysis around proximal poly(a ) sites show significant ug - rich clusters in r1 [ 100 .. 40 ] and r2 [ 40 .. 20 ] around repressed proximal poly(a ) sites . more distal binding in r3 [ 20 .. 80 ] results in enhancement.(b ) motif analysis around distal poly(a ) sites reveals less pronounced regulatory effects , mostly enhancement guided by uc and ua - rich clusters . motif analysis around tdp-43-regulated same - exon poly(a ) sites up to two of the most significant motif clusters are shown for each search region ( r1 , r2 , r3 ) . ( a ) motif analysis around proximal poly(a ) sites show significant ug - rich clusters in r1 [ 100 .. 40 ] and r2 [ 40 .. 20 ] around repressed proximal poly(a ) sites . ( b ) motif analysis around distal poly(a ) sites reveals less pronounced regulatory effects , mostly enhancement guided by uc and ua - rich clusters . to study whether the binding of tdp-43 alone is sufficient to regulate apa , we produced a minigene reporter with the 3 utr of structural maintenance of chromosomes 1a ( smc1a ) gene , which contains regulated poly(a ) sites from the same - exon class ( figure 5a ) . the 3 utr of smc1a gene was cloned downstream of the firefly luciferase orf in a modified pcdna3 plasmid , which does not contain any additional poly(a ) sites . qrt - pcr was then used to quantify the use of distal poly(a ) sites , which was normalized by the total amount of mrna that was produced . to distinguish the minigene expression from the endogenous smc1a gene , we quantified the total mrna with a forward primer that recognizes part of the luciferase coding sequence . to monitor the distal poly(a ) site use , the forward sequence was designed across the artificial junction that was introduced with production of the shortened smc1a 3 utr . this confirmed the significant increase in the use of the proximal poly(a ) site upon tdp-43 kd ( figure 5b).figure 5smc1a mini - gene(a ) iclip tdp-43 binding along smc1a 3-utr and zoomed in binding upstream of proximal poly(a ) site . the intact smca1 sequence around the proximal polya site is shown in the lower part ( wild - type [ wt ] smca1 ) , which includes the region upstream of the polya signal ( in bold ) that contains a ug - rich region ( in bold ) that crosslinks to tdp-43 ( shown above the sequence with blue bars ) . we introduced mutations ( mut ) or deletion of tg dimers ( del ) to prevent tdp-43 binding to the ug - rich region in the rna . the final minigene was designed with mutations that replaced g in tg dimers into t or a to convert the sequence into a binding site for tia proteins ( tia ) . the mutant nucleotides are marked in red.(b ) ratio in the use of distal vs. proximal polya site in control cells , tdp-43 kd or the double kd of tia1 and tial1 ( tia kd ) . ( a ) iclip tdp-43 binding along smc1a 3-utr and zoomed in binding upstream of proximal poly(a ) site . the intact smca1 sequence around the proximal polya site is shown in the lower part ( wild - type [ wt ] smca1 ) , which includes the region upstream of the polya signal ( in bold ) that contains a ug - rich region ( in bold ) that crosslinks to tdp-43 ( shown above the sequence with blue bars ) . we introduced mutations ( mut ) or deletion of tg dimers ( del ) to prevent tdp-43 binding to the ug - rich region in the rna . the final minigene was designed with mutations that replaced g in tg dimers into t or a to convert the sequence into a binding site for tia proteins ( tia ) . ( b ) ratio in the use of distal vs. proximal polya site in control cells , tdp-43 kd or the double kd of tia1 and tial1 ( tia kd ) . and thus it is theoretically possible that it binds to the longer mrna isoform and stabilizes this isoform , which could explain why the shorter mrna isoform is increased upon tdp-43 kd . to rule out this possibility , we mutated or deleted a tdp-43 binding site that is located upstream of the proximal poly(a ) site ( figure 5a ) . this binding site is present in both short and long isoforms , and it is unlikely that this site could lead to differential stability of the two isoforms . in contrast , the site is positioned upstream of the repressed poly(a ) site , thus representing the pattern where the rna map detects most binding enrichment ( figure 3a ) . tdp-43 binding in this region is ideally positioned to repress the nearby poly(a ) site by blocking the recruitment of cleavage and polyadenylation factors . indeed , disruption and deletion of the tdp-43 binding site in the minigene caused a strong increase in proximal poly(a ) site use under control conditions , which is comparable with the derepression of the site that is seen upon tdp-43 kd in the wild - type minigene ( figure 5b ) . tdp-43 kd caused no further effect on the mutated or deleted minigene , confirming that these mutations abolished the capacity of tdp-43 to regulate the proximal poly(a ) site . another protein , tia1/tial1 , can also bind to u - rich sequences and has been shown to bind to the 3 utr ( wang et al . , 2010 ) . to test whether the position - dependent activity of tdp-43 is shared by other rbps , we therefore replaced the ug - rich motifs with a ua - rich sequence that is designed to promote binding of cytotoxic granule - associated rna binding ( tia ) proteins based on its similarity to the tia - binding sites ( figure 5a ) identified previously by iclip ( wang et al . , 2010 ) . addition of this site restored repression of the proximal poly(a ) site , and kd of tia1/tial1 proteins confirmed that this repression is caused by binding of tia proteins rather than tdp-43 ( figure 5b ) . this demonstrates that diverse rbps can bind in the vicinity of the poly(a ) site to block its use . both tdp-43 and tia proteins are also important regulators of splicing , thus indicating that splicing and 3 end processing could often be regulated by the same proteins . to provide insight into shared mechanisms of splicing and 3 end processing , we used rnamotifs2 to further examine the poly(a ) sites and exons that are differentially used in the brain compared to other tissues . we first examined the poly(a ) sites that are differentially used between brain and universal human reference ( derti et al . , 2012 ) . this detected several types of motifs , including enrichment of tcat immediately downstream of the proximal poly(a ) sites that are repressed in the brain ( figure s4e ) , which agrees with the previous finding that nova ( neuro - oncological ventral antigen ) proteins generally repress poly(a ) sites when binding in close vicinity ( licatalosi et al . , 2008 ) and indicates that nova proteins promote formation of mrna isoforms with longer 3 utrs , which are known to be more common in neurons ( derti et al . , 2012 , miura et al . , 2013 ) . finally , we analyzed motifs at alternative exons that are differentially spliced between brain and heart as defined by splicing microarray ( arrayexpress emtab-1911 ) . as in our previous study , this detected tc - rich motifs and tcat - related motifs , which correspond to the tc - rich and tcat - related preferences of polypyrimidine tract - binding protein ( ptbp ) and nova proteins , respectively ( cereda et al . the positional enrichment agreed with the known rna maps of the two proteins , since ptbp proteins mainly bind upstream of brain - specific exons to repress them , while nova proteins bind upstream and within the exons that are repressed , and downstream of exons that are enhanced in the brain ( ule et al . , 2006 , witten and ule , 2011 ) . by detecting the nova - binding motifs both at the poly(a ) sites and exons that are differentially regulated in the brain , rnamotifs2 confirmed the important role of nova proteins in both splicing and 3 end processing.figure 6motif clusters regulating alternative splicing in the human brainsignificant motif clusters involved in alternative splicing ( comparing brain and heart tissue ) obtained with rnamotifs2 . alternative exons flanked by upstream tc and t - rich clusters ( ptbp targets ) are enhanced in the brain , where ptbp expression is low compared to other tissue . contrary , nova proteins are highly expressed in the brain , and exons flanked by upstream tcat - rich clusters ( nova targets ) are therefore repressed in this tissue . for previous analysis of data , motif clusters regulating alternative splicing in the human brain significant motif clusters involved in alternative splicing ( comparing brain and heart tissue ) obtained with rnamotifs2 . alternative exons flanked by upstream tc and t - rich clusters ( ptbp targets ) are enhanced in the brain , where ptbp expression is low compared to other tissue . contrary , nova proteins are highly expressed in the brain , and exons flanked by upstream tcat - rich clusters ( nova targets ) are therefore repressed in this tissue . for previous analysis of data , our upgraded rnamotifs2 can discover clusters of diverse motifs , and therefore it can provide insights into potential combinatorial regulation of poly(a ) sites or exons . indeed , it showed that ug - rich motifs tend to cluster close to both uc - rich and ucau - rich motifs around the regulated exons ( figure 6 ) and regulated poly(a ) sites ( figure s4e ) in the brain . to further examine this clustering , we plotted the co - occurrence of ug and uc - rich motif clusters at 3 splice sites of silenced alternative exons ( figure s4f ) in the brain . moreover , analysis of the alternative exons regulated by ptb , nova , and tdp-43 proteins discloses that ug and ucau - rich motifs tend to cluster together around the exons regulated by nova proteins ( figure s6 ) . these results indicate that proteins such as tdp-43 might cooperate with tissue - specific rbps to regulate pre - mrna processing ; however , this hypothesis will need further experimental validation . in this study , we developed several tools to examine how cis - acting elements recruit rbps to regulate splicing and apa in a transcriptome - wide manner ( figure s7 ) . we developed expressrna , a platform that processes 3 mrna sequencing data , classifies the sites where cleavage and polyadenylation takes place ( poly(a ) sites ) , and identifies the differentially regulated poly(a ) sites . we also integrated the expressrna platform with an upgraded rnamotifs2 software to identify enrichment of combinatorial motif clusters around regulated exons or poly(a ) sites . we used the motifs and the iclip data to define high - resolution rna maps of apa regulation by tdp-43 . this showed that tdp-43 can directly regulate both proximal and distal poly(a ) sites of same - exon or skipped classes ( figure 7 , table s6 , p value < 0.01 , brg > 10 ) . when binding close to the poly(a ) signal or the poly(a ) site , tdp-43 represses use of the site , whereas it can enhance the site when binding further downstream . similarly , tdp-43 generally represses splicing when binding close to 3 splice site or the exon , while enhancing when binding further downstream of the regulated alternative exon ( tollervey et al . , 2011 ) . this indicates common position - dependent regulatory principles of both mechanisms.figure 7summary of the main position - dependent modes of apa regulation by tdp-43(a ) the binding patterns that are most enriched for the same - exon type of apa , as shown in figures 3a3d and s3b - e.(b ) the binding patterns that are most enriched for the skipped - exon type of apa , as shown in figures s4c and s4d ; the arrow marks the position of the regulated poly(a ) site , and the circle marks the main position of tdp-43 binding . the blue color denotes the repressive , and the red the enhancing effect of tdp-43 , and the positions of the regulatory patterns can be found in the table s6 ( p value < 0.01 , brg > 10 ) . summary of the main position - dependent modes of apa regulation by tdp-43 ( a ) the binding patterns that are most enriched for the same - exon type of apa , as shown in figures 3a3d and s3b - e . ( b ) the binding patterns that are most enriched for the skipped - exon type of apa , as shown in figures s4c and s4d ; the arrow marks the position of the regulated poly(a ) site , and the circle marks the main position of tdp-43 binding . the blue color denotes the repressive , and the red the enhancing effect of tdp-43 , and the positions of the regulatory patterns can be found in the table s6 ( p value < 0.01 , brg > 10 ) . our findings are consistent with previous studies of rna maps for apa regulation by several other rbps ( batra et al . , 2014 , licatalosi et al . , 2008 , liu et al . , 2013 , masuda et al . , 2015 ) . we extend these findings by assessing the regulatory principles at high resolution and in a quantitative manner , which shows the strongest direct role of tdp-43 is in repression of proximal poly(a ) sites , especially by binding in close proximity upstream and downstream of the poly(a ) site . while pas is the primary element recruiting the cleavage and polyadenylation specificity factor ( cpsf ) , other auxiliary sequences tend to be ug or u - rich in mammalian transcripts ( yang and doubli , 2011 ) . these include the tgta motif upstream of the pas that recruits the cleavage factor i(m ) ( cfim ) and the downstream ug - rich motifs that recruit the cleavage stimulation factor ( cstf ) ( hoque et al . , 2013 , martin et al . , 2012 , yao et al . , 2012 ) tdp-43 thus appears to act as a competitor to displace cfi and cstf from the ug - rich sites on pre - mrnas . interestingly , we also find that tdp-43 binding is pronounced further downstream of the enhanced poly(a ) sites , in which cases it might be able to stabilize the binding of processing factors at the nearby poly(a ) site . the rna maps of splicing suggest that tdp-43 might compete with binding of u2af and other splicing factors when binding close to 3 splice sites or recruit u1 small nuclear ribonucleoprotein ( snrnp ) when binding downstream of 5 splice sites . it remains to be seen how tdp-43 manages to block or enhance pre - mrna binding of such a variety of factors . the use of rnamotifs2 confirms that apa is , like alternative splicing , often regulated by rbps that bind clusters of closely spaced short motifs on pre - mrnas . it is clear that the precise position of ug - rich motifs defines the function of tdp-43 in repressing or enhancing either alternative exons or poly(a ) sites ( tollervey et al . , 2011 ) . mutation of the ug to ua - rich motifs replaced the function of tdp-43 with tia proteins , which are also otherwise major regulators of splicing . this indicates that many rbps may have pleiotropic functions in splicing and apa , with the position of their rna - binding site being the primary determinant of their function . our preliminary analyses indicate that an integrative analysis of tissue - specific splicing and 3 end processing could uncover additional rbps that can regulate both processes . for example , we detected the known function of the cis - acting element tcat in the brain - specific splicing and apa . moreover , we find that ug - rich motifs are present within clusters of uc motifs at the tissue - specific exons , indicating a potential for crosstalk between proteins binding these motifs , such as tdp-43 , celf , and ptbp proteins . enrichment of ug - rich motifs was recently also identified next to binding sites of rbfox proteins ( damianov et al . , 2016 ) . since our software can detect clusters of diverse motifs , it is well suited for identification of such sites of overlapping binding motifs for multiple rbps , which might allow cooperative or competitive regulation of alternative splicing or alternative polyadenylation across various tissues and conditions . the platform can examine published poly(a ) sites and exons as well as discover additional ones . we apply state - of - the - art statistical methodology to identify differentially polyadenylated genes ( dexseq ) . in this context , the ability of successfully identifying the regulatory rna maps could also be an estimator ( benchmark ) of the validity and success of the presented approaches . therefore , expressrna provides a flexible ( modular ) data integrative research platform , making computational analysis highly reproducible and allowing user - friendly visualization with sharing of data and results . hek293 flpin t - rex cells were maintained in dmem with 10% fetal bovine serum ( fbs ) , supplemented with 3 g / ml blasticidine and 50 g / ml zeocin . for the small interfering rna ( sirna)-induced tdp-43 kd , 20 nm of tardbp stealth sirna ( invitrogen , a-012394 - 14 , 5-ggcucaagcauggauucua-3 ) was mixed with 10 l of rnaimax following the manufacturer s reverse transfection protocol and added to a 10-cm dish of hek293 flpin cells . for the non - targeting sirna ( stealth rnai sirna negative control med gc content , invitrogen 12935 - 300 ) , 20 nm was also used to distinguish off - target effects from biologically relevant ones . after the first 24 hr of transfection , the medium was replaced with dmem with 10% fbs , and , after an additional 24 hr , the cells were collected for analysis . rna was extracted using the direct - zol rna kit , and an in - column dnase digestion step was performed at room temperature for 15 min . the poly(a)seq libraries for samples that were transfected with either non - targeting sirna or tardbp sirna were generated using the reverse quantseq 3 mrna - seq kit . one fragment per transcript was generated , which resulted in extremely accurate gene expression values . for the initial step of this kit , oligodt priming , including illumina - compatible linker sequences , was carried out . single - end sequencing ( 60 nt ) was performed on a illumina ga-2 with a rapid run flow - cell . this kit makes use of a custom sequencing primer that anneals to a linker sequence previously introduced in the oligo(dt ) priming step for reverse transcription . we developed two independent research platforms , expressrna for computational analysis of post - transcriptional modifications including analysis of 3 end sequence data ( expressrna.apa ) and rnamotifs2 for clustered motif analysis ( see rnamotifs2 ) . the web application part of expressrna ( jquery and javascript ) allows the exploration of combined analysis results over an interactive web interface ( figure 1 ) . expressrna supports several open - source and commercial 3 end sequencing protocols ( lexogen forward / reverse , 3p - seq , pa - seq , polyaseq ) . the platform is modular and scalable and runs on desktop computers for small sequence samples and on multi - core machines for large datasets . it provides a complete analytical framework incorporating several tools for reads alignment , genome annotation , calling of differentially polyadenylated genes , and integration of rna - protein binding ( iclip ) datasets ( figure s1 ) . we established an online server ( http://expressrna.org/paper ) to provide interactive browsing of results presented in this publication . we processed each of the 12 experimental datasets ( table s1 ) by aligning the reads to the reference human genome ( hg19 ) using star aligner ( dobin et al . , 2013 ) with default parameters . tagging only one position per alignment ( the first 5 aligned nucleotide ) , we constructed the database of genome - wide polyadenylation events ( figure 2a ) . since internal priming ( annealing to the genomic sequence instead of the poly(a ) tail ) is a major problem in 3 end sequencing protocols , we checked the genomic sequence in the region [ 10 .. 10 ] surrounding the polyadenylation events and filtered out alignments containing stretches of six consecutive a or with 70% a coverage in any 10-nt sub - window in this region . published studies indicate that auxiliary rna motifs tend to be enriched in the region approximately up to 75 nt upstream ( ure ) and 50 nt downstream ( dre ) of each cleavage site ( beaudoing et al . we wished to focus our study on fully independent cleavage sites that contain their own pas and auxiliary motifs . therefore , we identified the dominant poly(a ) sites based on read count , such that all resulting sites were at least 125 nt apart . for this purpose , we ranked the polyadenylation events by read count in descending order and considered only the high - ranking events that are more than 125 nt apart . it has been shown that cleavage is not an exact process ( pauws et al . , 2001 ) , and we find that cleavage can occur within a small window of positions around the dominant cleavage site ( figure s2b ) . to allow for the variation in cleavage precision ( as seen in figure s2b ) , we computed the per - experiment expression of each poly(a ) site by summing the read counts that identify any position in the region [ 5 .. 5 ] . to identify regulated poly(a ) sites in genes , we input the read counts of all poly(a ) sites remaining after filtering into dexseq . the analogy to alternative exons counts usually input to dexseq are read counts at poly(a ) sites . we input count values for all replicates for control and tdp-43 kd 3 end sequencing experiments . dexseq returns fold change ( log2 ) and adjusted p value for each site . the genes where no poly(a ) site reaches significance ( p < 0.05 ) are classified as controls . in genes with more than one poly(a ) site , only two poly(a ) sites are selected for further analysis . in control genes , the two poly(a ) sites with highest read count two significantly changed sites ( adjusted p value < 0.05 ) with highest difference in fold change are selected for each gene , additionally requiring that fold changes are of opposite signs . if the gene was marked as control , the proximal and distal control poly(a ) sites are further labeled as control - down and control - up ( dependent on their fold change ) . for regulated sites , if a proximal site has fold change < 0 , the site is marked as repressed , and if fold change > 0 , marked as enhanced ( the reverse holds for distal sites ) . gene site pairs are then used for further analyses , including rna maps , rnamotifs2 , and gene ontology ( go ) term analysis . after we select the proximal and distal site for each gene , we classify the pair ( and consequently the gene ) into same exon , composite exon , and skipped exon . for this classification , we use the gene level annotation that is computed by linearizing the ensembl gene annotation by merging the transcript annotation . when no annotated splice site is present between the selected poly(a ) sites , then the gene is assigned to the same - exon category ( figure 2b ) . if there is a splice site present between the selected poly(a ) sites , further classification depends on the type of splice site preceding the proximal poly(a ) site . if there is a 5 splice site immediately upstream of the proximal site , the gene is assigned to the composite - exon category , and , if there is a 3 splice site immediately upstream of the proximal site , the gene is assigned to the skipped - exon category ( figure 2b ) . rbps play a significant role not only in the regulation of alternative splicing but also in the regulation of apa . we designed experiments with intact cells as controls and kd samples as test experiments . after identifying alternatively polyadenylated genes , we defined three sets of genes ( repressed , enhanced , and controls ) and cumulatively plotted the iclip data around poly(a ) sites of these three categories . the position of the polya site ( also referred to as the cleavage site ) is marked with a red line at the center of the rna maps ( figures 3 and s3s5 ) . the approach for visualizing an rna map of apa is analogous to any other region of interest , such as , for instance , the intron - exon boundaries in the context of alternative splicing ( ule et al . , 2006 ) . to display the variability of positional binding enrichment detected by rna maps , we performed 10,000 bootstraps ( sampling with replacement ) of each gene class to obtain the sd ( gray ) . the colored areas ( red , blue ) on rna maps are drawn between the signal ( repressed , enhanced ) and control regions , excluding sds , which conveys a picture of the most reliable enrichment of candidate regulatory binding sites ( figure 3a ) . to determine the number of regulated genes that contain tdp-43 binding within a specific set of positions , we calculated the brg , where the binding sites are defined either by the significant iclip crosslink clusters or rna motif clusters ( table s6 ) . brg corresponds to the number of genes that contain at least one binding site in a specific window within the rna map at a specific class of poly(a ) sites . the brg allows a comparative analysis of rna maps , for example , showing the brg in the entire map ( reported brg400 , since the entire rna map window is 400 nt ) . furthermore , to determine positions on the rna map that have the strongest regulatory significance ( fold change between repressed / enhanced and control genes ) , we performed 1 m bootstraps ( sampling with replacement ) on the entire dataset . we then used a 40-nt sliding window and computed the p value by considering data versus bootstrap fold changes . for same - exon proximal poly(a ) sites , we found that the most significant 40-nt window is between the 97 and 57 nucleotide relative to the repressed proximal poly(a ) sites ( p value = 1e-6 , log2 fold change = 2.04 , brg = 33 ) . the positions of the 40-nt most significant windows with respective brgs and fold changes are available for each type of poly(a ) site ( table s6 ) . we additionally plotted heatmaps of the repressed and enhanced top 20 bound genes , which demonstrate positional contributions of individual sites ( figures 3b , 3c , s3b , and s3c ; table s2 ) . iclip data were processed as described previously by icount web server ( http://icount.biolab.si ) ( wang et al . , 2010 ) . the crosslink clusters were identified considering all crosslink sites that were significant with a false discovery rate ( fdr ) < 0.05 at a maximum spacing of 20 nt between crosslink sites . each binding site position was then defined by the exact position of the bound gu - rich motifs ( gtgtg , tgtgt , tgcgt , tgtgc , cgtgt , atgtg , gtatg , gtgta , gcgtg , gtgcg , tgtga , tgtat , gtgtt , ctgtg , tatgt , ttgtg , tgagt , ggtgt , gagtg , gtgtc , tgtgg , agtgt , gtttg ) that were present in iclip crosslink clusters . rnamotifs2 is an upgraded version of the previously published rnamotifs software ( cereda et al . , 2014 ) . we extended the motif search from alternative splicing regions to three regions surrounding poly(a ) sites and modified the search algorithm with an iterative clustering method . the approach finds the motif with the strongest signal that differentiates up / downregulated sequences to control sequences first . next , it removes sequences where the motif signal is strongest and compensates the loss of signal by clustering the strongest motif with additional motifs , iteratively constructing a cluster consisting of up to four different motifs . the input of rnamotifs2 is a list of genomic positions , either alternative exons or alternative poly(a ) sites . each input needs to be assigned to one of the three classes : control , enhanced , or repressed . after computing cluster motif analysis , the region used for the smc1a minigene is composed of two sequences surrounding both poly(a ) sites . the first part of smc1a minigene contains an 812-bp - long sequence surrounding the proximal poly(a ) site ( positions 53406176 .. 53406987 ) , which is coupled to the 537-nt - long 3 utr sequence close to the distal poly(a ) site ( positions 53400970 .. 53401506 ) . the intervening part of the 3 utr was not incorporated in the minigene reporter due to the limiting size of the plasmid . the minigene ( 0.3 g ) was co - transfected into hela cells together with sirna against tdp-43 or control or tia , and the cells were harvested after 48 hr . reverse transcription was performed using revertaid ( fermentas ) , and qpcr was performed to assess the level of different poly(a ) site usage using sybrgreen according to manufacturer s protocol . the expression of 3 utr of minigene was assessed using forward primer in the luciferase gene and reverse primer in the short 3 utr . the use of distal poly(a ) site was assessed by using forward primer across the junction between proximal poly(a ) site and the downstream sequence surrounding the distal poly(a ) site , and reverse primer around the distal poly(a ) site . ratio between the distal and proximal poly(a ) site use in each condition was normalized to the ratio in the control cells .

summarymany rna - binding proteins ( rbps ) regulate both alternative exons and poly(a ) site selection . to understand their regulatory principles , we developed expressrna , a web platform encompassing computational tools for integration of iclip and rna motif analyses with rna - seq and 3 mrna sequencing . this reveals at nucleotide resolution the rna maps describing how the rna binding positions of rbps relate to their regulatory functions . we use this approach to examine how tdp-43 , an rbp involved in several neurodegenerative diseases , binds around its regulated poly(a ) sites . binding close to the poly(a ) site generally represses , whereas binding further downstream enhances use of the site , which is similar to tdp-43 binding around regulated exons . our rnamotifs2 software also identifies sequence motifs that cluster together with the binding motifs of tdp-43 . we conclude that tdp-43 directly regulates diverse types of pre - mrna processing according to common position - dependent principles .

Introduction Results Discussion Experimental Procedures Author Contributions

both alternative splicing and apa are regulated by rna - binding proteins ( rbps ) ( di giammartino et al . such position - dependent regulatory principles have been visualized at high - resolution in the form of rna maps ( ule et al . the nucleotide resolution that is obtained by the truncated cdnas in iclip , and the quantitative nature gained by the analysis of unique molecular identifiers ( umis ) , allowed us to examine the rna maps of apa regulation in much greater detail . our study examines how the tar ( transactive response ) dna - binding protein 43 ( tdp-43 , also referred to as tardbp ) regulates splicing and apa via position - dependent principles . tdp-43 is an rbp involved in several neurodegenerative diseases , including frontotemporal lobar degeneration ( ftld - tdp ) and amyotrophic lateral sclerosis ( als ) ( ratti and buratti , 2016 ) . tdp-43 regulates alternative splicing in a position - dependent manner by binding to intronic dinucleotide of uridine and guanosine ( ug)-rich motifs , such that binding close to the splice sites represses splicing , whereas binding further downstream of the exon enhances splicing ( tollervey et al . we identified poly(a ) sites regulated by tdp-43 in hek293 cells by 3 mrna sequencing and defined tdp-43 binding sites with individual nucleotide crosslinking and immunoprecipitation ( iclip ) . to perform 3 end data analysis , we developed expressrna , a modular bioinformatics research platform that manages data and inter - connects recently developed ( expressrna.apa module , rnamotifs2 ) and existing ( star , dexseq ) analysis software . the expressrna.apa module identifies the sites where cleavage and polyadenylation takes place ( poly(a ) sites ) , marks and annotates the differentially regulated poly(a ) sites , and visualizes binding patterns of rbps around these sites . the resulting rna maps defined the binding positions where tdp-43 can enhance or repress multiple types of poly(a ) sites , and we validated its direct action with a minigene reporter . , 2014 ) this identified enriched clusters composed of multiple types of motifs around tissue - specific exons or poly(a ) sites , which can indicate a potential for combinatorial regulation of splicing and apa . the 3 ends of mrnas were amplified with the quantseq rev 3 mrna sequencing ( mrna - seq ) library prep kit ( lexogen ) , which uses a poly(t ) primer to reverse transcribe the mrnas . the first step of expressrna is to process and map the 3 mrna - seq data to the genome , classify the sites where cleavage and polyadenylation takes place ( poly(a ) sites ) , and identify the differentially regulated poly(a ) sites . the identified or provided regulated features ( poly(a ) sites , alternative exons ) are then combined with rna protein binding information ( iclip ) . the identified or provided regulated features ( poly(a ) sites , alternative exons ) since the longest 3 utr isoforms are in some cases not fully annotated , we added 5 kb of the intergenic region downstream of each gene . we found that alternative positions of cleavage and polyadenylation can be clustered in close proximity of a dominant poly(a ) site , with most variation occurring within 5 nt of the dominant site , indicating that the cleavage position is not always precise and can vary by a few nucleotides ( figure s2b ) . for quantification of poly(a ) sites , we therefore summed up the counts of reads that identified cleavage up to 5 nt away from each dominant poly(a ) site ( figure s2c ) . quantseq relies on annealing a poly(t ) primer to the poly(a ) tail of mrnas to identify the 3 end of the mrnas . to ensure robust analysis , poly(a ) sites with less than ten read counts in either control or kd experiments we then identified the poly(a ) site in each gene that contained the highest read count , which we consider as the major poly(a ) site . to avoid poorly used sites , we then discarded those poly(a ) sites that have less than 5% of reads compared to the number of reads present at the major poly(a ) site in the same gene . the overlapping sites have the characteristic nucleotide signature of a - rich pas upstream of the cleavage site , which is followed by t - rich sequences , in agreement with past studies ( sheets et al . however , the sites identified in our study have similar nucleotide signature to the ones that overlap with the published sites , indicating that the stringent filtering criteria applied by our study are sufficient to ensure that most of the identified poly(a ) sites are valid ( figure s2 g ) . we further tested the validity of our detected poly(a ) sites by plotting the coverage of rna sequencing ( rna - seq ) experiments from published studies performed in hek293 cells ( pham et al . we find a peak of increased rna - seq average coverage upstream of both types of poly(a ) sites , and a drop to negligible levels of rna - seq coverage downstream of the distal poly(a ) sites ( figure s2j ) . for further comparison , we also plotted heatmaps of rna - seq read coverage around the 50 known and poly(a ) sites uncovered by this analysis that contain most quantseq reads by displaying the quantseq 3 end targeted sequencing coverage ( data from our study ) alongside the rna - seq coverage data ( figures s2k and s2l ) . this shows that the rna - seq read distribution at individual poly(a ) sites is similar for known and sites uncovered by this analysis , since most sites contain enrichment of rna - seq reads upstream , and lack the reads downstream of the poly(a ) sites . to define the regulatory principles with high fidelity , we focused our analyses on 3,291 genes where we can robustly annotate multiple poly(a ) sites . this represents about 33% of detected genes , which is slightly lower compared to most past studies , which reported approximately 40% of protein coding genes with > 1 poly(a ) site ( ni et al . next , we wished to identify those genes with multiple poly(a ) sites where the use of the poly(a ) sites changes upon tdp-43 kd . our approach identified 3,291 genes ( poly(a ) site pairs ) that we then classified based on the position of the two poly(a ) sites in the gene ( figure 2b ) . if the proximal poly(a ) site is part of a composite exon that contains an internal 5 splice site , and the two poly(a ) sites were generated by alternative 5 splice site use , we classified them as a composite exon . to identify changes in apa between control and tdp-43 kd , we examined the changes in relative read counts at the proximal and distal poly(a ) sites in each gene , which is referred to as fold change ( reported by dexseq ) . we report the number of iclip cdnas around each regulated poly(a ) site ( table s2 ) . we also examined the ug - rich binding motifs ( see defining tdp-43 binding positions with iclip ) , which are known to bind tdp-43 . reassuringly , crosslink clusters and enriched ug - rich motifs are abundant at similar positions ( figures 3a and s3a s3d ) , further indicating that these two independent approaches correctly define the binding sites of tdp-43.figure 3rna maps of tdp-43 around proximal and distal same - exon poly(a ) sites with iclip clusters(a ) proximal poly(a ) site rna map of tdp-43 iclip at regulated genes . rna maps of tdp-43 around proximal and distal same - exon poly(a ) sites with iclip clusters ( a ) proximal poly(a ) site rna map of tdp-43 iclip at regulated genes . to further assess the specificity of the rna map , we redrew it by using just the 13,034 poly(a ) sites that overlap with the ones identified also with the 3p - seq method in the previous study ( figure 2d ) . the resulting same - exon proximal rna map is almost identical to the map that used all the sites ( figures 3a and s3e ) , which demonstrates that the map is robust and largely unaffected by the identification of additional poly(a ) sites . moreover , we compared iclip binding of tdp-43 , tial1 , and stau1 around the same - exon proximal poly(a ) sites regulated by tdp-43 ( figure s3f ) . we plotted the enrichment of crosslink clusters for each protein by comparing regulated versus control poly(a ) sites , which demonstrated much stronger and position - specific enrichment for tdp-43 compared to the other control rbps . since tdp-43 is a known regulator of splicing , we also examined whether use of the poly(a ) sites might be indirectly affected due to co - regulation ( binding ) at nearby splice sites . we found position - dependent tdp-43 crosslinking at the regulated composite - exon and skipped - exon poly(a ) sites ( figures s4a s4d ) and much less at splice sites flanking these poly(a ) sites ( figures s5a s5f ; table s6 ) . this indicates that tdp-43 rarely regulates apa indirectly via splicing but rather directly regulates both types of competing poly(a ) sites . to demonstrate in a simple way that tdp-43 binds at different positions to repress or enhance the poly(a ) sites , we defined the 40-nt window around each type of regulated poly(a ) sites that had the strongest enrichment of crosslink clusters compared to controls ( repressed / enhanced versus control genes ) ( table s6 ) . to determine the number of genes that contain a specific class of alternative poly(a ) site that is directly regulated by tdp-43 , we counted the number of iclip crosslink clusters at this 40-nt window , which allowed us to calculate the bound regulated genes score ( brg ) ( see experimental procedures and table s6 ) . this shows that tdp-43 most often binds next to the proximal same - exon poly(a ) sites ( 97 .. 57 relative to proximal poly(a ) site , brg = 33 , p value 1e-6 , figure 3 ) , while enriched binding is also seen further downstream of the enhanced sites ( 72 .. 112 relative to proximal poly(a ) site , brg = 19 , p value 3e-5 , figure 3 ) . to understand whether the rna map of apa regulation by tdp-43 can be discovered without any knowledge of its binding specificity so that it could assess regulated poly(a ) sites , in addition to alternative exons . to study whether the binding of tdp-43 alone is sufficient to regulate apa , we produced a minigene reporter with the 3 utr of structural maintenance of chromosomes 1a ( smc1a ) gene , which contains regulated poly(a ) sites from the same - exon class ( figure 5a ) . this confirmed the significant increase in the use of the proximal poly(a ) site upon tdp-43 kd ( figure 5b).figure 5smc1a mini - gene(a ) iclip tdp-43 binding along smc1a 3-utr and zoomed in binding upstream of proximal poly(a ) site . to rule out this possibility , we mutated or deleted a tdp-43 binding site that is located upstream of the proximal poly(a ) site ( figure 5a ) . in contrast , the site is positioned upstream of the repressed poly(a ) site , thus representing the pattern where the rna map detects most binding enrichment ( figure 3a ) . indeed , disruption and deletion of the tdp-43 binding site in the minigene caused a strong increase in proximal poly(a ) site use under control conditions , which is comparable with the derepression of the site that is seen upon tdp-43 kd in the wild - type minigene ( figure 5b ) . to test whether the position - dependent activity of tdp-43 is shared by other rbps , we therefore replaced the ug - rich motifs with a ua - rich sequence that is designed to promote binding of cytotoxic granule - associated rna binding ( tia ) proteins based on its similarity to the tia - binding sites ( figure 5a ) identified previously by iclip ( wang et al . this demonstrates that diverse rbps can bind in the vicinity of the poly(a ) site to block its use . to provide insight into shared mechanisms of splicing and 3 end processing , we used rnamotifs2 to further examine the poly(a ) sites and exons that are differentially used in the brain compared to other tissues . this detected several types of motifs , including enrichment of tcat immediately downstream of the proximal poly(a ) sites that are repressed in the brain ( figure s4e ) , which agrees with the previous finding that nova ( neuro - oncological ventral antigen ) proteins generally repress poly(a ) sites when binding in close vicinity ( licatalosi et al . by detecting the nova - binding motifs both at the poly(a ) sites and exons that are differentially regulated in the brain , rnamotifs2 confirmed the important role of nova proteins in both splicing and 3 end processing.figure 6motif clusters regulating alternative splicing in the human brainsignificant motif clusters involved in alternative splicing ( comparing brain and heart tissue ) obtained with rnamotifs2 . indeed , it showed that ug - rich motifs tend to cluster close to both uc - rich and ucau - rich motifs around the regulated exons ( figure 6 ) and regulated poly(a ) sites ( figure s4e ) in the brain . in this study , we developed several tools to examine how cis - acting elements recruit rbps to regulate splicing and apa in a transcriptome - wide manner ( figure s7 ) . we developed expressrna , a platform that processes 3 mrna sequencing data , classifies the sites where cleavage and polyadenylation takes place ( poly(a ) sites ) , and identifies the differentially regulated poly(a ) sites . we also integrated the expressrna platform with an upgraded rnamotifs2 software to identify enrichment of combinatorial motif clusters around regulated exons or poly(a ) sites . when binding close to the poly(a ) signal or the poly(a ) site , tdp-43 represses use of the site , whereas it can enhance the site when binding further downstream . similarly , tdp-43 generally represses splicing when binding close to 3 splice site or the exon , while enhancing when binding further downstream of the regulated alternative exon ( tollervey et al . this indicates common position - dependent regulatory principles of both mechanisms.figure 7summary of the main position - dependent modes of apa regulation by tdp-43(a ) the binding patterns that are most enriched for the same - exon type of apa , as shown in figures 3a3d and s3b - e.(b ) the binding patterns that are most enriched for the skipped - exon type of apa , as shown in figures s4c and s4d ; the arrow marks the position of the regulated poly(a ) site , and the circle marks the main position of tdp-43 binding . the blue color denotes the repressive , and the red the enhancing effect of tdp-43 , and the positions of the regulatory patterns can be found in the table s6 ( p value < 0.01 , brg > 10 ) . summary of the main position - dependent modes of apa regulation by tdp-43 ( a ) the binding patterns that are most enriched for the same - exon type of apa , as shown in figures 3a3d and s3b - e . ( b ) the binding patterns that are most enriched for the skipped - exon type of apa , as shown in figures s4c and s4d ; the arrow marks the position of the regulated poly(a ) site , and the circle marks the main position of tdp-43 binding . we extend these findings by assessing the regulatory principles at high resolution and in a quantitative manner , which shows the strongest direct role of tdp-43 is in repression of proximal poly(a ) sites , especially by binding in close proximity upstream and downstream of the poly(a ) site . interestingly , we also find that tdp-43 binding is pronounced further downstream of the enhanced poly(a ) sites , in which cases it might be able to stabilize the binding of processing factors at the nearby poly(a ) site . the rna maps of splicing suggest that tdp-43 might compete with binding of u2af and other splicing factors when binding close to 3 splice sites or recruit u1 small nuclear ribonucleoprotein ( snrnp ) when binding downstream of 5 splice sites . it is clear that the precise position of ug - rich motifs defines the function of tdp-43 in repressing or enhancing either alternative exons or poly(a ) sites ( tollervey et al . since internal priming ( annealing to the genomic sequence instead of the poly(a ) tail ) is a major problem in 3 end sequencing protocols , we checked the genomic sequence in the region [ 10 .. 10 ] surrounding the polyadenylation events and filtered out alignments containing stretches of six consecutive a or with 70% a coverage in any 10-nt sub - window in this region . to identify regulated poly(a ) sites in genes , we input the read counts of all poly(a ) sites remaining after filtering into dexseq . in genes with more than one poly(a ) site , only two poly(a ) sites are selected for further analysis . to determine the number of regulated genes that contain tdp-43 binding within a specific set of positions , we calculated the brg , where the binding sites are defined either by the significant iclip crosslink clusters or rna motif clusters ( table s6 ) . for same - exon proximal poly(a ) sites , we found that the most significant 40-nt window is between the 97 and 57 nucleotide relative to the repressed proximal poly(a ) sites ( p value = 1e-6 , log2 fold change = 2.04 , brg = 33 ) . the positions of the 40-nt most significant windows with respective brgs and fold changes are available for each type of poly(a ) site ( table s6 ) . the first part of smc1a minigene contains an 812-bp - long sequence surrounding the proximal poly(a ) site ( positions 53406176 .. 53406987 ) , which is coupled to the 537-nt - long 3 utr sequence close to the distal poly(a ) site ( positions 53400970 .. 53401506 ) .

tuberculosis ( tb ) is one of the most common communicable diseases world and continues to be a major global health problem . it causes disease among millions of people each year , and , after hiv , it ranks as the second leading cause of death from an infectious disease worldwide . in the last 30 years , migration and emigration , negligence of tb control programs , and above all , the emergence of the hiv / aids epidemic , have produced an increase of tb rates in several countries . although the principles of tb management are well defined , this infectious disease is often not properly diagnosed and treated . insufficient knowledge among doctors about tb is one of the reasons for this failure . despite the abundance of information on all content areas of tb , there is the need for accurate undergraduate training in tb and of a comprehensive educational strategy is essential to provide medical students with the appropriate knowledge , skills , and attitudes necessary for the effective management of tb to promote effective prevention , early diagnosis , and successful treatment . in the usa , the national institutes of health funded the national tuberculosis curriculum consortium ( ntcc ) to meet this need . the goal of the ntcc is to improve skills and instill appropriate attitudes in the management and control of tb among medical students in their formative years , and to establish a foundation by which complex issues relating to tb can be continually revisited throughout the span of their careers . compared knowledge and practices regarding tb among final - year medical students at schools in endemic and non - endemic areas ( canada , india , and uganda ) , revealing significant differences in undergraduate knowledge and practice competency in regards to tb at 3 medical schools . kilicaslan et al . conducted a study at the istanbul medical school to assess whether tb - related questions asked during chest medicine examinations complied with the world health organization s ( who ) learning objectives for tb training and to investigate students skills in interpreting tb radiology and smears . the study showed that the examination questions did not adequately reflect who learning objectives , and that the students skills suggested that their practical training on tb was insufficient . performed a self - administered survey among students in ntcc schools to establish a baseline level of knowledge , attitudes , and confidence about tb , and they concluded that there was room for improvement in these 3 areas . in brazil , teixeira et al . performed a cross - sectional survey of undergraduate medical students in preclinical , early clinical and late clinical years about previous lectures on tb , knowledge about tb transmission , exposure to patients with active pulmonary tb , and the use of protective respiratory masks . they concluded that many medical students were not aware of the main routes of tb infection and that lectures on tb were not sufficient to change knowledge and practices . regardless of knowledge about tb transmission , students engaged in risky behaviors : more than two - thirds did not use a protective mask when examining an active tb case . little is known about the effectiveness of training programs for the treatment of tb in europe . with this background , we performed a survey to assess knowledge , attitudes , and competencies about tb among fifth - year medical students at the school of medicine of the catholic university in rome , italy . the aim of this study was 2-fold : 1 ) to determine knowledge , experiences , and attitudes of medical students regarding tb in a university teaching hospital and 2 ) to establish which individual factors are associated with a greater knowledge about tb . a cross - sectional survey was conducted at the school of medicine of the catholic university in rome among fifth - year medical students . this group of students was selected because the course on infectious diseases takes place in the fourth year . the survey was carried out with a self - administered questionnaire designed on the basis of ntcc contents . the questionnaire consisted of 39 multiple - choice questions divided by area : attitudes and experiences ( 7 questions ) , knowledge about epidemiology and prevention ( 13 questions ) , diagnosis ( 14 questions ) , and treatment ( 5 questions ) . each question about knowledge had 5 possible answers , of which only 1 was correct . to test the questionnaire , a pilot study was conducted from april to june 2012 among second- and third - year midwifery students and first- , second- , and third - year medical residents in hygiene , occupational medicine , and forensic medicine . thirty - seven students and residents were enrolled in this pilot phase and all completed the questionnaire . the survey was conducted from july to october 2012 on fifth - year medical students . to increase the number of respondents , students were enrolled before being examined in public health . students were introduced to the aims , objectives , and the methodology of the study , and they were invited to participate on a voluntary basis . after obtaining informed verbal consent , the students were asked to complete the questionnaire anonymously . an overall score was computed for all questions related to knowledge ( total score ) . separate scores were calculated for questions related to epidemiology and prevention , diagnosis , and treatment . mean percentage scores were computed for different subgroups on the basis of demographic variables , experiences , and attitudes . multivariable linear regression analysis was performed to estimate the effect on tb knowledge of each variable , adjusting for the effect of the other variables in the model . multivariable regression models were built for epidemiology and prevention , diagnosis , and treatment , respectively . results of these models are shown using coefficients with 95% confidence intervals ( 95% ci ) , which describe the expected change in the percentage of correct answers associated with each variable . a cross - sectional survey was conducted at the school of medicine of the catholic university in rome among fifth - year medical students . this group of students was selected because the course on infectious diseases takes place in the fourth year . the survey was carried out with a self - administered questionnaire designed on the basis of ntcc contents . the questionnaire consisted of 39 multiple - choice questions divided by area : attitudes and experiences ( 7 questions ) , knowledge about epidemiology and prevention ( 13 questions ) , diagnosis ( 14 questions ) , and treatment ( 5 questions ) . each question about knowledge had 5 possible answers , of which only 1 was correct . to test the questionnaire , a pilot study was conducted from april to june 2012 among second- and third - year midwifery students and first- , second- , and third - year medical residents in hygiene , occupational medicine , and forensic medicine . thirty - seven students and residents were enrolled in this pilot phase and all completed the questionnaire . the survey was conducted from july to october 2012 on fifth - year medical students . to increase the number of respondents , students were enrolled before being examined in public health . students were introduced to the aims , objectives , and the methodology of the study , and they were invited to participate on a voluntary basis . after obtaining informed verbal consent , the students were asked to complete the questionnaire anonymously . an overall score was computed for all questions related to knowledge ( total score ) . separate scores were calculated for questions related to epidemiology and prevention , diagnosis , and treatment . mean percentage scores were computed for different subgroups on the basis of demographic variables , experiences , and attitudes . multivariable linear regression analysis was performed to estimate the effect on tb knowledge of each variable , adjusting for the effect of the other variables in the model . multivariable regression models were built for epidemiology and prevention , diagnosis , and treatment , respectively . results of these models are shown using coefficients with 95% confidence intervals ( 95% ci ) , which describe the expected change in the percentage of correct answers associated with each variable . among 220 fifth - year medical students enrolled in the university , 186 students were present at the time of data collection . of these , 183 ( 83.1% of fifth - year students ) completed the questionnaire . the mean age was 24 years ( sd 0.12 , range 2230 ) . about half were female ( 52% , n=95 ) , and more than half ( 58.5% , n=107 ) reported attending a clinical unit . about one - fifth ( 21.9% , n=40 ) had performed at least 1 mantoux test . two - thirds of the students ( 66.1% , n=121 ) observed at least 1 tb case during their medical education and 83.1% ( n=152 ) had observed at least 1 tb patient x - ray . only two - thirds ( 66.7% , n=122 ) were aware of being at risk for tb infection . among the 32 multiple - choice questions designed to evaluate student knowledge of tb , the mean percentage of correct answers was the mean percentage of correct answers was 63.5% ( sd 16.3% ) for epidemiology and prevention , 54.1% ( sd 12.4% ) for diagnosis , and 45.7% ( sd 20.4% ) for treatment . figure 1 shows the distribution of the mean percentage of correct answers by area of knowledge . in the epidemiology and prevention area ( figure 2 ) , the highest and the lowest mean scores were reported for the following questions , respectively : the etiologic agent of tb is transmitted by _ _ _ _ ( 98.9% correct answers ) ; and currently the prevalence of tb is highest in the following areas of the world : _ _ _ _ ( 8.2% correct answers ) . in the diagnosis area ( figure 3 ) , the questions with the highest and the lowest mean scores , respectively , were : which organ could be affected by m. tuberculosis ( 95.6% ) ; and the tine test is _ _ _ _ ( 10.4% ) . in the treatment area ( figure 4 ) , the questions with the highest and the lowest mean scores , respectively , were : which of these antibiotics are not useful for tb treatment : _ _ _ _ ( 82.0% ) ; and prophylactic treatment with isoniazid is implemented to _ _ _ _ ( 13.1% ) . table 2 shows that younger students ( age < 24 ) had on average greater tb knowledge ( 58.1% of correct answers vs. 54.7% , p=0.05 ) , whereas no association was found between gender and the mean percentage of correct answers . there was a significant association between internship in units and departments and greater knowledge about tb diagnosis ( 55.9% vs. 51.6% , p=0.02 ) , treatment ( 48.4% vs. 41.8% , p=0.03 ) , and total score ( 58.1% vs. 54.5% , p=0.04 ) . students who reported receiving the mantoux test had greater knowledge about tb epidemiology and prevention ( 65.4% vs. 53.3% , p=0.001 ) , diagnosis ( 55.2% vs. 48.3% , p=0.005 ) , and total score ( 58.0% vs. 49.1% , p=0.001 ) . students who reported observing at least 1 active pulmonary tb case had a higher percentage of correct answers about diagnosis ( 55.5% vs. 51.4% , p=0.03 ) and total score ( 57.9% vs. 54.0% , p=0.03 ) . students who reported observing at least 1 x - ray of a tb patient had a higher percentage of correct answers about epidemiology and prevention ( 64.7% vs. 57.3% , p=0.02 ) , diagnosis ( 55.1% vs. 49.5% , p=0.02 ) , and total score ( 57.7% vs. 51.2% , p=0.004 ) . students who considered themselves to be at risk for tb reported a similar percentage of correct answers as other students . the multivariable linear regression analysis ( table 3 ) confirmed the association between receiving the mantoux test and greater knowledge about tb epidemiology and prevention ( coefficient=10.8 ; 95% ci 4.217.3 ) , diagnosis ( coefficient=5.0 ; 95% ci 0.110.0 ) , and total score ( coefficient=7.2 ; 95% ci 2.611.7 ) . in addition , having observed at least 1 x - ray of a tb patient was associated with a higher total score ( coefficient=5.3 ; 95% ci 1.09.7 ) . the analysis did not reveal any association between greater knowledge and gender , age , and feeling at risk . among the 32 multiple - choice questions designed to evaluate student knowledge of tb , the mean percentage of correct answers was 56.6% ( sd 11.6% ) . the mean percentage of correct answers was 63.5% ( sd 16.3% ) for epidemiology and prevention , 54.1% ( sd 12.4% ) for diagnosis , and 45.7% ( sd 20.4% ) for treatment . figure 1 shows the distribution of the mean percentage of correct answers by area of knowledge . in the epidemiology and prevention area ( figure 2 ) , the highest and the lowest mean scores were reported for the following questions , respectively : the etiologic agent of tb is transmitted by _ _ _ _ ( 98.9% correct answers ) ; and currently the prevalence of tb is highest in the following areas of the world : _ _ _ _ ( 8.2% correct answers ) . in the diagnosis area ( figure 3 ) , the questions with the highest and the lowest mean scores , respectively , were : which organ could be affected by m. tuberculosis ( 95.6% ) ; and the tine test is _ _ _ _ ( 10.4% ) . in the treatment area ( figure 4 ) , the questions with the highest and the lowest mean scores , respectively , were : which of these antibiotics are not useful for tb treatment : _ _ _ _ ( 82.0% ) ; and prophylactic treatment with isoniazid is implemented to _ _ _ _ ( 13.1% ) . table 2 shows that younger students ( age < 24 ) had on average greater tb knowledge ( 58.1% of correct answers vs. 54.7% , p=0.05 ) , whereas no association was found between gender and the mean percentage of correct answers . there was a significant association between internship in units and departments and greater knowledge about tb diagnosis ( 55.9% vs. 51.6% , p=0.02 ) , treatment ( 48.4% vs. 41.8% , p=0.03 ) , and total score ( 58.1% vs. 54.5% , p=0.04 ) . students who reported receiving the mantoux test had greater knowledge about tb epidemiology and prevention ( 65.4% vs. 53.3% , p=0.001 ) , diagnosis ( 55.2% vs. 48.3% , p=0.005 ) , and total score ( 58.0% vs. 49.1% , p=0.001 ) . students who reported observing at least 1 active pulmonary tb case had a higher percentage of correct answers about diagnosis ( 55.5% vs. 51.4% , p=0.03 ) and total score ( 57.9% vs. 54.0% , p=0.03 ) . students who reported observing at least 1 x - ray of a tb patient had a higher percentage of correct answers about epidemiology and prevention ( 64.7% vs. 57.3% , p=0.02 ) , diagnosis ( 55.1% vs. 49.5% , p=0.02 ) , and total score ( 57.7% vs. 51.2% , p=0.004 ) . students who considered themselves to be at risk for tb reported a similar percentage of correct answers as other students . the multivariable linear regression analysis ( table 3 ) confirmed the association between receiving the mantoux test and greater knowledge about tb epidemiology and prevention ( coefficient=10.8 ; 95% ci 4.217.3 ) , diagnosis ( coefficient=5.0 ; 95% ci 0.110.0 ) , and total score ( coefficient=7.2 ; 95% ci 2.611.7 ) . in addition , having observed at least 1 x - ray of a tb patient was associated with a higher total score ( coefficient=5.3 ; 95% ci 1.09.7 ) . the analysis did not reveal any association between greater knowledge and gender , age , and feeling at risk . this study showed that more than half of the questions about knowledge of tb treatment were wrong . this is in line with other surveys carried out among medical students [ 79 ] . one - sixth of the students did not remember receiving the mantoux test , which was compulsory for the investigated medical students . furthermore , medical students who remembered receiving the mantoux test demonstrated a better grasp of knowledge about tb . specifically , we found an association between memory receiving the mantoux test and greater knowledge of epidemiology and prevention , diagnosis , and the total score , which strengthens the role of the memory effect . the relationship between recall of preventive medicine activities and greater knowledge about tb is consistent with previous studies that reported an association between hpv and influenza vaccination and a greater knowledge of those diseases [ 1012 ] . we also found evidence of a significant association between greater clinical experience , indicated by those who had seen cases and a chest x - ray of a tb patient and/or those who had attended an internship in clinical wards and had greater knowledge of tb diagnosis and treatment . this may be related to the time devoted to personal insights about tb and the number of patients with tb seen by medical students . in recent years , the role of problem - based learning in medical education was highlighted by empirical studies that found strong evidence that this method had positive effects on student knowledge , stressing the benefits of problem - based learning integrated within a traditional curriculum . appropriate practice and attitudes in the treatment of tb are among the main goals that can be achieved through the adoption of competency - based programs that are responsive to rapidly changing needs , as well as promoting a new instrument of learning that includes the capacity to integrate knowledge and good practices . studies like ours , which are designed to evaluate the training of medical students and to identify inadequacies in their education , are needed to implement changes because failure to address these issues can affect both patient and public health . according to the who , medical professionals should know about national and international expansion of the tb burden , national tb prevention policies such as control strategies , and the bcg vaccination recommendations . knowledge of these topics was investigated in the present study and a poor level of knowledge was reported . in recent years , the centers for disease control and prevention and ntcc have proposed curricula offering the basics of training undergraduate students and doctors about the fundamentals of tb prevention , diagnosis , and treatment . our survey results highlight the need to develop educational tools using active learning strategies to improve knowledge and ensure social accountability of medical schools . the questionnaire was administered prior to the public health course examination ; consequently , some medical students might have answered haphazardly because they were more concerned about the upcoming examination . however , this timing of data collection allowed us to obtain a very large sample and high compliance . on the other hand , the preparation for the public health exam may have increased the students general level of knowledge about tb . we investigated not only comprehensive knowledge , but also experience and attitudes about tb among students and related these to knowledge . the cross - sectional design of this survey does not allow the evaluation of causal relationships , but it does make associations between several factors that represent , in any case , the basis for testing causal hypothesis in further studies . our survey represents a good starting point for future longitudinal studies aimed at assessing the impact of changes in curricula on student knowledge . our findings show a moderate knowledge about tb among fifth - year medical students . internship was found to be strongly associated with a greater knowledge of tb diagnosis , epidemiology , and prevention . we provide a clear snapshot of the differences in tb learning between the more sensitized and experienced students who apply what they have learned , and others who are more dedicated to theoretical learning . if you are a student of medicine , in which department you are ? _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ have you ever attended courses in which tb was discussed ? i received mantoux test : i performed at least one mantoux test : i observed at least one tb case : i observed at least one x - ray of tb patient : i think i am at risk for tb : if you are a student of medicine , in which department you are ? _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ i received mantoux test : i performed at least one mantoux test : i observed at least one tb case : i observed at least one x - ray of tb patient : i think i am at risk for tb : currently the prevalence of tb is highest in the following areas of the world : central and south america which of these microorganisms is strongly related with the tb bacillus ? the current tb vaccine consists of : live and attenuated bacteria it is a recombinant / purified protein vaccine what is the name of tb vaccine ? is it necessary to isolate an open pulmonary tb patient ? yes , using a positive pressure room yes , using a negative pressure room yes , only if the patient coughs the prevalence of tb in italy is currently : 0.60.7 cases per 100,000 people 67 cases per 100,000 people 6070 cases per 100,000 people 600700 cases per 100,000 people the risk of developing active tb in a lifetime : exceeds 20% in patients with latent tb infection and hiv infection is not related to tb latent infection is not related to hiv infection is not related to the exposure and the recent acquisition of infection which action strategy is not useful in controlling the spread of tb ? tb vaccination in high prevalence populations identification , isolation and treatment of active disease cases diagnosis and treatment of latent tb infection tb vaccination in low prevalence populations which groups are considered at risk of tb ? immigrants from high prevalence countries elderly , children , immunosuppressed patients all the above answers the most frequent aetiologic agent of tb in immunosuppressed patients is : mycobacterium tuberculosis 2 . mycobacterium avium complex the aetiologic agent of tb is transmitted by : higher incidence and delay in diagnosis of tb in disadvantaged populations can be related to : socio - economic issues low resources of native country all the above answers which ppes ( personal protective equipment ) are needed in a room where an open tb patient stays : all the above answers 14 ) the immune response to tb is : caused by circulating immune complexes a multiple puncture test an intradermal injection an intramuscular injection an intradermal injection an intradermal response 17 ) which one is not a limit of tuberculin skin test : it is a test in vivo that requires a return visit it is an operator - dependent test it shows a number of false positive results due to cross - reactivity with other antigenic components it is an expression of immune response to mycobacterial antigens 18 ) which of the following about quantiferon tb gold test is false : it is a test performed in vitro on a sample of peripheral venous blood it measures the production of ifn by antigen - specific for mtb lymphocytes t using an elisa methodology in 2001 this test has been approved by the fda as a support in the diagnosis of latent tuberculosis infection it does not differentiate infection from other reasons for testing positive 19 ) mantoux test is positive from an induration diameter of : 20 ) positive intradermal reaction test indicates : diagnosis of tb infection increased sensitization to m. tuberculosis increased sensitization to drugs 21 ) in case of a suspected pulmonary tb it is necessary to collect the following biological specimen : 22 ) first contact with bacteria causes : change of the antibody titer 23 ) in case of positive tuberculin skin test it is recommended : 24 ) which organ could be affected by m. tuberculosis ? all the above answers 25 ) koch bacillus identification is always performed through : hematoxylin and eosin stain 26 ) is it recommended mantoux test to patients and health operators which come in contact with a tb patient ? no , they should be immediately vaccinated yes , if they are not vaccinated 27 ) how long after the primary infection does tuberculin test result positive ? 28 ) which of these antibiotics are not useful for tb treatment ? 29 ) consider an active tb woman in homecare therapy who has recently discovered to be pregnant . tuberculosis does not increase the risk of miscarriage you reassure her because there s not risk for the child you recommend her to change treatment because there is a potential risk of toxicity for the child 30 ) how long is a tb patient supposed to be hospitalized for an effective treatment ? 31 ) prophylactic treatment with isoniazid is implemented to : all health care workers of the ward close contacts resulted negative to the test unvaccinated contacts 32 ) in case of positive mantoux test in an exposed health operator , should he / she be assessed for further investigations and for prophylactic treatment with isoniazid ? no , he / she should not it s not necessary , he / she should be immediately vaccinated yes , only if he / she has specific symptoms

backgroundtuberculosis is the second leading cause of death from infectious disease . insufficient knowledge among doctors about tuberculosis is one of the reasons for the increased tuberculosis rates in several low - endemic countries . the purpose of this study was to assess knowledge , experience , and attitude about tuberculosis among medical students.material/methodsafter a pilot study , a cross - sectional survey was performed on fifth - year medical students at the catholic university of rome ( italy ) , using a self - administered questionnaire on attitude , experience and knowledge about epidemiology , diagnosis , and treatment of tuberculosis . the t test and multivariable linear regression analysis were performed to estimate the association between tb knowledge and investigated variables.resultsamong 220 fifth - year medical students , the response rate was 83.1% . the mean percentage of correct answers was 56.6% ( 63.5% for epidemiology and prevention , 54.1% for diagnosis , and 45.7% for treatment ) . associations between internships in wards and greater knowledge of tuberculosis diagnosis ( 55.9% vs. 51.6% , p=0.02 ) , treatment ( 48.4% vs. 41.8% , p=0.03 ) and total score ( 58.1% vs. 54.5% , p=0.04 ) were found.students who reported receiving the mantoux test had higher knowledge of tuberculosis epidemiology and prevention ( 65.4% vs. 53.3% , p=0.001 ) , diagnosis ( 55.2% vs. 48.3% , p=0.005 ) , and total score ( 58.0% vs. 49.1% , p=0.001 ) . students who had observed at least 1 active pulmonary tuberculosis case had a higher percentage of correct answers about diagnosis ( 55.5% vs. 51.4% , p=0.03 ) and total score ( 57.9% vs. 54.0% , p=0.03).the multivariable linear regression confirmed the association between higher knowledge and receiving the mantoux test ( coefficient=7.2 ; 95% ci 2.611.7 ) , as well as having observed at least 1 x - ray of a tb patient ( coefficient=5.3 ; 95% ci 1.09.7).conclusionsa moderate level of general knowledge about tuberculosis was found , which suggests the need to modify current programs of infectious diseases in the curriculum of medical schools .

Background Material and Methods Study design Statistical analyses Results Knowledge Factors associated with knowledge Discussion Conclusions Questionnaire General information Experiences and attitudes Knowledge Epidemiology and prevention Diagnosis Treatment

tuberculosis ( tb ) is one of the most common communicable diseases world and continues to be a major global health problem . it causes disease among millions of people each year , and , after hiv , it ranks as the second leading cause of death from an infectious disease worldwide . in the last 30 years , migration and emigration , negligence of tb control programs , and above all , the emergence of the hiv / aids epidemic , have produced an increase of tb rates in several countries . insufficient knowledge among doctors about tb is one of the reasons for this failure . despite the abundance of information on all content areas of tb , there is the need for accurate undergraduate training in tb and of a comprehensive educational strategy is essential to provide medical students with the appropriate knowledge , skills , and attitudes necessary for the effective management of tb to promote effective prevention , early diagnosis , and successful treatment . the goal of the ntcc is to improve skills and instill appropriate attitudes in the management and control of tb among medical students in their formative years , and to establish a foundation by which complex issues relating to tb can be continually revisited throughout the span of their careers . compared knowledge and practices regarding tb among final - year medical students at schools in endemic and non - endemic areas ( canada , india , and uganda ) , revealing significant differences in undergraduate knowledge and practice competency in regards to tb at 3 medical schools . performed a self - administered survey among students in ntcc schools to establish a baseline level of knowledge , attitudes , and confidence about tb , and they concluded that there was room for improvement in these 3 areas . performed a cross - sectional survey of undergraduate medical students in preclinical , early clinical and late clinical years about previous lectures on tb , knowledge about tb transmission , exposure to patients with active pulmonary tb , and the use of protective respiratory masks . they concluded that many medical students were not aware of the main routes of tb infection and that lectures on tb were not sufficient to change knowledge and practices . with this background , we performed a survey to assess knowledge , attitudes , and competencies about tb among fifth - year medical students at the school of medicine of the catholic university in rome , italy . the aim of this study was 2-fold : 1 ) to determine knowledge , experiences , and attitudes of medical students regarding tb in a university teaching hospital and 2 ) to establish which individual factors are associated with a greater knowledge about tb . a cross - sectional survey was conducted at the school of medicine of the catholic university in rome among fifth - year medical students . the survey was carried out with a self - administered questionnaire designed on the basis of ntcc contents . the questionnaire consisted of 39 multiple - choice questions divided by area : attitudes and experiences ( 7 questions ) , knowledge about epidemiology and prevention ( 13 questions ) , diagnosis ( 14 questions ) , and treatment ( 5 questions ) . to test the questionnaire , a pilot study was conducted from april to june 2012 among second- and third - year midwifery students and first- , second- , and third - year medical residents in hygiene , occupational medicine , and forensic medicine . the survey was conducted from july to october 2012 on fifth - year medical students . students were introduced to the aims , objectives , and the methodology of the study , and they were invited to participate on a voluntary basis . separate scores were calculated for questions related to epidemiology and prevention , diagnosis , and treatment . multivariable linear regression analysis was performed to estimate the effect on tb knowledge of each variable , adjusting for the effect of the other variables in the model . multivariable regression models were built for epidemiology and prevention , diagnosis , and treatment , respectively . results of these models are shown using coefficients with 95% confidence intervals ( 95% ci ) , which describe the expected change in the percentage of correct answers associated with each variable . a cross - sectional survey was conducted at the school of medicine of the catholic university in rome among fifth - year medical students . the survey was carried out with a self - administered questionnaire designed on the basis of ntcc contents . the questionnaire consisted of 39 multiple - choice questions divided by area : attitudes and experiences ( 7 questions ) , knowledge about epidemiology and prevention ( 13 questions ) , diagnosis ( 14 questions ) , and treatment ( 5 questions ) . to test the questionnaire , a pilot study was conducted from april to june 2012 among second- and third - year midwifery students and first- , second- , and third - year medical residents in hygiene , occupational medicine , and forensic medicine . the survey was conducted from july to october 2012 on fifth - year medical students . students were introduced to the aims , objectives , and the methodology of the study , and they were invited to participate on a voluntary basis . separate scores were calculated for questions related to epidemiology and prevention , diagnosis , and treatment . multivariable linear regression analysis was performed to estimate the effect on tb knowledge of each variable , adjusting for the effect of the other variables in the model . multivariable regression models were built for epidemiology and prevention , diagnosis , and treatment , respectively . results of these models are shown using coefficients with 95% confidence intervals ( 95% ci ) , which describe the expected change in the percentage of correct answers associated with each variable . among 220 fifth - year medical students enrolled in the university , 186 students were present at the time of data collection . of these , 183 ( 83.1% of fifth - year students ) completed the questionnaire . about half were female ( 52% , n=95 ) , and more than half ( 58.5% , n=107 ) reported attending a clinical unit . about one - fifth ( 21.9% , n=40 ) had performed at least 1 mantoux test . two - thirds of the students ( 66.1% , n=121 ) observed at least 1 tb case during their medical education and 83.1% ( n=152 ) had observed at least 1 tb patient x - ray . among the 32 multiple - choice questions designed to evaluate student knowledge of tb , the mean percentage of correct answers was the mean percentage of correct answers was 63.5% ( sd 16.3% ) for epidemiology and prevention , 54.1% ( sd 12.4% ) for diagnosis , and 45.7% ( sd 20.4% ) for treatment . figure 1 shows the distribution of the mean percentage of correct answers by area of knowledge . in the epidemiology and prevention area ( figure 2 ) , the highest and the lowest mean scores were reported for the following questions , respectively : the etiologic agent of tb is transmitted by _ _ _ _ ( 98.9% correct answers ) ; and currently the prevalence of tb is highest in the following areas of the world : _ _ _ _ ( 8.2% correct answers ) . in the diagnosis area ( figure 3 ) , the questions with the highest and the lowest mean scores , respectively , were : which organ could be affected by m. tuberculosis ( 95.6% ) ; and the tine test is _ _ _ _ ( 10.4% ) . in the treatment area ( figure 4 ) , the questions with the highest and the lowest mean scores , respectively , were : which of these antibiotics are not useful for tb treatment : _ _ _ _ ( 82.0% ) ; and prophylactic treatment with isoniazid is implemented to _ _ _ _ ( 13.1% ) . table 2 shows that younger students ( age < 24 ) had on average greater tb knowledge ( 58.1% of correct answers vs. 54.7% , p=0.05 ) , whereas no association was found between gender and the mean percentage of correct answers . there was a significant association between internship in units and departments and greater knowledge about tb diagnosis ( 55.9% vs. 51.6% , p=0.02 ) , treatment ( 48.4% vs. 41.8% , p=0.03 ) , and total score ( 58.1% vs. 54.5% , p=0.04 ) . students who reported receiving the mantoux test had greater knowledge about tb epidemiology and prevention ( 65.4% vs. 53.3% , p=0.001 ) , diagnosis ( 55.2% vs. 48.3% , p=0.005 ) , and total score ( 58.0% vs. 49.1% , p=0.001 ) . students who reported observing at least 1 active pulmonary tb case had a higher percentage of correct answers about diagnosis ( 55.5% vs. 51.4% , p=0.03 ) and total score ( 57.9% vs. 54.0% , p=0.03 ) . students who reported observing at least 1 x - ray of a tb patient had a higher percentage of correct answers about epidemiology and prevention ( 64.7% vs. 57.3% , p=0.02 ) , diagnosis ( 55.1% vs. 49.5% , p=0.02 ) , and total score ( 57.7% vs. 51.2% , p=0.004 ) . students who considered themselves to be at risk for tb reported a similar percentage of correct answers as other students . the multivariable linear regression analysis ( table 3 ) confirmed the association between receiving the mantoux test and greater knowledge about tb epidemiology and prevention ( coefficient=10.8 ; 95% ci 4.217.3 ) , diagnosis ( coefficient=5.0 ; 95% ci 0.110.0 ) , and total score ( coefficient=7.2 ; 95% ci 2.611.7 ) . in addition , having observed at least 1 x - ray of a tb patient was associated with a higher total score ( coefficient=5.3 ; 95% ci 1.09.7 ) . the analysis did not reveal any association between greater knowledge and gender , age , and feeling at risk . among the 32 multiple - choice questions designed to evaluate student knowledge of tb , the mean percentage of correct answers was 56.6% ( sd 11.6% ) . the mean percentage of correct answers was 63.5% ( sd 16.3% ) for epidemiology and prevention , 54.1% ( sd 12.4% ) for diagnosis , and 45.7% ( sd 20.4% ) for treatment . figure 1 shows the distribution of the mean percentage of correct answers by area of knowledge . in the epidemiology and prevention area ( figure 2 ) , the highest and the lowest mean scores were reported for the following questions , respectively : the etiologic agent of tb is transmitted by _ _ _ _ ( 98.9% correct answers ) ; and currently the prevalence of tb is highest in the following areas of the world : _ _ _ _ ( 8.2% correct answers ) . in the diagnosis area ( figure 3 ) , the questions with the highest and the lowest mean scores , respectively , were : which organ could be affected by m. tuberculosis ( 95.6% ) ; and the tine test is _ _ _ _ ( 10.4% ) . in the treatment area ( figure 4 ) , the questions with the highest and the lowest mean scores , respectively , were : which of these antibiotics are not useful for tb treatment : _ _ _ _ ( 82.0% ) ; and prophylactic treatment with isoniazid is implemented to _ _ _ _ ( 13.1% ) . table 2 shows that younger students ( age < 24 ) had on average greater tb knowledge ( 58.1% of correct answers vs. 54.7% , p=0.05 ) , whereas no association was found between gender and the mean percentage of correct answers . there was a significant association between internship in units and departments and greater knowledge about tb diagnosis ( 55.9% vs. 51.6% , p=0.02 ) , treatment ( 48.4% vs. 41.8% , p=0.03 ) , and total score ( 58.1% vs. 54.5% , p=0.04 ) . students who reported receiving the mantoux test had greater knowledge about tb epidemiology and prevention ( 65.4% vs. 53.3% , p=0.001 ) , diagnosis ( 55.2% vs. 48.3% , p=0.005 ) , and total score ( 58.0% vs. 49.1% , p=0.001 ) . students who reported observing at least 1 active pulmonary tb case had a higher percentage of correct answers about diagnosis ( 55.5% vs. 51.4% , p=0.03 ) and total score ( 57.9% vs. 54.0% , p=0.03 ) . students who reported observing at least 1 x - ray of a tb patient had a higher percentage of correct answers about epidemiology and prevention ( 64.7% vs. 57.3% , p=0.02 ) , diagnosis ( 55.1% vs. 49.5% , p=0.02 ) , and total score ( 57.7% vs. 51.2% , p=0.004 ) . students who considered themselves to be at risk for tb reported a similar percentage of correct answers as other students . the multivariable linear regression analysis ( table 3 ) confirmed the association between receiving the mantoux test and greater knowledge about tb epidemiology and prevention ( coefficient=10.8 ; 95% ci 4.217.3 ) , diagnosis ( coefficient=5.0 ; 95% ci 0.110.0 ) , and total score ( coefficient=7.2 ; 95% ci 2.611.7 ) . in addition , having observed at least 1 x - ray of a tb patient was associated with a higher total score ( coefficient=5.3 ; 95% ci 1.09.7 ) . the analysis did not reveal any association between greater knowledge and gender , age , and feeling at risk . this study showed that more than half of the questions about knowledge of tb treatment were wrong . one - sixth of the students did not remember receiving the mantoux test , which was compulsory for the investigated medical students . furthermore , medical students who remembered receiving the mantoux test demonstrated a better grasp of knowledge about tb . specifically , we found an association between memory receiving the mantoux test and greater knowledge of epidemiology and prevention , diagnosis , and the total score , which strengthens the role of the memory effect . the relationship between recall of preventive medicine activities and greater knowledge about tb is consistent with previous studies that reported an association between hpv and influenza vaccination and a greater knowledge of those diseases [ 1012 ] . we also found evidence of a significant association between greater clinical experience , indicated by those who had seen cases and a chest x - ray of a tb patient and/or those who had attended an internship in clinical wards and had greater knowledge of tb diagnosis and treatment . appropriate practice and attitudes in the treatment of tb are among the main goals that can be achieved through the adoption of competency - based programs that are responsive to rapidly changing needs , as well as promoting a new instrument of learning that includes the capacity to integrate knowledge and good practices . studies like ours , which are designed to evaluate the training of medical students and to identify inadequacies in their education , are needed to implement changes because failure to address these issues can affect both patient and public health . knowledge of these topics was investigated in the present study and a poor level of knowledge was reported . in recent years , the centers for disease control and prevention and ntcc have proposed curricula offering the basics of training undergraduate students and doctors about the fundamentals of tb prevention , diagnosis , and treatment . our survey results highlight the need to develop educational tools using active learning strategies to improve knowledge and ensure social accountability of medical schools . on the other hand , the preparation for the public health exam may have increased the students general level of knowledge about tb . the cross - sectional design of this survey does not allow the evaluation of causal relationships , but it does make associations between several factors that represent , in any case , the basis for testing causal hypothesis in further studies . our findings show a moderate knowledge about tb among fifth - year medical students . internship was found to be strongly associated with a greater knowledge of tb diagnosis , epidemiology , and prevention . we provide a clear snapshot of the differences in tb learning between the more sensitized and experienced students who apply what they have learned , and others who are more dedicated to theoretical learning . i received mantoux test : i performed at least one mantoux test : i observed at least one tb case : i observed at least one x - ray of tb patient : i think i am at risk for tb : if you are a student of medicine , in which department you are ? _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ i received mantoux test : i performed at least one mantoux test : i observed at least one tb case : i observed at least one x - ray of tb patient : i think i am at risk for tb : currently the prevalence of tb is highest in the following areas of the world : central and south america which of these microorganisms is strongly related with the tb bacillus ? mycobacterium avium complex the aetiologic agent of tb is transmitted by : higher incidence and delay in diagnosis of tb in disadvantaged populations can be related to : socio - economic issues low resources of native country all the above answers which ppes ( personal protective equipment ) are needed in a room where an open tb patient stays : all the above answers 14 ) the immune response to tb is : caused by circulating immune complexes a multiple puncture test an intradermal injection an intramuscular injection an intradermal injection an intradermal response 17 ) which one is not a limit of tuberculin skin test : it is a test in vivo that requires a return visit it is an operator - dependent test it shows a number of false positive results due to cross - reactivity with other antigenic components it is an expression of immune response to mycobacterial antigens 18 ) which of the following about quantiferon tb gold test is false : it is a test performed in vitro on a sample of peripheral venous blood it measures the production of ifn by antigen - specific for mtb lymphocytes t using an elisa methodology in 2001 this test has been approved by the fda as a support in the diagnosis of latent tuberculosis infection it does not differentiate infection from other reasons for testing positive 19 ) mantoux test is positive from an induration diameter of : 20 ) positive intradermal reaction test indicates : diagnosis of tb infection increased sensitization to m. tuberculosis increased sensitization to drugs 21 ) in case of a suspected pulmonary tb it is necessary to collect the following biological specimen : 22 ) first contact with bacteria causes : change of the antibody titer 23 ) in case of positive tuberculin skin test it is recommended : 24 ) which organ could be affected by m. tuberculosis ? tuberculosis does not increase the risk of miscarriage you reassure her because there s not risk for the child you recommend her to change treatment because there is a potential risk of toxicity for the child 30 ) how long is a tb patient supposed to be hospitalized for an effective treatment ?

neural stem cells ( nscs ) are multipotent cells that can give rise to various cell types in the central nervous system ( cns ) , including neurons , astrocytes , and oligodendrocytes ( alvarez - buylla et al . , 2002 ) . in adult mammals , neurogenesis is limited to two brain regions ; the subventricular zone ( svz ) by the lateral ventricles and the subgranular zone ( sgz ) of the dentate gyrus in the hippocampus ( reviewed in zhao et al . , 2008 ) . nscs in the svz , so called type - b cells , are of astrocytic lineage and generate transient amplifying type - c cells . neuroblasts then migrate along a defined pathway , the rostral migratory stream , to mature and form interneurons in the olfactory bulb ( doetsch et al . , 1999 ) . they generate type 2 intermediate proliferating progenitors that subsequently give rise to type 3 neuroblasts migrating into the subgranular layer , to become mature functional glutamatergic granule neurons ( kempermann et al . , 2004 ) . although the functional role of adult neurogenesis still is debated , it is undoubtedly a complex and highly regulated process . recently , micrornas ( mirnas ) have been suggested as players of neurogenesis , controlling expression of key regulatory genes ( gao , 2010 ; shi et al . , 2010 ; luikart et al . , 2011 ) . mirnas are one of the central determinants of mrna abundance , functioning as standby mediators of mrna regulation . they are small ( 2123 nucleotides long ) , non - coding endogenously expressed rna that bind to complementary mrna targets , resulting in a decrease in target mrna activity ( bartel , 2009 ) . since the discovery of the first mirna in 1993 , they have been identified in animals , plants , and viruses and more than 1000 mirna sequences have so far been found in humans ( www.mirbase.org ) . many are conserved across species . some have a general expression pattern , others are specifically expressed in certain tissues or cell types and expression can be spatially and temporally restricted . several labs have utilized conditional deletion of dicer , a key enzyme in mirna biogenesis , as a tool to study the role of mirnas in neurogenesis . disruption of the function of dicer during brain development results in gross anatomical changes and in some cases it is embryonic lethal ( giraldez et al . , 2005 ; davis et al . , 2008 ; de pietri tonelli et al . , nscs can be cultured in the absence of dicer but are unable to generate neurons or astrocytes upon differentiation ( andersson et al . , 2010 ) . although , dicer knockout ( ko ) studies indicate that mirnas are important regulators of neurogenesis and nsc self - renewal , they are difficult to interpret . not only because the simultaneous deficiency of all mirnas , but the long half - life of mature mirna makes conditional dicer ko experiments difficult to control from a temporal aspect , moreover there are concerns that non - mirna related functions of dicer contribute to the observed phenotypes ( konopka et al . . for example , when dicer was conditionally deleted in the retinal pigmented epithelium , cell death occurred via a mechanism that is independent on mirna , rather depending on the accumulation of retrotransposon transcripts ( kaneko et al . , 2011 ) . with this in mind , studies of the functional role of individual mirnas are a necessity . in 2007 there were four simultaneous reports of mirna ko mice ( rodriguez et al . , 2007 ; thai et al . , 2007 ; van rooij et al . , 2007 ; zhao et al . , 2007 ) , which enabled loss - of - function studies of specific mirnas . however , for several mirna , including many of those involved in neurogenesis , a classic ko strategy is complicated to apply due to single mirna species being located in multiple copies in separate regions of the genome , in clusters or located within other genes . one example of such a mirna family is mir-124 , which is transcribed as three different primary transcripts from three independent loci . mir-124 - 1 , mir-124 - 2 , and mir-124 - 3 all produce the same mature mirna ( griffiths - jones , 2006 ) . another example is mir-9 which is expressed from three independent loci , mir-9 - 1 , mir-9 - 2 , and mir-9 - 3 . the mature sequence is identical , and conserved in vertebrates and mammals . with let-7 the situation is even more complex with 12 human let-7 genes encoding for nine distinct , but closely related , mature forms of the mirna ( griffiths - jones , 2006 ) . recently , a ko mouse for mir-124 - 1 was described showing that reduction of mir-124 has severe consequences for neuronal survival and axonal outgrowth ( sanuki et al . , 2011 ) . while this study demonstrates the importance of mir-124 , it also highlights the problems of using a classic ko strategy . ( 2011 ) that compensation of mir-124 - 2 and mir-124 - 3 influence the phenotype of the mir-124 - 1 ko mouse . likewise , a ko mice that have two of the three copies of mir-9 deleted has been reported ( shibata et al . , these two reports suggest that conditional deletion of all three copies of either mir-9 or mir-124 is necessary in order to fully understand the role of these mirnas in adult neurogenesis . although challenging and time consuming , such a strategy may be feasible for mir-9 and mir-124 . however , for let-7 such a strategy appears unlikely to be successful given the large size of the let-7-family . adding to this , in situ analysis of the expression profile of individual mirna is difficult due to the small size of the mature mirna , which leads to poor resolution obtained in the brain with current histological techniques . as such , the study of the functional role of individual mirnas in vivo is complicated and makes interpretation and comparison between different studies challenging . we describe the current status of the field , existing attempts to study loss of mirna function , and point out technical limitations that need to be circumvented in order to move the field forward . it is fairly straightforward to profile mirna - expression patterns from bulk rna samples , either at single species resolution using for example northern blot or pcr - techniques , or at a global level using mirna arrays , pcr - array , deep sequencing of small rnas or other more specialized platforms . these methods all have their innate differences and parallel analysis of the same samples using different techniques may give significantly different results ( see , e.g. , hebert and nelson , 2011 for a discussion on this matter ) . since there is currently no gold standard for transcriptional profiling of mirna , the use of independent techniques to verify results is therefore necessary . nevertheless , these approaches have revealed the complexity of mirna - expression patterns among different cell types and have allowed identification of a number of candidate mirnas that appear to be enriched in cultured nscs . however , the technical difficulties of purifying populations of nscs and progenitors from in vivo material , using for example fluorescence activated cell sorting , make these approaches problematic to transfer to the in vivo setting ( see table 1 ) . histological approaches to study mirna expression in brain tissue have to a great extent relied on in situ hybridization ( ish ) techniques . due to the small size of the mirna it is not possible to use standard ish protocols ; an additional fixation step of the mirna is needed and probe hybridization must be optimal ( pena et al . , 2009 ) . locked nucleic acid ( lna ) modified oligonucleotides is preferable to use , since the melting temperature of the lna probe / mirna duplex is increased , resulting in stringent hybridization conditions , which in turn increases specificity and sensitivity ( reviewed in obernosterer et al . , 2007 ; wheeler et al . , 2007 ) . to do so , additional probes that target all the various precursor transcripts need to be used ( obernosterer et al . , however , this can be technically challenging when analyzing mirnas with multiple precursor transcripts ( such as mir-9 or mir-124 ) . furthermore , the results from this method are of limited resolution , thereby making it difficult to distinguish between two adjacent cells . in addition , ish is also problematic to use in combination with other labeling techniques that are routinely used in nsc - research . we have in our lab not been able to adopt protocols that allow the use of mirna ish in combination with , for example brdu - labeling , which is widely used in this field . this is primarily due to the stringent treatment of the tissue that is necessary for ish , which is incompatible with the tissue treatments for brdu - labeling . the problem of in situ mirna - expression analysis is a major concern for the study of mirna in the nervous system where it is essential to understand the cellular localization with regards to functionality . more recently , mirna reporter or sensor vectors have been used to visualize the expression pattern of endogenous mirna in cells . these are gene transfer vectors that contain a reporter gene ( i.e. , gfp ) along with binding sites for specific mirna ( mansfield et al . . in the case that a cell is actively expressing the specific mirna , the expression of the reporter gene will be suppressed by the binding of the mirna to the complimentary binding sites . thus , this system reports the absence of the target mirna , and cells that do not express it will be gfp - positive . this technique is highly specific , simple , and robust and makes it possible to study mirna expression as cells differentiate . injection of a mir-124 reporter vector into the brain will separate reporter gene expression between different cell types such as neurons and astrocytes ( colin et al . , 2009 ) . the system has been used to segregate differentiated neural cells in pluripotent cell cultures , based on the expression of mir-292 that is expressed in embryonic stem cells but not in nscs ( sachdeva et al . , 2010 ) , as well as the opposite where a mirna let-7a reporter was used to select undifferentiated cells from more differentiated cells ( di stefano et al . , 2011 ) . in nscs in vivo , sensor vectors have been used to track the expression of mir-132 in the dg ( luikart et al . the use of sensor vectors has the advantage that they measure the activity of the mirna rather than expression per se allowing easy determination if the mature mirna is present . also , the use of a fluorescent reporter gene allows excellent morphological analysis of cells in vivo . still , the technique is time - consuming including the generation of viral vectors followed by experimentation in cell culture or in vivo models . however , the versatility of the technique opens up the possibility of generating transgenic reporter animals , making it possible to visualize the expression pattern of a specific mirna throughout an organism over time ( see , e.g. , gentner et al . , 2010 ) . although this approach remains to be tested in the cns , it may be an attractive alternative in order to achieve sensitive , high - quality expression analysis of mirnas in vivo . as mentioned above , generation of ko mice for individual mirnas is often complicated since many mirnas are present in several copies or in clusters while some are present within introns of genes . to circumvent this issue several knockdown or inhibition approaches an early approach to analyze specific mirnas was by using anti - mirna oligonucleotide ( amo ) which are nucleic acids that are antisense to the mirna , thus hindering the interaction between the mirna and target mrna . this technique was first used to inactivate mir-2 and mir-13 in drosophila , in the search for mirna - target genes ( boutla et al . , 2003 ) . it was soon found that unmodified oligonucleotides are ineffective since the cellular machinery degrades them . 2-o - methyl amo is a simple chemical modification where the methyl group reduces the chances of endonucleolytic cleavage , and improves binding affinity to the mirna ( weiler et al . , 2006 ) . another variant is 2-o - methoxyethyl amo , which are fully modified oligonucleotides , with higher affinity and specificity than 2-o - methyl amo variants ( davis et al . , 2006 ; esau et al . , recently , lna ( locked nucleic acid ) modified oligonucleotides allow for even further stabilization of the mirna / target duplex structure improving silencing and also making it possible to the use small oligonucleotides that enables targeting of entire mirna - families ( vester and wengel , 2004 ; obad et al . , 2011 ) . anti - mirna oligonucleotide targets single mirnas with high specificity , as they are completely complementary to the mature sequence of the mirna ( boutla et al . , 2003 ) . despite the successful knockdown of mirna in vitro and in vivo using this method , it has several limitations . first , a direct measurement of the down regulation of mirna is difficult , because amo binds to the mirna and sequesters it from its target rather than inducing its degradation ( krutzfeldt et al . , 2005 ; davis et al . , 2009 ) . therefore the only possible way to confirm the decrease of mirna is to use indirect methods , whereby one can measure the level of expression of a reporter gene containing a target sequence of the mirna or analyzing upregulation of endogenous target genes . secondly it is not possible to identify the cells in which the amos are active as they do not carry a reporter . on top of that , the level of amo should preferably be kept constant to allow a continuous sequestration of the mirna . oligonucleotide antagomirs are chemically modified ; cholesterol conjugated single stranded rna analogs complementary to a target mirna ( krutzfeldt et al . , 2005 ) . the modification includes a partial phosphorothioate backbone in addition to 2-o - methoxyethyl to inhibit ago-2-mediated cleavage . in vivo , antagomirs have been given through intravenous injection where they appear to efficiently target mirna in various tissues . however , antagomirs do not cross the blood brain barrier , which means that in the brain , antagomirs have the same limitations as amos have . microrna sponges are transcripts expressed from strong promoters , containing multiple , tandem binding sites to a selected member of the mirna seed family of interest ( ebert et al . , 2007 ) . the binding site is imperfect , containing a bulge , for preventing rna interference cleavage and degradation of the sponge rna through endonucleolytic cleavage by ago-2 . the main advantage of mirna sponges is the possibility to achieve stable expression from integrated transgenes in vivo ( gentner et al . , 2009 ) . this can be used for studying long - term effects of mirna loss - of - function and also allows for stably expressing cell lines to be generated . the use of vector - mediated delivery also enables the incorporation of a reporter gene in order to identify the modified cells . another advantage is that sponges complementarily bind to the seed sequence of the mirna , which means that one sponge can target an entire family of mirnas . in summary , these features make sponge vectors an attractive approach to study mirna function in vivo in nscs . recently ; this technology was employed to demonstrate that mir-132 affects the integration of new - born neurons in the adult hippocampus ( luikart et al . , 2011 ) . a previous report used a retrovirus expressing the cre - recombinase to delete a floxed mir-132 gene allowing a side - by - side comparison of sponge vs. conditional ko ( magill et al . , 2010 ; luikart et al . , 2011 ) . the sponge recapitulates some , but not all , defects detected following a complete deletion of mir-132 suggesting that the use of a sponge reduces rather than eliminates levels of mir-132 ( magill et al . , 2010 ; luikart et al . , 2011 ) . as with the use of amos , the sponge vectors also have other limitations , including the difficulty of validating the down regulation of a specific mirna and the only possible way to confirm the decrease of mirna is to use indirect methods as described above . taken together , it is evident that is technically challenging to perform loss - of - function studies of mirnas in vivo in nscs . the problem of validating the inhibition , the use of transient systems together with the appearance of potential off - target effects makes the interpretation of several studies challenging . an example of this are two studies performed in the developing chick , one report suggested that mir-124 plays no role in neurogenesis while another found that mir-124 modestly promotes neurogenesis ( cao et al . , 2007 ; visvanathan et al . , 2007 ) . direct delivery of mirna - duplexes or the use of various plasmid based approaches and viral vectors have been effectively used to overexpress mirna . there are several relatively simple designs of vectors enabling stable expression that all appear to work efficiently ( krutzfeldt et al . , 2006 ; the statement that gain of function studies are easier to perform than loss of function studies is reflected in the literature and a great extent of the insight gained in the mirna field is from overexpression studies . in the last part of this review we discuss in detail three mirnas that have been functionally implicated in neurogenesis ; mir-9 , mir-124 , and the let-7 family . we give an overview of the current understanding of how these mirnas influence neurogenesis and also highlight the technical shortcomings that still prevent a full understanding of the role of these mirnas in vivo . in addition to the three above - mentioned mirna , there is a growing literature of other mirnas , including for example mir-125b , mir-132 , mir-137 , and mir-184 that influence neurogenesis ( le et al . , 2009 ; liu et al . , 2010 ; magill et al . , 2010 ; szulwach et al . , 2010 ; luikart et al . , 2011 ; sun et al . , 2011 ) . several of the technical problems that limit our understanding of mir-9 , mir-124 , and the let-7 family also hold true for other mirnas . mir-124 is perhaps the best characterized brain - specific mirna , and accounts for 2548% of all brain mirna ( lagos - quintana et al . , 2002 ) . mir-124 is expressed in neurons and have been proposed to suppress non - neural transcripts to promote neural identity ( visvanathan et al . , 2007 ; de pietri tonelli et al . , 2008 ; maiorano and mallamaci , 2009 ; farrell et al . , 2011 ; liu et al . , 2011a ; yoo et al . , 2011 ) several studies suggest that mir-124 is not expressed in other cells of the cns such as astrocytes ( see , e.g. , smirnova et al . , 2005 ) while ponomarev et al . ( 2010 ) also found it to be expressed in microglia and downregulated in activated microglia , being a key regulator of microglia quiescence . mir-124 is not expressed in nscs but is suggested to regulate the temporal progression of neurogenesis in svz . it is upregulated in the transition between type - c and type - a cells and further upregulated as the neuroblasts exit the cell cycle ( cheng et al . , 2009 ) . delivery of mir-124 to mouse or human cells in vitro causes the global expression profile of mrna to shift toward that of the brain . ( 2008 ) used a dna plasmid with primary mir-124 transcript whereas yoo et al . ( 2011 ) delivered mir-124 with a lentiviral vector harboring a mir-124 precursor , all resulting in promoting a neural phenotype . in addition , transfection of mir-124 duplexes into glioblastoma cells inhibits proliferation and induces differentiation ( silber et al . , 2008 ) . in vitro loss of function , by using antisense 2-o - methyl amo , results in delayed neurite outgrowth ( yu et al . , 2008 ) and upregulation of non - neural transcripts ( conaco et al . , 2006 ) . blocking mir-124 in svz cell populations in vivo by delivering antisense 2-o - methyl amo by a micro - osmotic pump into the ventricle , maintains neural progenitors as dividing precursors ( cheng et al . , 2009 ) . on the contrary , injecting a retrovirus overexpressing mir-124 into the svz promotes precocious neural maturation ( cheng et al . , 2009 ) . although mir-124 has been reported to be an important regulator of neurogenesis both in the developing and the adult brain , contradictory findings have been published suggesting mir-124 to be less important for neurogenesis in the developing spinal cord ( cao et al . based on in vitro mir-124 suppression or overexpression experiments , numerous mir-124 target genes have been found and validated . mir-124 has been shown to suppress several components of the re1 silencing transcription factor ( rest ) pathway ( conaco et al . , 2006 ; rest is a master regulator of neuronal phenotype ( lunyak and rosenfeld , 2005 ) and together with co - repressors it recruits histone deacetylases to suppress non - neural genes . mir-124 and rest act reciprocally ; mir-124 represses rest in neurons to promote expression of neural genes , whereas rest downregulates mir-124 in non - neural cells to inhibit expression of neural genes ( conaco et al . , 2006 ) . another mir-124 target is ptbp1 ( makeyev et al . , 2007 ) , a repressor of alternative splicing in non - neural cells demonstrating that mir-124 promotes a neuronal transcriptome by altering splicing . these targets suggest that mir-124 expression maintains a neuronal transcriptome by repressing non - neuronal genes at several levels . other targets include jagged1 in the notch signaling pathway ( cheng et al . , 2009 ; liu et al . , these targets suggest that mir-124 also plays a role in regulating the exit of a self - renewing state of nsc . however , these are only a few examples since computational algorithms suggest more than 1000 mir-124 targets ( griffiths - jones , 2006 ) . the large number of mir-124 target genes together with the observation that overexpression of mir-124 can induce a neuronal gene program suggest that mir-124 plays a crucial role in establishing and maintaining a neuronal transcription network . in light of this , it is surprising that the in vivo phenotypes found when blocking mir-124 using antisense technology is subtle , characterized mainly by a delay in differentiation or without detectable malformations ( cao et al . , 2007 ; however , a recent report using a classic ko strategy shows that deletion of mir-124 - 1 leads to major developmental phenotypes including small brain size , defective axonal outgrowth , and cell death confirming a crucial role for mir-124 in neurogenesis ( sanuki et al . , 2011 ) . as mentioned above , it is clear that compensation of mir-124 - 2 and mir-124 - 3 influence the phenotype of the mir-124-ko mouse , which highlights the need for generation of a conditional triple - mir-124-ko mouse . although this will be challenging and time consuming , such a strategy is necessary in order to understand the role of mir-124 during brain development and will assist the understanding of mir-124 in adult neurogenesis . however , since the use of retroviral or lentiviral vectors allow targeting of nscs in vivo , the application of stable inhibition vectors using for example mirna sponges to reassess the role of mir-124 in adult nscs is an interesting alternative . another well - studied brain - specific mirna involved in neurogenesis is mir-9 that is expressed in nscs and upregulated upon neural differentiation ( krichevsky et al . , 2006 ; de pietri tonelli et al . , 2008 ; zhao et al . , 2008 ; laneve et al . , 2010 ; bonev et al . , 2011 ; shibata et al . , 2011 ; initial studies , done in zebrafish , showed that mir-9 directs late organizer activity of the midbrain hindbrain boundary ( mhb ; leucht et al . , 2008 ) . the mhb is an organizing center in the vertebrate neural tube essential for proper development of the midbrain and anterior hindbrain ( wurst and bally - cuif , 2001 ) . in human cells , mir-9 was found to have an important role in migration and proliferation of nscs . knockdown experiments in neurospheres , using an lna antisense probe , led to reduced proliferation , and an increased migration ( delaloy et al . , 2010 ) . in contrast , zhao et al . found reduced proliferation accompanied with increased differentiation of mouse nscs when overexpressing mir-9 by rna duplexes . when they knocked down mir-9 with 2-o - methyl amo , proliferation increased ( zhao et al . , 2009 ) . in mouse , mir-9 has been shown to have a regional diversity along the anterior / posterior - axis ; knockdown in hindbrain leads to a failure of cell cycle , promoting proliferation of neural progenitor cells , whereas cells lacking mir-9 in the forebrain undergo p53-dependent apoptosis ( bonev et al . , , mir-9 has reciprocal actions with rest ; rest inhibits mir-9 - 2 in undifferentiated neuroblastoma cells , rest and creb inactivation triggers mir-9 - 2 activation ( laneve et al . , 2010 ) . other mir-9 targets include stathmin that increases microtubule instability ( delaloy et al . , 2010 ) and tlx that regulate stem cell fate ( zhao et al . , 2009 ) . tlx is suppressed by mir-9 to negatively regulate stem cell proliferation and accelerate neural differentiation ( zhao et al . , 2009 ) . hairy1 has also been suggested to mediate the effects of mir-9 on proliferation ( bonev et al . , 2011 ) . mir-9 expression pattern during mouse development has been investigated by ish ; it is expressed in the developing medial pallium although it is most abundant in cortex . when a mir-9 amo was electroporated into e11.5 cerebral cortex , deficiencies in differentiation of cajal retzius cells and early born neurons were seen , suggested to be due to the increased expression of the target gene foxg1 ( shibata et al . , 2008 ) . generation of a mir-9 - 2 and mir-9 - 3 double ko mouse , that is the two most abundant forms during telencephalon development , resulted in major phenotypic brain defects . cortical layers and vz were reduced , lateral ventricles expanded , the proliferative zones hyperplasic and differentiated structures reduced . in addition , mice suffered from growth retardation and died within 1 week , demonstrating the importance for mir-9 in neurogenesis ( shibata et al . , 2011 ) . these reports clearly demonstrate that mir-9 influence nscs , perhaps by regulating self - renewal and migration . still , much remains unclear regarding the functional role of mir-9 in nscs in vivo . as with mir-124 , it will be interesting to follow the generation of a conditional triple - mir-9-ko mouse or the application of stable mirna sponges to study the functional role of mir-9 . let-7 is one of the first mirna discovered in c. elegans , the first known human mirna and it is conserved over species . there are 12 human let-7 genes encoding for nine distinct mature forms of the mirna , let-7a through to let-7i . increased let-7 expression is seen in early neurogenesis and neural differentiation while it is decreased in the adult brain . ish shows induction of let-7 already at e9.5 in the developing cns ( wulczyn et al . , 2007 ) . let-7 expression closely resembles the expression of other brain - enriched mirnas ( wulczyn et al . , 2007 ) . in utero electroporation of let-7b duplexes injected into the lateral ventricles of e13.5 mice causes a reduction in cell cycle progression in nscs ( zhao et al . , overexpression of let-7b in nscs causes reduced proliferation and an increase in neural differentiation ( zhao et al . , 2010 ) . suppression of let-7 , using a mirna - sponge , causes an increase in levels of lin28 protein ( rybak et al . , 2008 ) . lin28 is a protein that specifically binds and blocks processing of let-7 , thereby inducing pluripotency ( rybak et al . , 2008 ; balzer et al . , 2010 ) lin28 is expressed broadly throughout the neural tube early during development where neural differentiation has not begun ( balzer et al . , 2010 ) , at this stage it co - localizes with sox2 , a maker for nscs . it has also been shown that let-7b suppresses the expression of tlx ( zhao et al . , 2010 ) . these reports suggest that the let-7-family serves as key regulators of nsc proliferation and accelerated neural differentiation ( wulczyn et al . , 2007 ; . however , the large size of the let-7 family posses a technical hurdle for the generation of loss - of - function mutants , which limits our understanding of the role of let-7 . in the future it appears likely that the use of sponge - vectors allowing stable inhibition of the entire family may be the most feasible choice to study the functional role of let-7 in nscs in vivo . it is likely that we have so far only begun to realize the complexity of mirna - mediated regulation of nscs . the multitude of mirna complimentary targets in the genome implicates the complexity of mirna gene regulation . therefore studies of mirna - target regulation in specific cell types at various developmental time points are essential . other questions that need to be answered are ; can several mirnas act to suppress the same mrna simultaneously and do they have compensatory , collaborative or competitive effects ? the development of new biotechnological tools such as mirna sponges and transgenic reporter systems will enable new types of studies that will clarify the functional properties of individual mirnas . in the coming years it will be extremely interesting to follow this field as it matures and unravels the full role of mirnas in nscs . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

in adult mammals , neural stem cells ( nscs ) are found in two niches of the brain ; the subventricular zone by the lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus . neurogenesis is a complex process that is tightly controlled on a molecular level . recently , micrornas ( mirnas ) have been implicated to play a central role in the regulation of ncss . mirnas are small , endogenously expressed rnas that regulate gene expression at the post - transcriptional level . however , functional studies of mirnas are complicated due to current technical limitations . in this review we describe recent findings about mirnas in nscs looking closely at mir-124 , mir-9 , and let-7 . in addition , we highlight technical strategies used to investigate mirna function , accentuating limitations , and potentials .

Introduction Expression Profiling of miRNA in Neural Stem Cells Loss of Function Studies of miRNAs miR-124 miR-9 The Let-7-Family Conclusion Conflict of Interest Statement

neural stem cells ( nscs ) are multipotent cells that can give rise to various cell types in the central nervous system ( cns ) , including neurons , astrocytes , and oligodendrocytes ( alvarez - buylla et al . in adult mammals , neurogenesis is limited to two brain regions ; the subventricular zone ( svz ) by the lateral ventricles and the subgranular zone ( sgz ) of the dentate gyrus in the hippocampus ( reviewed in zhao et al . neuroblasts then migrate along a defined pathway , the rostral migratory stream , to mature and form interneurons in the olfactory bulb ( doetsch et al . although the functional role of adult neurogenesis still is debated , it is undoubtedly a complex and highly regulated process . recently , micrornas ( mirnas ) have been suggested as players of neurogenesis , controlling expression of key regulatory genes ( gao , 2010 ; shi et al . mirnas are one of the central determinants of mrna abundance , functioning as standby mediators of mrna regulation . they are small ( 2123 nucleotides long ) , non - coding endogenously expressed rna that bind to complementary mrna targets , resulting in a decrease in target mrna activity ( bartel , 2009 ) . since the discovery of the first mirna in 1993 , they have been identified in animals , plants , and viruses and more than 1000 mirna sequences have so far been found in humans ( www.mirbase.org ) . several labs have utilized conditional deletion of dicer , a key enzyme in mirna biogenesis , as a tool to study the role of mirnas in neurogenesis . , nscs can be cultured in the absence of dicer but are unable to generate neurons or astrocytes upon differentiation ( andersson et al . although , dicer knockout ( ko ) studies indicate that mirnas are important regulators of neurogenesis and nsc self - renewal , they are difficult to interpret . for example , when dicer was conditionally deleted in the retinal pigmented epithelium , cell death occurred via a mechanism that is independent on mirna , rather depending on the accumulation of retrotransposon transcripts ( kaneko et al . with this in mind , studies of the functional role of individual mirnas are a necessity . , 2007 ) , which enabled loss - of - function studies of specific mirnas . however , for several mirna , including many of those involved in neurogenesis , a classic ko strategy is complicated to apply due to single mirna species being located in multiple copies in separate regions of the genome , in clusters or located within other genes . one example of such a mirna family is mir-124 , which is transcribed as three different primary transcripts from three independent loci . another example is mir-9 which is expressed from three independent loci , mir-9 - 1 , mir-9 - 2 , and mir-9 - 3 . the mature sequence is identical , and conserved in vertebrates and mammals . with let-7 the situation is even more complex with 12 human let-7 genes encoding for nine distinct , but closely related , mature forms of the mirna ( griffiths - jones , 2006 ) . recently , a ko mouse for mir-124 - 1 was described showing that reduction of mir-124 has severe consequences for neuronal survival and axonal outgrowth ( sanuki et al . while this study demonstrates the importance of mir-124 , it also highlights the problems of using a classic ko strategy . ( 2011 ) that compensation of mir-124 - 2 and mir-124 - 3 influence the phenotype of the mir-124 - 1 ko mouse . likewise , a ko mice that have two of the three copies of mir-9 deleted has been reported ( shibata et al . , these two reports suggest that conditional deletion of all three copies of either mir-9 or mir-124 is necessary in order to fully understand the role of these mirnas in adult neurogenesis . however , for let-7 such a strategy appears unlikely to be successful given the large size of the let-7-family . adding to this , in situ analysis of the expression profile of individual mirna is difficult due to the small size of the mature mirna , which leads to poor resolution obtained in the brain with current histological techniques . as such , the study of the functional role of individual mirnas in vivo is complicated and makes interpretation and comparison between different studies challenging . we describe the current status of the field , existing attempts to study loss of mirna function , and point out technical limitations that need to be circumvented in order to move the field forward . these methods all have their innate differences and parallel analysis of the same samples using different techniques may give significantly different results ( see , e.g. however , the technical difficulties of purifying populations of nscs and progenitors from in vivo material , using for example fluorescence activated cell sorting , make these approaches problematic to transfer to the in vivo setting ( see table 1 ) . due to the small size of the mirna it is not possible to use standard ish protocols ; an additional fixation step of the mirna is needed and probe hybridization must be optimal ( pena et al . locked nucleic acid ( lna ) modified oligonucleotides is preferable to use , since the melting temperature of the lna probe / mirna duplex is increased , resulting in stringent hybridization conditions , which in turn increases specificity and sensitivity ( reviewed in obernosterer et al . , however , this can be technically challenging when analyzing mirnas with multiple precursor transcripts ( such as mir-9 or mir-124 ) . in addition , ish is also problematic to use in combination with other labeling techniques that are routinely used in nsc - research . we have in our lab not been able to adopt protocols that allow the use of mirna ish in combination with , for example brdu - labeling , which is widely used in this field . this is primarily due to the stringent treatment of the tissue that is necessary for ish , which is incompatible with the tissue treatments for brdu - labeling . the problem of in situ mirna - expression analysis is a major concern for the study of mirna in the nervous system where it is essential to understand the cellular localization with regards to functionality . more recently , mirna reporter or sensor vectors have been used to visualize the expression pattern of endogenous mirna in cells . in the case that a cell is actively expressing the specific mirna , the expression of the reporter gene will be suppressed by the binding of the mirna to the complimentary binding sites . thus , this system reports the absence of the target mirna , and cells that do not express it will be gfp - positive . this technique is highly specific , simple , and robust and makes it possible to study mirna expression as cells differentiate . injection of a mir-124 reporter vector into the brain will separate reporter gene expression between different cell types such as neurons and astrocytes ( colin et al . the system has been used to segregate differentiated neural cells in pluripotent cell cultures , based on the expression of mir-292 that is expressed in embryonic stem cells but not in nscs ( sachdeva et al . , 2010 ) , as well as the opposite where a mirna let-7a reporter was used to select undifferentiated cells from more differentiated cells ( di stefano et al . in nscs in vivo , sensor vectors have been used to track the expression of mir-132 in the dg ( luikart et al . the use of sensor vectors has the advantage that they measure the activity of the mirna rather than expression per se allowing easy determination if the mature mirna is present . however , the versatility of the technique opens up the possibility of generating transgenic reporter animals , making it possible to visualize the expression pattern of a specific mirna throughout an organism over time ( see , e.g. although this approach remains to be tested in the cns , it may be an attractive alternative in order to achieve sensitive , high - quality expression analysis of mirnas in vivo . as mentioned above , generation of ko mice for individual mirnas is often complicated since many mirnas are present in several copies or in clusters while some are present within introns of genes . this technique was first used to inactivate mir-2 and mir-13 in drosophila , in the search for mirna - target genes ( boutla et al . 2-o - methyl amo is a simple chemical modification where the methyl group reduces the chances of endonucleolytic cleavage , and improves binding affinity to the mirna ( weiler et al . , recently , lna ( locked nucleic acid ) modified oligonucleotides allow for even further stabilization of the mirna / target duplex structure improving silencing and also making it possible to the use small oligonucleotides that enables targeting of entire mirna - families ( vester and wengel , 2004 ; obad et al . anti - mirna oligonucleotide targets single mirnas with high specificity , as they are completely complementary to the mature sequence of the mirna ( boutla et al . first , a direct measurement of the down regulation of mirna is difficult , because amo binds to the mirna and sequesters it from its target rather than inducing its degradation ( krutzfeldt et al . on top of that , the level of amo should preferably be kept constant to allow a continuous sequestration of the mirna . the modification includes a partial phosphorothioate backbone in addition to 2-o - methoxyethyl to inhibit ago-2-mediated cleavage . in vivo , antagomirs have been given through intravenous injection where they appear to efficiently target mirna in various tissues . however , antagomirs do not cross the blood brain barrier , which means that in the brain , antagomirs have the same limitations as amos have . the binding site is imperfect , containing a bulge , for preventing rna interference cleavage and degradation of the sponge rna through endonucleolytic cleavage by ago-2 . another advantage is that sponges complementarily bind to the seed sequence of the mirna , which means that one sponge can target an entire family of mirnas . in summary , these features make sponge vectors an attractive approach to study mirna function in vivo in nscs . recently ; this technology was employed to demonstrate that mir-132 affects the integration of new - born neurons in the adult hippocampus ( luikart et al . as with the use of amos , the sponge vectors also have other limitations , including the difficulty of validating the down regulation of a specific mirna and the only possible way to confirm the decrease of mirna is to use indirect methods as described above . taken together , it is evident that is technically challenging to perform loss - of - function studies of mirnas in vivo in nscs . an example of this are two studies performed in the developing chick , one report suggested that mir-124 plays no role in neurogenesis while another found that mir-124 modestly promotes neurogenesis ( cao et al . direct delivery of mirna - duplexes or the use of various plasmid based approaches and viral vectors have been effectively used to overexpress mirna . , 2006 ; the statement that gain of function studies are easier to perform than loss of function studies is reflected in the literature and a great extent of the insight gained in the mirna field is from overexpression studies . in the last part of this review we discuss in detail three mirnas that have been functionally implicated in neurogenesis ; mir-9 , mir-124 , and the let-7 family . we give an overview of the current understanding of how these mirnas influence neurogenesis and also highlight the technical shortcomings that still prevent a full understanding of the role of these mirnas in vivo . in addition to the three above - mentioned mirna , there is a growing literature of other mirnas , including for example mir-125b , mir-132 , mir-137 , and mir-184 that influence neurogenesis ( le et al . several of the technical problems that limit our understanding of mir-9 , mir-124 , and the let-7 family also hold true for other mirnas . mir-124 is perhaps the best characterized brain - specific mirna , and accounts for 2548% of all brain mirna ( lagos - quintana et al . mir-124 is expressed in neurons and have been proposed to suppress non - neural transcripts to promote neural identity ( visvanathan et al . , 2011 ) several studies suggest that mir-124 is not expressed in other cells of the cns such as astrocytes ( see , e.g. mir-124 is not expressed in nscs but is suggested to regulate the temporal progression of neurogenesis in svz . it is upregulated in the transition between type - c and type - a cells and further upregulated as the neuroblasts exit the cell cycle ( cheng et al . delivery of mir-124 to mouse or human cells in vitro causes the global expression profile of mrna to shift toward that of the brain . in addition , transfection of mir-124 duplexes into glioblastoma cells inhibits proliferation and induces differentiation ( silber et al . although mir-124 has been reported to be an important regulator of neurogenesis both in the developing and the adult brain , contradictory findings have been published suggesting mir-124 to be less important for neurogenesis in the developing spinal cord ( cao et al . mir-124 has been shown to suppress several components of the re1 silencing transcription factor ( rest ) pathway ( conaco et al . other targets include jagged1 in the notch signaling pathway ( cheng et al . , these targets suggest that mir-124 also plays a role in regulating the exit of a self - renewing state of nsc . however , these are only a few examples since computational algorithms suggest more than 1000 mir-124 targets ( griffiths - jones , 2006 ) . the large number of mir-124 target genes together with the observation that overexpression of mir-124 can induce a neuronal gene program suggest that mir-124 plays a crucial role in establishing and maintaining a neuronal transcription network . , 2007 ; however , a recent report using a classic ko strategy shows that deletion of mir-124 - 1 leads to major developmental phenotypes including small brain size , defective axonal outgrowth , and cell death confirming a crucial role for mir-124 in neurogenesis ( sanuki et al . as mentioned above , it is clear that compensation of mir-124 - 2 and mir-124 - 3 influence the phenotype of the mir-124-ko mouse , which highlights the need for generation of a conditional triple - mir-124-ko mouse . although this will be challenging and time consuming , such a strategy is necessary in order to understand the role of mir-124 during brain development and will assist the understanding of mir-124 in adult neurogenesis . however , since the use of retroviral or lentiviral vectors allow targeting of nscs in vivo , the application of stable inhibition vectors using for example mirna sponges to reassess the role of mir-124 in adult nscs is an interesting alternative . another well - studied brain - specific mirna involved in neurogenesis is mir-9 that is expressed in nscs and upregulated upon neural differentiation ( krichevsky et al . , 2011 ; initial studies , done in zebrafish , showed that mir-9 directs late organizer activity of the midbrain hindbrain boundary ( mhb ; leucht et al . the mhb is an organizing center in the vertebrate neural tube essential for proper development of the midbrain and anterior hindbrain ( wurst and bally - cuif , 2001 ) . in human cells , mir-9 was found to have an important role in migration and proliferation of nscs . knockdown experiments in neurospheres , using an lna antisense probe , led to reduced proliferation , and an increased migration ( delaloy et al . in mouse , mir-9 has been shown to have a regional diversity along the anterior / posterior - axis ; knockdown in hindbrain leads to a failure of cell cycle , promoting proliferation of neural progenitor cells , whereas cells lacking mir-9 in the forebrain undergo p53-dependent apoptosis ( bonev et al . , , mir-9 has reciprocal actions with rest ; rest inhibits mir-9 - 2 in undifferentiated neuroblastoma cells , rest and creb inactivation triggers mir-9 - 2 activation ( laneve et al . , 2010 ) and tlx that regulate stem cell fate ( zhao et al . mir-9 expression pattern during mouse development has been investigated by ish ; it is expressed in the developing medial pallium although it is most abundant in cortex . when a mir-9 amo was electroporated into e11.5 cerebral cortex , deficiencies in differentiation of cajal retzius cells and early born neurons were seen , suggested to be due to the increased expression of the target gene foxg1 ( shibata et al . generation of a mir-9 - 2 and mir-9 - 3 double ko mouse , that is the two most abundant forms during telencephalon development , resulted in major phenotypic brain defects . cortical layers and vz were reduced , lateral ventricles expanded , the proliferative zones hyperplasic and differentiated structures reduced . in addition , mice suffered from growth retardation and died within 1 week , demonstrating the importance for mir-9 in neurogenesis ( shibata et al . still , much remains unclear regarding the functional role of mir-9 in nscs in vivo . let-7 is one of the first mirna discovered in c. elegans , the first known human mirna and it is conserved over species . increased let-7 expression is seen in early neurogenesis and neural differentiation while it is decreased in the adult brain . ish shows induction of let-7 already at e9.5 in the developing cns ( wulczyn et al . in utero electroporation of let-7b duplexes injected into the lateral ventricles of e13.5 mice causes a reduction in cell cycle progression in nscs ( zhao et al . , overexpression of let-7b in nscs causes reduced proliferation and an increase in neural differentiation ( zhao et al . lin28 is a protein that specifically binds and blocks processing of let-7 , thereby inducing pluripotency ( rybak et al . however , the large size of the let-7 family posses a technical hurdle for the generation of loss - of - function mutants , which limits our understanding of the role of let-7 . in the future it appears likely that the use of sponge - vectors allowing stable inhibition of the entire family may be the most feasible choice to study the functional role of let-7 in nscs in vivo . it is likely that we have so far only begun to realize the complexity of mirna - mediated regulation of nscs . therefore studies of mirna - target regulation in specific cell types at various developmental time points are essential . in the coming years it will be extremely interesting to follow this field as it matures and unravels the full role of mirnas in nscs . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

a total of 422 patients were recruited between 1998 and 2010 ; 168 were recruited at the london centre of pediatric endocrinology , based at great ormond street hospital for children and university college london ( ucl ) hospitals in london ; the remainder were referred from national ( n = 157 ) and international ( n = 97 ) centers . ethical committee approval was obtained from the ucl institute of child health / great ormond street hospital for children joint research ethics committee , and informed written consent was obtained from patients and/or parents . of the 422 patients screened , 375 ( 89% ) had sod and its variants , whereas 47 ( 11% ) had hpe or midline clefts . primers for the pcr amplification ( 35 cycles ) of the coding region of human prok2 ( nm_001126128 ) and prokr2 ( nm_144773 ) were designed using the online primer3 program ( http://frodo.wi.mit.edu/primer3 ) . amplified dna was then analyzed for mutations by direct sequencing , using bigdye version 1.1 sequencing chemistry ( applied biosystems , foster city , california ) and analysis on a 3730x1 dna analyzer ( applied biosystems / hitachi , tokyo , japan ) . for any novel mutations / sequence variations detected in either gene , 480 ethnically matched controls ( if available ) changes were checked with reference to the dbsnp database ( www.ncbi.nlm.nih.gov/snp ) and 1000 genomes project ( www.1000genomes.org ) . for cell surface quantification by elisa , hek-293 cells were cultivated in dmem supplemented with 10% fetal calf serum and transfected by electroporation with a gene pulser xcell eukaryotic system ( bio - rad , hercules , california ) as described previously ( 13 ) . then , 10 cells were transfected with 2 g of recombinant prk5 plasmid vectors coding for the n - terminal hemagglutinin ( ha)-tagged wild - type or mutant prokr2 and made up to a total amount of 10 g plasmid dna with empty vector . twenty hours after transfection , hek-293 cells were washed with pbs and fixed with 4% paraformaldehyde in pbs for 5 min . triton x-100 for 5 min , or not permeabilized as previously described ( 13 ) . ha - tagged proteins were detected using monoclonal anti - ha peroxidase antibody 12ca5 ( roche diagnostics , mannheim , germany ) at 0.5 g / ml . because prokr2 is a gq - coupled receptor , we examined the signaling properties of novel variants by measuring intracellular calcium release and the accumulation of d - myo - inositol monophosphate ( ip1 ) in hek-293 cells in response to prok2 ligand using ip - one htrf assay kit ( cisbio bioassays , condolet , france ) , as previously described ( 13 , 22 ) . prokr2 can also couple to gs protein to generate camp , so we additionally evaluated this pathway to better characterize the functionality of novel prokr2 variants using camp htrf assay kit as previously described ( 13 , 22 ) . prokr2 null and wild - type embryos were collected at embryonic day ( e ) 18.5 and fixed with 4% paraformaldehyde ( sigma , st louis , missouri ) and dehydrated to 100% ethanol to be embedded in paraffin . paraffin sections ( 7 m ) were used for both immunohistochemistry and in situ hybridization . in short , immunohistochemistry was performed by dewaxing sections with histoclear , followed by hydration and antigen retrieval using microwave with citric acid buffer ( 10 mm citric acid , .05% tween 20 [ ph 6.0 ] ) . antibodies were obtained from hybridoma bank [ developmental studies hybridoma bank ( university of iowa ) and national hormone and peptide program ( harbor - university of california , los angeles medical center ) ] and used at 1:1000 concentration in 5% inactivated sheep serum . for immunofluorescence , secondary goat antirabbit biotinylated antibody 1:300 ( dako , carpinteria , california ) was used , followed by 1:500 streptavidin ( sigma ) . 3,3-diaminobenzidine ( dab ; vector laboratories , burlingame , california ) staining was used in accordance with the manufacturer 's protocol . slide in situ hybridization on paraffin sections was performed as described in gaston - massuet et al ( 23 ) . to quantify the number of lh cells , 3 different sections at different axial levels were selected from 3 embryos per genotype . quantification of arginine vasopressin ( avp ) , oxytocin ( ot ) , and ghrh neurons was performed using 3 sections of equivalent axial level between mutant and wild - type from the supraoptic to the tubular area of the hypothalamus . data are presented as mean number of cells sem , with student 's t test used for statistical analysis and a p < .05 value considered statistically significant . a total of 422 patients were recruited between 1998 and 2010 ; 168 were recruited at the london centre of pediatric endocrinology , based at great ormond street hospital for children and university college london ( ucl ) hospitals in london ; the remainder were referred from national ( n = 157 ) and international ( n = 97 ) centers . ethical committee approval was obtained from the ucl institute of child health / great ormond street hospital for children joint research ethics committee , and informed written consent was obtained from patients and/or parents . of the 422 patients screened , 375 ( 89% ) had sod and its variants , whereas 47 ( 11% ) had hpe or midline clefts . primers for the pcr amplification ( 35 cycles ) of the coding region of human prok2 ( nm_001126128 ) and prokr2 ( nm_144773 ) were designed using the online primer3 program ( http://frodo.wi.mit.edu/primer3 ) . amplified dna was then analyzed for mutations by direct sequencing , using bigdye version 1.1 sequencing chemistry ( applied biosystems , foster city , california ) and analysis on a 3730x1 dna analyzer ( applied biosystems / hitachi , tokyo , japan ) . for any novel mutations / sequence variations detected in either gene , 480 ethnically matched controls ( if available ) were then screened at the corresponding residue . changes were checked with reference to the dbsnp database ( www.ncbi.nlm.nih.gov/snp ) and 1000 genomes project ( www.1000genomes.org ) . for cell surface quantification by elisa , hek-293 cells were cultivated in dmem supplemented with 10% fetal calf serum and transfected by electroporation with a gene pulser xcell eukaryotic system ( bio - rad , hercules , california ) as described previously ( 13 ) . then , 10 cells were transfected with 2 g of recombinant prk5 plasmid vectors coding for the n - terminal hemagglutinin ( ha)-tagged wild - type or mutant prokr2 and made up to a total amount of 10 g plasmid dna with empty vector . twenty hours after transfection , hek-293 cells were washed with pbs and fixed with 4% paraformaldehyde in pbs for 5 min . triton x-100 for 5 min , or not permeabilized as previously described ( 13 ) . ha - tagged proteins were detected using monoclonal anti - ha peroxidase antibody 12ca5 ( roche diagnostics , mannheim , germany ) at 0.5 g / ml . because prokr2 is a gq - coupled receptor , we examined the signaling properties of novel variants by measuring intracellular calcium release and the accumulation of d - myo - inositol monophosphate ( ip1 ) in hek-293 cells in response to prok2 ligand using ip - one htrf assay kit ( cisbio bioassays , condolet , france ) , as previously described ( 13 , 22 ) . prokr2 can also couple to gs protein to generate camp , so we additionally evaluated this pathway to better characterize the functionality of novel prokr2 variants using camp htrf assay kit as previously described ( 13 , 22 ) . prokr2 null and wild - type embryos were collected at embryonic day ( e ) 18.5 and fixed with 4% paraformaldehyde ( sigma , st louis , missouri ) and dehydrated to 100% ethanol to be embedded in paraffin . paraffin sections ( 7 m ) were used for both immunohistochemistry and in situ hybridization . in short , immunohistochemistry was performed by dewaxing sections with histoclear , followed by hydration and antigen retrieval using microwave with citric acid buffer ( 10 mm citric acid , .05% tween 20 [ ph 6.0 ] ) . antibodies were obtained from hybridoma bank [ developmental studies hybridoma bank ( university of iowa ) and national hormone and peptide program ( harbor - university of california , los angeles medical center ) ] and used at 1:1000 concentration in 5% inactivated sheep serum . for immunofluorescence , secondary goat antirabbit biotinylated antibody 1:300 ( dako , carpinteria , california ) was used , followed by 1:500 streptavidin ( sigma ) . 3,3-diaminobenzidine ( dab ; vector laboratories , burlingame , california ) staining was used in accordance with the manufacturer 's protocol . slide in situ hybridization on paraffin sections was performed as described in gaston - massuet et al ( 23 ) . to quantify the number of lh cells , 3 different sections at different axial levels were selected from 3 embryos per genotype . quantification of arginine vasopressin ( avp ) , oxytocin ( ot ) , and ghrh neurons was performed using 3 sections of equivalent axial level between mutant and wild - type from the supraoptic to the tubular area of the hypothalamus . data are presented as mean number of cells sem , with student 's t test used for statistical analysis and a p < .05 value considered statistically significant . no mutations / variations were found in prok2 , whereas 11 unrelated patients exhibited mutations / variations within the coding region of prokr2 , 9 of whom have variations that have been previously described in ks and shown to be functionally significant ( p.l173r [ n = 4 ] , p.r268c [ n = 4 ] , and p.r85l [ n = 1 ] ) ( supplemental figure 1 ) . p.l173r is known to disrupt cell - surface targeting of the receptor , whereas the latter 2 variants affect g protein coupling ( 13 ) . none of the 11 patients with prokr2 variations had changes in fgf8 , fgfr1 , kal1 , nelf , chd7 , wdr11 , hesx1 , sox3 , or shh . the c.254g > t , p.r85l variant was detected in heterozygosity in a male caucasian patient ( ii ) who presented at 6 years of age with combined pituitary hormone deficiency ( cphd ; gh deficiency [ ghd ] , tsh deficiency [ tshd ] , and acth deficiency [ acthd ] ) . he had normal visual acuity and normosmia , and magnetic resonance imaging ( mri ) of the brain showed an absent anterior pituitary and an ectopic / undescended posterior pituitary . puberty was induced with gonadotropins at the age of 23 years , and the patient has since remained on testosterone ( table 1 and supplemental table 1 ) . phenotypes in patients with prokr2 variations abbreviations : ht , heterozygous ; hm , homozygous ; m , male ; f , female ; ht , height ; wt , weight ; sds , sd score ; gnd , gonadotrophin deficiency ; di , diabetes insipidus ; apa , anterior pituitary aplasia ; aph , anterior pituitary hypoplasia ; epp , ectopic posterior pituitary ; pp , posterior pituitary ; cc , corpus callosum ; ods , optic discs ; onh , optic nerve hypoplasia ; bl , bilateral ; l , left ; rt , right ; phpv , persistent hyperplastic vitreous ; gord , gastroesophageal reflux disease . the table shows endocrine deficits , ocular phenotypes , and results of mri in patients with prokr2 variations . initial presentation with neonatal hypoglycemia ; commenced hydrocortisone and t4 treatment on day 7 of life . the 4 patients ( iii , iv , v , vi ) carrying the heterozygous c.518t > g , p.l173r variant were all caucasian and presented with variable phenotypes . two patients ( iv , vi ) had sod , and all four patients had multiple anterior pituitary hormone deficiencies , with the additional diagnosis of diabetes insipidus in patient iv ( table 1 and supplemental table 1 ) . patient iii presented in the neonatal period with profound hypoglycemia and was diagnosed with cortisol deficiency ( cortisol < 30 nmol / l ) . despite hydrocortisone treatment , she had increasing glucose requirements to maintain normoglycemia ( up to 15 mg / kg / min ) and had an inappropriately increased insulin concentration ( 11.4 hyperinsulinism resolved by the age of 1 year , and the diagnosis of ghd was subsequently confirmed by glucagon provocation with commencement of recombinant human gh . she has since been diagnosed as having gastrointestinal ( gi ) dysmotility . in 3 of the 4 patients , . only 1 of the parents manifested a phenotype ; the heterozygous mother of patient v exhibited mild anosmia and delayed menarche with reported normal fertility . surprisingly , the mother of patient vi was homozygous for the p.l173r variation and yet asymptomatic ( figure 2 ) , with no evidence of abnormal gonadotropin secretion ( peak lh , 30.3 iu / l ; peak fsh , 8.6 iu / l , in response to gnrh ; estradiol , 593 she had 3 other children ( without fertility treatment ) who were also heterozygous for the variation ( data not shown ) . one male sibling of patient vi had a sleep disorder with behavioral problems , whereas another male sibling had epilepsy ; their older sister was phenotypically normal . a , direct sequencing of prokr2 in patient vi and his parents shows that the patient is heterozygous for the p.l173r variation ( bottom panel , arrow ) , whereas the mother is homozygous ( top panel ) and the father has the wild - type sequence ( middle panel ) . b , the genotypes were confirmed by restriction digest of pcr products using the bsawi enzyme that does not cut in the wt sequence , thus leaving a band of 596 bp . incubation with the patient 's dna results in 3 fragments , including the full - length product plus the cleaved 342- and 254-bp products , whereas the parent homozygous for the variant only shows the 342- and 254-bp products . the proband ( ii.4 ) was heterozygous for the p.l173r variation , whereas his mother ( i.2 ) was the unaffected , homozygous carrier . she had 3 other children ( ii.13 ) , all of whom were heterozygous for the variation but lacked a hypopituitary phenotype . the prokr2 c.802c > t , p.r268c variant was detected both in heterozygosity ( vii , ix , x ) and in homozygosity ( viii ) . three patients had optic nerve hypoplasia and a hypoplastic anterior pituitary on mri , whereas patient x had sod with agenesis of the corpus callosum and small optic discs , but no anterior pituitary hypoplasia at the time of presentation ( table 1 and supplemental table 1 ) . patients vii and x had additional brain abnormalities ( cerebellar hypoplasia , dandy - walker cyst , focal abnormality of mesial frontal cortex ) with / without epilepsy and developmental delay . although patient vii has not yet developed any pituitary hormone deficiencies , he is under regular clinical follow - up . both parental dna samples were only available for patient ix ; the unaffected father was the heterozygous carrier . only the maternal dna sample for patient x was available , and she was not a carrier ; parental dna could not be obtained for patients vii or viii . patient i is a female of chinese origin with sod who first presented at the age of 11 months with bilateral optic nerve hypoplasia and pigmentary changes of the right optic nerve . she developed ghd ( peak gh to stimulation , 3.3 g / l ) by the age of 6 years and commenced treatment with recombinant human gh . she entered puberty ( breast stage 2 ) at the age of 10.3 years , and she is followed up regularly . to date , no other pituitary hormone deficiencies have been identified ( table 1 and supplemental table 1 ) . sequencing analysis revealed a heterozygous missense variation in prokr2 ( c.151g > a , p.a51 t ) . although p.a51 t occurs at a highly conserved residue , it has also been detected in 1 of our 480 controls and has recently been determined as functionally benign ( 21 ) . therefore , no further functional work was conducted . the novel prokr2 sequence variant ( c.1111c > g , p.g371r ) he presented with sod including unilateral optic nerve hypoplasia , anterior pituitary hypoplasia , and ghd ( table 1 and supplemental table 1 ) . the sequence variant occurred at a highly conserved residue located within the intracellular c - terminal region of prokr2 and was not detected in any of our 480 controls . the total amount and the amount at the cell surface of the variant are similar ( figure 3a ) , indicating that the trafficking properties of the p.g371r variant are not impaired compared to the wild - type receptor . further functional analysis showed that the signaling activity of the variant was similar to that of the wild - type receptor for both ca release and ip1 accumulation ( figure 3 , b and c ) . as shown in figure 3d , the accumulation of camp from the variant after prok2 stimulation was also comparable to that of the wild - type prokr2 . these results showed that the p.g371r variant does not alter either gq or gs signaling pathways , although we can not exclude the possibility that this variation may cause defects in other aspects of prokr2 signaling , such as gi - protein coupling , which were not investigated in this study . amounts of ha - tagged receptors at the cell surface ( nonpermeabilized cells , gray histograms ) and in permeabilized cells ( black histograms ) were quantified by elisa . d , functional assays comparing wt prokr2 and the prokr2 p.g371r variant against negative control ( mock ) . induction of protein signaling through either the novel mutant or wt prokr2 receptor by ligand prok2 treatment resulted in similar levels ( p > .05 ) of intracellular ca turnover ( b ) and ip1 ( c ) and camp ( d ) accumulation , indicating that p.g371r is not a pathogenic variant . using immunohistochemistry against a variety of pituitary terminal differentiation markers ( gh , acth , tsh , and -glycoprotein subunit [ gonadotropes and thyrotropes ] ) , we aimed to investigate whether the phenotype in prokr2 knockout mice at e18.5 would be comparable to the phenotypes we observed in our patients ( figure 4 ) . extensive prokr2 expression has been reported in the developing preoptic region of the brain that contains the hypothalamic nuclei ( 24 ) , whereas in humans , expression of prokr2 has been shown by rt - pcr in the pituitary gland and central nervous system postnatally ( 25 , 26 ) . expression of each of the markers was consistent between wild - type and mutant mice with a reduction in pituitary size , consistent with a reported reduction in global embryo size in the mutants ( 17 ) . lh - immunoreactive cell number was significantly reduced in prokr2 embryos ( figure 4 , k , l , k , l , and m ) . in situ hybridization using specific probes to the endocrine hypothalamic neurons ( median eminence and paraventricular / supraoptic nuclei ) expressing avp , ot , and ghrh showed no overt differences between wild - type and prokr2 embryos at e18.5 either morphologically ( figure 5 , a our results indicate that mprokr2 is dispensable for proper formation of the hypothalamic - pituitary axis . the down - regulation in lh in the prokr2-null embryos agrees with the role of mprokr2 in regulating gnrh neuronal migration previously reported by matsumoto et al ( 16 ) . coronal sections through the pituitary gland of the wild - type , prokr2 ( a , c , e , g , i , k ) and prokr2 ( b , d , f , h , j , l ) embryos immunostained against gh ( a , b ) , acth ( c , d ) , tsh ( e , f ) , -glycoprotein subunit hormone ( g , h ) , prolactin ( i , j ) , and lh ( k , l ) . immunoreactivity of gh , acth , tsh , -gsu , and prl show no difference between wild - type and prokr2-deficient embryos . although the pituitary glands of prokr2 embryos appear smaller , this is due to an overall reduction in head size of these mutant embryos . the number of lh - expressing cells appears reduced in prokr2 embryos ( k , l , and k , l , enlarged boxed areas in k and l , respectively ) , and quantification of lh cells demonstrates a statistically significant reduction in prokr2 p < .05 ( m ) . coronal sections through the brain of mouse embryos ( represented in g ) at e18.5 , wild - type prokr2 ( a , c , e ) , and mutant prokr2 ( b , d , f ) , hybridized with avp ( a , b ) , ot ( c , d ) , and ghrh ( e , f ) . avp- and ot - expressing neurons in the paraventricular and supraoptic nuclei appear similar between prokr2 and prokr2 embryos , suggesting that prokr2 is not required for the development of these structures . expression of ghrhr in the arcuate nucleus at the level of the median eminence appears unaltered between genotypes ( e , f ) . h , plotted graphs representing numbers of avp , ot , and ghrh neurons indicate no difference in the number of avp , ot , and ghrh neurons between wild - type ( white columns ) and prokr2 embryos . arc , arcuate nucleus ; pvn , paraventricular nuclei ; son , supraoptic nuclei ; me , median eminence . no mutations / variations were found in prok2 , whereas 11 unrelated patients exhibited mutations / variations within the coding region of prokr2 , 9 of whom have variations that have been previously described in ks and shown to be functionally significant ( p.l173r [ n = 4 ] , p.r268c [ n = 4 ] , and p.r85l [ n = 1 ] ) ( supplemental figure 1 ) . p.l173r is known to disrupt cell - surface targeting of the receptor , whereas the latter 2 variants affect g protein coupling ( 13 ) . none of the 11 patients with prokr2 variations had changes in fgf8 , fgfr1 , kal1 , nelf , chd7 , wdr11 , hesx1 , sox3 , or shh . the c.254g > t , p.r85l variant was detected in heterozygosity in a male caucasian patient ( ii ) who presented at 6 years of age with combined pituitary hormone deficiency ( cphd ; gh deficiency [ ghd ] , tsh deficiency [ tshd ] , and acth deficiency [ acthd ] ) . he had normal visual acuity and normosmia , and magnetic resonance imaging ( mri ) of the brain showed an absent anterior pituitary and an ectopic / undescended posterior pituitary . puberty was induced with gonadotropins at the age of 23 years , and the patient has since remained on testosterone ( table 1 and supplemental table 1 ) . phenotypes in patients with prokr2 variations abbreviations : ht , heterozygous ; hm , homozygous ; m , male ; f , female ; ht , height ; wt , weight ; sds , sd score ; gnd , gonadotrophin deficiency ; di , diabetes insipidus ; apa , anterior pituitary aplasia ; aph , anterior pituitary hypoplasia ; epp , ectopic posterior pituitary ; pp , posterior pituitary ; cc , corpus callosum ; ods , optic discs ; onh , optic nerve hypoplasia ; bl , bilateral ; l , left ; rt , right ; phpv , persistent hyperplastic vitreous ; gord , gastroesophageal reflux disease . the table shows endocrine deficits , ocular phenotypes , and results of mri in patients with prokr2 variations . initial presentation with neonatal hypoglycemia ; commenced hydrocortisone and t4 treatment on day 7 of life . the 4 patients ( iii , iv , v , vi ) carrying the heterozygous c.518t > g , p.l173r variant were all caucasian and presented with variable phenotypes . two patients ( iv , vi ) had sod , and all four patients had multiple anterior pituitary hormone deficiencies , with the additional diagnosis of diabetes insipidus in patient iv ( table 1 and supplemental table 1 ) . patient iii presented in the neonatal period with profound hypoglycemia and was diagnosed with cortisol deficiency ( cortisol < 30 nmol / l ) . despite hydrocortisone treatment , she had increasing glucose requirements to maintain normoglycemia ( up to 15 mg / kg / min ) and had an inappropriately increased insulin concentration ( 11.4 hyperinsulinism resolved by the age of 1 year , and the diagnosis of ghd was subsequently confirmed by glucagon provocation with commencement of recombinant human gh . she has since been diagnosed as having gastrointestinal ( gi ) dysmotility . in 3 of the 4 patients , only 1 of the parents manifested a phenotype ; the heterozygous mother of patient v exhibited mild anosmia and delayed menarche with reported normal fertility . surprisingly , the mother of patient vi was homozygous for the p.l173r variation and yet asymptomatic ( figure 2 ) , with no evidence of abnormal gonadotropin secretion ( peak lh , 30.3 iu / l ; peak fsh , 8.6 iu / l , in response to gnrh ; estradiol , 593 pmol / l ) . she had 3 other children ( without fertility treatment ) who were also heterozygous for the variation ( data not shown ) . one male sibling of patient vi had a sleep disorder with behavioral problems , whereas another male sibling had epilepsy ; their older sister was phenotypically normal . a , direct sequencing of prokr2 in patient vi and his parents shows that the patient is heterozygous for the p.l173r variation ( bottom panel , arrow ) , whereas the mother is homozygous ( top panel ) and the father has the wild - type sequence ( middle panel ) . b , the genotypes were confirmed by restriction digest of pcr products using the bsawi enzyme that does not cut in the wt sequence , thus leaving a band of 596 bp . incubation with the patient 's dna results in 3 fragments , including the full - length product plus the cleaved 342- and 254-bp products , whereas the parent homozygous for the variant only shows the 342- and 254-bp products . the proband ( ii.4 ) was heterozygous for the p.l173r variation , whereas his mother ( i.2 ) was the unaffected , homozygous carrier . she had 3 other children ( ii.13 ) , all of whom were heterozygous for the variation but lacked a hypopituitary phenotype . the prokr2 c.802c > t , p.r268c variant was detected both in heterozygosity ( vii , ix , x ) and in homozygosity ( viii ) . three patients had optic nerve hypoplasia and a hypoplastic anterior pituitary on mri , whereas patient x had sod with agenesis of the corpus callosum and small optic discs , but no anterior pituitary hypoplasia at the time of presentation ( table 1 and supplemental table 1 ) . patients vii and x had additional brain abnormalities ( cerebellar hypoplasia , dandy - walker cyst , focal abnormality of mesial frontal cortex ) with / without epilepsy and developmental delay . although patient vii has not yet developed any pituitary hormone deficiencies , he is under regular clinical follow - up . both parental dna samples were only available for patient ix ; the unaffected father was the heterozygous carrier . only the maternal dna sample for patient x was available , and she was not a carrier ; parental dna could not be obtained for patients vii or viii . patient i is a female of chinese origin with sod who first presented at the age of 11 months with bilateral optic nerve hypoplasia and pigmentary changes of the right optic nerve . she developed ghd ( peak gh to stimulation , 3.3 g / l ) by the age of 6 years and commenced treatment with recombinant human gh . she entered puberty ( breast stage 2 ) at the age of 10.3 years , and she is followed up regularly . to date , no other pituitary hormone deficiencies have been identified ( table 1 and supplemental table 1 ) . sequencing analysis revealed a heterozygous missense variation in prokr2 ( c.151g > a , p.a51 t ) . although p.a51 t occurs at a highly conserved residue , it has also been detected in 1 of our 480 controls and has recently been determined as functionally benign ( 21 ) . therefore , no further functional work was conducted . the novel prokr2 sequence variant ( c.1111c > g , p.g371r ) he presented with sod including unilateral optic nerve hypoplasia , anterior pituitary hypoplasia , and ghd ( table 1 and supplemental table 1 ) . the sequence variant occurred at a highly conserved residue located within the intracellular c - terminal region of prokr2 and was not detected in any of our 480 controls . the total amount and the amount at the cell surface of the variant are similar ( figure 3a ) , indicating that the trafficking properties of the p.g371r variant are not impaired compared to the wild - type receptor . further functional analysis showed that the signaling activity of the variant was similar to that of the wild - type receptor for both ca release and ip1 accumulation ( figure 3 , b and c ) . as shown in figure 3d , the accumulation of camp from the variant after prok2 stimulation was also comparable to that of the wild - type prokr2 . these results showed that the p.g371r variant does not alter either gq or gs signaling pathways , although we can not exclude the possibility that this variation may cause defects in other aspects of prokr2 signaling , such as gi - protein coupling , which were not investigated in this study . amounts of ha - tagged receptors at the cell surface ( nonpermeabilized cells , gray histograms ) and in permeabilized cells ( black histograms ) were quantified by elisa . d , functional assays comparing wt prokr2 and the prokr2 p.g371r variant against negative control ( mock ) . induction of protein signaling through either the novel mutant or wt prokr2 receptor by ligand prok2 treatment resulted in similar levels ( p > .05 ) of intracellular ca turnover ( b ) and ip1 ( c ) and camp ( d ) accumulation , indicating that p.g371r is not a pathogenic variant . using immunohistochemistry against a variety of pituitary terminal differentiation markers ( gh , acth , tsh , and -glycoprotein subunit [ gonadotropes and thyrotropes ] ) , we aimed to investigate whether the phenotype in prokr2 knockout mice at e18.5 would be comparable to the phenotypes we observed in our patients ( figure 4 ) . extensive prokr2 expression has been reported in the developing preoptic region of the brain that contains the hypothalamic nuclei ( 24 ) , whereas in humans , expression of prokr2 has been shown by rt - pcr in the pituitary gland and central nervous system postnatally ( 25 , 26 ) . expression of each of the markers was consistent between wild - type and mutant mice with a reduction in pituitary size , consistent with a reported reduction in global embryo size in the mutants ( 17 ) . lh - immunoreactive cell number was significantly reduced in prokr2 embryos ( figure 4 , k , l , k , l , and m ) . in situ hybridization using specific probes to the endocrine hypothalamic neurons ( median eminence and paraventricular / supraoptic nuclei ) expressing avp , ot , and ghrh showed no overt differences between wild - type and prokr2 embryos at e18.5 either morphologically ( figure 5 , a our results indicate that mprokr2 is dispensable for proper formation of the hypothalamic - pituitary axis . the down - regulation in lh in the prokr2-null embryos agrees with the role of mprokr2 in regulating gnrh neuronal migration previously reported by matsumoto et al ( 16 ) . coronal sections through the pituitary gland of the wild - type , prokr2 ( a , c , e , g , i , k ) and prokr2 ( b , d , f , h , j , l ) embryos immunostained against gh ( a , b ) , acth ( c , d ) , tsh ( e , f ) , -glycoprotein subunit hormone ( g , h ) , prolactin ( i , j ) , and lh ( k , l ) . immunoreactivity of gh , acth , tsh , -gsu , and prl show no difference between wild - type and prokr2-deficient embryos . although the pituitary glands of prokr2 embryos appear smaller , this is due to an overall reduction in head size of these mutant embryos . the number of lh - expressing cells appears reduced in prokr2 embryos ( k , l , and k , l , enlarged boxed areas in k and l , respectively ) , and quantification of lh cells demonstrates a statistically significant reduction in prokr2 p < .05 ( m ) . coronal sections through the brain of mouse embryos ( represented in g ) at e18.5 , wild - type prokr2 ( a , c , e ) , and mutant prokr2 ( b , d , f ) , hybridized with avp ( a , b ) , ot ( c , d ) , and ghrh ( e , f ) . avp- and ot - expressing neurons in the paraventricular and supraoptic nuclei appear similar between prokr2 and prokr2 embryos , suggesting that prokr2 is not required for the development of these structures . expression of ghrhr in the arcuate nucleus at the level of the median eminence appears unaltered between genotypes ( e , f ) . h , plotted graphs representing numbers of avp , ot , and ghrh neurons indicate no difference in the number of avp , ot , and ghrh neurons between wild - type ( white columns ) and prokr2 embryos . arc , arcuate nucleus ; pvn , paraventricular nuclei ; son , supraoptic nuclei ; me , median eminence . in this study , we have identified 11 patients with variable congenital hypopituitarism / sod , who presented with sequence variations in prokr2 ( table 1 ) . because the parental carriers included both maternal and paternal carriers , there is no suggestion of a parent of origin effect . despite its established role in ks ( 14 ) our results of the largest cohort of patients with congenital hypopituitarism , including both cphd and sod , screened to date are consistent with our recently published data ( 20 ) . here , we report the identification of prokr2 variants in patients with craniofacial / midline disorders and hypopituitarism , thus suggesting an overlap in genotypes / phenotypes between these conditions and ks ( 20 ) . in our cohort , 9 of 11 patients were found to harbor previously described prokr2 variations that had been shown to be functionally deleterious in vitro ; the lack of dominant - negative effects of these variants suggests that their functional significance in vivo remains debatable ( 13 ) . these variations are present in approximately 2% of our cohort with sod ; thus prokr2 variations occur more frequently than any other genetic abnormalities identified in association with sod to date ( 27 ) . however , the extent to which these variations contribute to the phenotype is yet to be established . of the 9 patients above , prokr2 variations were detected both in homozygosity ( n = 1 ) and in heterozygosity ( n = 8) . interestingly , 1 patient ( vi ) with sod / cphd and structural pituitary abnormalities was heterozygous for the prokr2 p.l173r variation , whereas his phenotypically normal mother was homozygous for the same variant ; this variant has previously been identified in several patients with ks and was shown to disrupt cell - surface targeting of the receptor in vitro ( 12 , 13 , 28 ) . we sought to better understand the phenotypes observed in our patients and the lack of a phenotype in the healthy homozygous mother by investigating a possible role for mprokr2 in the development and integrity of the hypothalamic - pituitary axis , using prokr2 null embryos as a model . our study of the homozygous knockout prokr2 mice revealed a morphologically normal pituitary and hypothalamus with normal hormone - secreting cells except for lh gonadotrophs , which were significantly reduced in the mutants and consistent with the previously reported ks - like phenotypes that these mice exhibit ( 16 , 17 ) . these observations suggest that in the absence of prokr2 , the murine hypothalamic - pituitary axis develops normally . although this may not be a human model , the parallels between the normal hypothalamic - pituitary axis between our knockout mice and the healthy mother of patient vi are compelling and suggest that the variants identified in this report may not be sufficient to cause a hypothalamic - pituitary phenotype in isolation . any contributory mechanisms of prokr2/prokr2 in the pathogenicity of hypopituitarism remain to be proven . in this cohort , the mother who was homozygous for the prokr2 p.l173r variation clearly did not have ks ( normosmic and fertile with 4 children that were conceived without assistance , and with normal gonadotropins and estrogen ) . in addition , 2 of the 4 patients who had reached pubertal age ( ii , viii ) required the induction of puberty and remained on testosterone treatment into adulthood , whereas the other 2 ( iv , v ) progressed through puberty spontaneously and remained off sex steroid treatment . therefore , the possibility that prokr2/prokr2 is not , in isolation , causative of ks either must be considered ; it may , however , contribute by modifying a phenotype caused by a defect in another gene(s ) or environmental factor(s ) , as recently postulated by raivio et al ( 20 ) . first , we detected functionally significant variants at the p.r85 and p.r268 alleles in heterozygosity or homozygosity in patients with phenotypes ranging from cphd to sod . they had previously only been detected in ks and healthy controls ( as had the p.l173r variant ) , albeit only in heterozygosity in the latter ( 12 ) . our data therefore strongly suggest that another gene or environmental factor is causing the more severe cphd - sod phenotypes . indeed , although not proven in any of our hypopituitary patients , digenic cases of ks involving prokr2 have been reported previously ( 12 , 15 , 2830 ) . in a recent study by sarfati et al ( 28 ) , male ks patients presenting with homozygous variations in prokr2 were significantly more likely to exhibit cryptorchidism , microphallus , lower mean testicular volumes , and circulating gonadotropins than their heterozygous counterparts ( 28 ) . this difference in the gene - dosage of prokr2 supports a contributory role to the pathogenesis of ks . no differences were observed between homozygous or heterozygous females , although this was attributed to the very low number of cases of the former ( n = 4 ) . thus , the extent to which prokr2 variants contribute to either hypopituitarism or ks - associated phenotypes remains to be established . care must be taken with the interpretation of results when patients presenting with such disorders also exhibit variations in the prokr2 gene , particularly with respect to genetic counseling . other genes known to be associated with these disorders should be screened and , should these be negative for mutations , then one can not rule out the presence of an as yet unidentified mutated gene or possible environmental factors . in addition to presenting with cphd / sod , some of our patients with prokr2 variants had additional manifestations including epilepsy , sleep abnormalities , gi dysmotility , and diabetes insipidus . although such variants have been detected for the first time in patients with the latter 2 disorders herein , a possible association is not altogether surprising given that prokineticin ligands and receptors are involved in various systems and processes including circadian rhythms in the brain , angiogenesis , neurogenesis , pain perception , immune responses , hematopoiesis , reproduction , and gi smooth muscle contraction ( 3137 ) . epilepsy , sleep disorders , and diabetes insipidus are suggestive of a forebrain / hypothalamic phenotype ( 35 , 36 , 38 ) . avp and prok2 mrna both colocalize to a significant number of hypothalamic neurons in rats , and there is evidence of interaction between these pathways through avp receptor - null mice ( 39 , 40 ) . however , prokr2 knockout mice exhibited a morphologically normal hypothalamus with quantitatively normal expression of avp , ot , and ghrh . these data do not , however , exclude a role for prokr2 in postnatal development of these hypothalamic disorders in our patients . however , considering that our patient with diabetes insipidus and epilepsy ( iv ) was heterozygous for the same variant that the unaffected mother of patient vi had in homozygosity , it appears unlikely that mutated prokr2 is causative of these phenotypes . additionally , we can not definitively state that the gi dysmotility phenotype in patient iii was caused by her heterozygous prokr2 variation , this again being the same as the mother of patient vi . any contributions of prokr2 variants to these phenotypes would necessitate further studies , particularly with the aid of a postnatal murine model . we have identified variations in prokr2 at a higher frequency in sod than any other previously described genetic factor ; we also describe other clinical features in association with these variations , including gi and hypothalamic disorders ( eg , diabetes insipidus ) . however , the role of prokr2 is controversial ; heterozygous and homozygous variants occurring across the protein induce comparable phenotypes or , as we have shown , more severe phenotypes in cases of the former than the latter . this is compounded by the incidence of homozygosity in the healthy mother of a heterozygous child with a severe form of hypopituitarism in the form of sod . our analyses of the pituitary and hypothalamus in prokr2 knockout mice are largely inconsistent with the patient phenotypes , yet strongly support the normal presentation of the mother and her 3 unaffected ( with respect to hypopituitarism ) heterozygous children , although one needs to note the reduced lh in the murine null mutants . the number of genetically assigned causes of both ks and hypopituitarism is low , accounting for 30% of ks and 510% of sod and related midline disorders , and given that none of our patients harbored mutations in any of the known genes in these disorders , there are clearly other genetic / environmental factors yet to be discovered . subjects with sequence variants in prokr2 represent a unique cohort ; further careful genetic investigation is likely to aid in the identification of the missing genetic or epigenetic modifiers that interact with this pathway in humans to account for the phenotypic heterogeneity . further research into uncovering these additional factors would help define the role , if any , of prokr2 in these and other disorders discussed herein .

context : loss - of - function mutations in prok2 and prokr2 have been implicated in kallmann syndrome ( ks ) , characterized by hypogonadotropic hypogonadism and anosmia . recent data suggest overlapping phenotypes / genotypes between ks and congenital hypopituitarism ( ch ) , including septo - optic dysplasia ( sod).objective : we screened a cohort of patients with complex forms of ch ( n = 422 ) for mutations in prok2 and prokr2.results:we detected 5 prokr2 variants in 11 patients with sod / ch : novel p.g371r and previously reported p.a51 t , p.r85l , p.l173r , and p.r268c the latter 3 being known functionally deleterious variants . surprisingly , 1 patient with sod was heterozygous for the p.l173r variant , whereas his phenotypically unaffected mother was homozygous for the variant . we sought to clarify the role of prokr2 in hypothalamopituitary development through analysis of prokr2/ mice . interestingly , these revealed predominantly normal hypothalamopituitary development and terminal cell differentiation , with the exception of reduced lh ; this was inconsistent with patient phenotypes and more analogous to the healthy mother , although she did not have ks , unlike the prokr2/ mice.conclusions:the role of prokr2 in the etiology of ch , sod , and ks is uncertain , as demonstrated by no clear phenotype - genotype correlation ; loss - of - function variants in heterozygosity or homozygosity can be associated with these disorders . however , we report a phenotypically normal parent , homozygous for p.l173r . our data suggest that the variants identified herein are unlikely to be implicated in isolation in these disorders ; other genetic or environmental modifiers may also impact on the etiology . given the phenotypic variability , genetic counseling may presently be inappropriate .

Patients and Methods Patients Mutation analysis Functional analysis of novel variants Immunohistochemistry and in situ hybridization of Results Mutation analysis Patient with Patients with Patients with Patients with other Analysis of the hypothalamic-pituitary axis in the Discussion Supplementary Material

because prokr2 is a gq - coupled receptor , we examined the signaling properties of novel variants by measuring intracellular calcium release and the accumulation of d - myo - inositol monophosphate ( ip1 ) in hek-293 cells in response to prok2 ligand using ip - one htrf assay kit ( cisbio bioassays , condolet , france ) , as previously described ( 13 , 22 ) . quantification of arginine vasopressin ( avp ) , oxytocin ( ot ) , and ghrh neurons was performed using 3 sections of equivalent axial level between mutant and wild - type from the supraoptic to the tubular area of the hypothalamus . because prokr2 is a gq - coupled receptor , we examined the signaling properties of novel variants by measuring intracellular calcium release and the accumulation of d - myo - inositol monophosphate ( ip1 ) in hek-293 cells in response to prok2 ligand using ip - one htrf assay kit ( cisbio bioassays , condolet , france ) , as previously described ( 13 , 22 ) . quantification of arginine vasopressin ( avp ) , oxytocin ( ot ) , and ghrh neurons was performed using 3 sections of equivalent axial level between mutant and wild - type from the supraoptic to the tubular area of the hypothalamus . no mutations / variations were found in prok2 , whereas 11 unrelated patients exhibited mutations / variations within the coding region of prokr2 , 9 of whom have variations that have been previously described in ks and shown to be functionally significant ( p.l173r [ n = 4 ] , p.r268c [ n = 4 ] , and p.r85l [ n = 1 ] ) ( supplemental figure 1 ) . p.l173r is known to disrupt cell - surface targeting of the receptor , whereas the latter 2 variants affect g protein coupling ( 13 ) . the c.254g > t , p.r85l variant was detected in heterozygosity in a male caucasian patient ( ii ) who presented at 6 years of age with combined pituitary hormone deficiency ( cphd ; gh deficiency [ ghd ] , tsh deficiency [ tshd ] , and acth deficiency [ acthd ] ) . the 4 patients ( iii , iv , v , vi ) carrying the heterozygous c.518t > g , p.l173r variant were all caucasian and presented with variable phenotypes . two patients ( iv , vi ) had sod , and all four patients had multiple anterior pituitary hormone deficiencies , with the additional diagnosis of diabetes insipidus in patient iv ( table 1 and supplemental table 1 ) . surprisingly , the mother of patient vi was homozygous for the p.l173r variation and yet asymptomatic ( figure 2 ) , with no evidence of abnormal gonadotropin secretion ( peak lh , 30.3 iu / l ; peak fsh , 8.6 iu / l , in response to gnrh ; estradiol , 593 she had 3 other children ( without fertility treatment ) who were also heterozygous for the variation ( data not shown ) . a , direct sequencing of prokr2 in patient vi and his parents shows that the patient is heterozygous for the p.l173r variation ( bottom panel , arrow ) , whereas the mother is homozygous ( top panel ) and the father has the wild - type sequence ( middle panel ) . incubation with the patient 's dna results in 3 fragments , including the full - length product plus the cleaved 342- and 254-bp products , whereas the parent homozygous for the variant only shows the 342- and 254-bp products . the proband ( ii.4 ) was heterozygous for the p.l173r variation , whereas his mother ( i.2 ) was the unaffected , homozygous carrier . she had 3 other children ( ii.13 ) , all of whom were heterozygous for the variation but lacked a hypopituitary phenotype . the prokr2 c.802c > t , p.r268c variant was detected both in heterozygosity ( vii , ix , x ) and in homozygosity ( viii ) . sequencing analysis revealed a heterozygous missense variation in prokr2 ( c.151g > a , p.a51 t ) . the novel prokr2 sequence variant ( c.1111c > g , p.g371r ) he presented with sod including unilateral optic nerve hypoplasia , anterior pituitary hypoplasia , and ghd ( table 1 and supplemental table 1 ) . the total amount and the amount at the cell surface of the variant are similar ( figure 3a ) , indicating that the trafficking properties of the p.g371r variant are not impaired compared to the wild - type receptor . these results showed that the p.g371r variant does not alter either gq or gs signaling pathways , although we can not exclude the possibility that this variation may cause defects in other aspects of prokr2 signaling , such as gi - protein coupling , which were not investigated in this study . using immunohistochemistry against a variety of pituitary terminal differentiation markers ( gh , acth , tsh , and -glycoprotein subunit [ gonadotropes and thyrotropes ] ) , we aimed to investigate whether the phenotype in prokr2 knockout mice at e18.5 would be comparable to the phenotypes we observed in our patients ( figure 4 ) . extensive prokr2 expression has been reported in the developing preoptic region of the brain that contains the hypothalamic nuclei ( 24 ) , whereas in humans , expression of prokr2 has been shown by rt - pcr in the pituitary gland and central nervous system postnatally ( 25 , 26 ) . in situ hybridization using specific probes to the endocrine hypothalamic neurons ( median eminence and paraventricular / supraoptic nuclei ) expressing avp , ot , and ghrh showed no overt differences between wild - type and prokr2 embryos at e18.5 either morphologically ( figure 5 , a our results indicate that mprokr2 is dispensable for proper formation of the hypothalamic - pituitary axis . the down - regulation in lh in the prokr2-null embryos agrees with the role of mprokr2 in regulating gnrh neuronal migration previously reported by matsumoto et al ( 16 ) . coronal sections through the pituitary gland of the wild - type , prokr2 ( a , c , e , g , i , k ) and prokr2 ( b , d , f , h , j , l ) embryos immunostained against gh ( a , b ) , acth ( c , d ) , tsh ( e , f ) , -glycoprotein subunit hormone ( g , h ) , prolactin ( i , j ) , and lh ( k , l ) . avp- and ot - expressing neurons in the paraventricular and supraoptic nuclei appear similar between prokr2 and prokr2 embryos , suggesting that prokr2 is not required for the development of these structures . h , plotted graphs representing numbers of avp , ot , and ghrh neurons indicate no difference in the number of avp , ot , and ghrh neurons between wild - type ( white columns ) and prokr2 embryos . no mutations / variations were found in prok2 , whereas 11 unrelated patients exhibited mutations / variations within the coding region of prokr2 , 9 of whom have variations that have been previously described in ks and shown to be functionally significant ( p.l173r [ n = 4 ] , p.r268c [ n = 4 ] , and p.r85l [ n = 1 ] ) ( supplemental figure 1 ) . p.l173r is known to disrupt cell - surface targeting of the receptor , whereas the latter 2 variants affect g protein coupling ( 13 ) . the c.254g > t , p.r85l variant was detected in heterozygosity in a male caucasian patient ( ii ) who presented at 6 years of age with combined pituitary hormone deficiency ( cphd ; gh deficiency [ ghd ] , tsh deficiency [ tshd ] , and acth deficiency [ acthd ] ) . the 4 patients ( iii , iv , v , vi ) carrying the heterozygous c.518t > g , p.l173r variant were all caucasian and presented with variable phenotypes . two patients ( iv , vi ) had sod , and all four patients had multiple anterior pituitary hormone deficiencies , with the additional diagnosis of diabetes insipidus in patient iv ( table 1 and supplemental table 1 ) . surprisingly , the mother of patient vi was homozygous for the p.l173r variation and yet asymptomatic ( figure 2 ) , with no evidence of abnormal gonadotropin secretion ( peak lh , 30.3 iu / l ; peak fsh , 8.6 iu / l , in response to gnrh ; estradiol , 593 pmol / l ) . a , direct sequencing of prokr2 in patient vi and his parents shows that the patient is heterozygous for the p.l173r variation ( bottom panel , arrow ) , whereas the mother is homozygous ( top panel ) and the father has the wild - type sequence ( middle panel ) . incubation with the patient 's dna results in 3 fragments , including the full - length product plus the cleaved 342- and 254-bp products , whereas the parent homozygous for the variant only shows the 342- and 254-bp products . the proband ( ii.4 ) was heterozygous for the p.l173r variation , whereas his mother ( i.2 ) was the unaffected , homozygous carrier . she had 3 other children ( ii.13 ) , all of whom were heterozygous for the variation but lacked a hypopituitary phenotype . the prokr2 c.802c > t , p.r268c variant was detected both in heterozygosity ( vii , ix , x ) and in homozygosity ( viii ) . sequencing analysis revealed a heterozygous missense variation in prokr2 ( c.151g > a , p.a51 t ) . the novel prokr2 sequence variant ( c.1111c > g , p.g371r ) he presented with sod including unilateral optic nerve hypoplasia , anterior pituitary hypoplasia , and ghd ( table 1 and supplemental table 1 ) . the total amount and the amount at the cell surface of the variant are similar ( figure 3a ) , indicating that the trafficking properties of the p.g371r variant are not impaired compared to the wild - type receptor . these results showed that the p.g371r variant does not alter either gq or gs signaling pathways , although we can not exclude the possibility that this variation may cause defects in other aspects of prokr2 signaling , such as gi - protein coupling , which were not investigated in this study . using immunohistochemistry against a variety of pituitary terminal differentiation markers ( gh , acth , tsh , and -glycoprotein subunit [ gonadotropes and thyrotropes ] ) , we aimed to investigate whether the phenotype in prokr2 knockout mice at e18.5 would be comparable to the phenotypes we observed in our patients ( figure 4 ) . extensive prokr2 expression has been reported in the developing preoptic region of the brain that contains the hypothalamic nuclei ( 24 ) , whereas in humans , expression of prokr2 has been shown by rt - pcr in the pituitary gland and central nervous system postnatally ( 25 , 26 ) . in situ hybridization using specific probes to the endocrine hypothalamic neurons ( median eminence and paraventricular / supraoptic nuclei ) expressing avp , ot , and ghrh showed no overt differences between wild - type and prokr2 embryos at e18.5 either morphologically ( figure 5 , a our results indicate that mprokr2 is dispensable for proper formation of the hypothalamic - pituitary axis . the down - regulation in lh in the prokr2-null embryos agrees with the role of mprokr2 in regulating gnrh neuronal migration previously reported by matsumoto et al ( 16 ) . avp- and ot - expressing neurons in the paraventricular and supraoptic nuclei appear similar between prokr2 and prokr2 embryos , suggesting that prokr2 is not required for the development of these structures . h , plotted graphs representing numbers of avp , ot , and ghrh neurons indicate no difference in the number of avp , ot , and ghrh neurons between wild - type ( white columns ) and prokr2 embryos . in this study , we have identified 11 patients with variable congenital hypopituitarism / sod , who presented with sequence variations in prokr2 ( table 1 ) . despite its established role in ks ( 14 ) our results of the largest cohort of patients with congenital hypopituitarism , including both cphd and sod , screened to date are consistent with our recently published data ( 20 ) . here , we report the identification of prokr2 variants in patients with craniofacial / midline disorders and hypopituitarism , thus suggesting an overlap in genotypes / phenotypes between these conditions and ks ( 20 ) . in our cohort , 9 of 11 patients were found to harbor previously described prokr2 variations that had been shown to be functionally deleterious in vitro ; the lack of dominant - negative effects of these variants suggests that their functional significance in vivo remains debatable ( 13 ) . however , the extent to which these variations contribute to the phenotype is yet to be established . of the 9 patients above , prokr2 variations were detected both in homozygosity ( n = 1 ) and in heterozygosity ( n = 8) . interestingly , 1 patient ( vi ) with sod / cphd and structural pituitary abnormalities was heterozygous for the prokr2 p.l173r variation , whereas his phenotypically normal mother was homozygous for the same variant ; this variant has previously been identified in several patients with ks and was shown to disrupt cell - surface targeting of the receptor in vitro ( 12 , 13 , 28 ) . we sought to better understand the phenotypes observed in our patients and the lack of a phenotype in the healthy homozygous mother by investigating a possible role for mprokr2 in the development and integrity of the hypothalamic - pituitary axis , using prokr2 null embryos as a model . our study of the homozygous knockout prokr2 mice revealed a morphologically normal pituitary and hypothalamus with normal hormone - secreting cells except for lh gonadotrophs , which were significantly reduced in the mutants and consistent with the previously reported ks - like phenotypes that these mice exhibit ( 16 , 17 ) . although this may not be a human model , the parallels between the normal hypothalamic - pituitary axis between our knockout mice and the healthy mother of patient vi are compelling and suggest that the variants identified in this report may not be sufficient to cause a hypothalamic - pituitary phenotype in isolation . in this cohort , the mother who was homozygous for the prokr2 p.l173r variation clearly did not have ks ( normosmic and fertile with 4 children that were conceived without assistance , and with normal gonadotropins and estrogen ) . therefore , the possibility that prokr2/prokr2 is not , in isolation , causative of ks either must be considered ; it may , however , contribute by modifying a phenotype caused by a defect in another gene(s ) or environmental factor(s ) , as recently postulated by raivio et al ( 20 ) . first , we detected functionally significant variants at the p.r85 and p.r268 alleles in heterozygosity or homozygosity in patients with phenotypes ranging from cphd to sod . they had previously only been detected in ks and healthy controls ( as had the p.l173r variant ) , albeit only in heterozygosity in the latter ( 12 ) . our data therefore strongly suggest that another gene or environmental factor is causing the more severe cphd - sod phenotypes . indeed , although not proven in any of our hypopituitary patients , digenic cases of ks involving prokr2 have been reported previously ( 12 , 15 , 2830 ) . this difference in the gene - dosage of prokr2 supports a contributory role to the pathogenesis of ks . no differences were observed between homozygous or heterozygous females , although this was attributed to the very low number of cases of the former ( n = 4 ) . care must be taken with the interpretation of results when patients presenting with such disorders also exhibit variations in the prokr2 gene , particularly with respect to genetic counseling . other genes known to be associated with these disorders should be screened and , should these be negative for mutations , then one can not rule out the presence of an as yet unidentified mutated gene or possible environmental factors . in addition to presenting with cphd / sod , some of our patients with prokr2 variants had additional manifestations including epilepsy , sleep abnormalities , gi dysmotility , and diabetes insipidus . although such variants have been detected for the first time in patients with the latter 2 disorders herein , a possible association is not altogether surprising given that prokineticin ligands and receptors are involved in various systems and processes including circadian rhythms in the brain , angiogenesis , neurogenesis , pain perception , immune responses , hematopoiesis , reproduction , and gi smooth muscle contraction ( 3137 ) . however , considering that our patient with diabetes insipidus and epilepsy ( iv ) was heterozygous for the same variant that the unaffected mother of patient vi had in homozygosity , it appears unlikely that mutated prokr2 is causative of these phenotypes . any contributions of prokr2 variants to these phenotypes would necessitate further studies , particularly with the aid of a postnatal murine model . however , the role of prokr2 is controversial ; heterozygous and homozygous variants occurring across the protein induce comparable phenotypes or , as we have shown , more severe phenotypes in cases of the former than the latter . this is compounded by the incidence of homozygosity in the healthy mother of a heterozygous child with a severe form of hypopituitarism in the form of sod . our analyses of the pituitary and hypothalamus in prokr2 knockout mice are largely inconsistent with the patient phenotypes , yet strongly support the normal presentation of the mother and her 3 unaffected ( with respect to hypopituitarism ) heterozygous children , although one needs to note the reduced lh in the murine null mutants . the number of genetically assigned causes of both ks and hypopituitarism is low , accounting for 30% of ks and 510% of sod and related midline disorders , and given that none of our patients harbored mutations in any of the known genes in these disorders , there are clearly other genetic / environmental factors yet to be discovered . subjects with sequence variants in prokr2 represent a unique cohort ; further careful genetic investigation is likely to aid in the identification of the missing genetic or epigenetic modifiers that interact with this pathway in humans to account for the phenotypic heterogeneity . further research into uncovering these additional factors would help define the role , if any , of prokr2 in these and other disorders discussed herein .

human cadaveric donor corneas not suitable for transplantation and preserved in optisol - gs ( baush & lomb , rochester , ny , usa ) were procured from the lions eye institute for transplant and research ( tampa , fl , usa ) , the national disease research interchange ( ndri ; philadelphia , pa , usa ) , eversight ( ann arbor , mi , usa ) , and the san diego eye bank ( san diego , ca , usa ) . donor confidentiality was maintained by the eye banks and by this laboratory according to the tenets of the declaration of helsinki . the age of donors ranged from 2 to 77 years , with preference for donors under 50-years old and endothelial cell counts above 2000 cells / mm . corneas from donors undergoing chemotherapy at the time of death and with history of diabetes or sepsis were excluded . the time from death to preservation was on average 13 hours and primary cultures of hcecs were initiated within 14 days of preservation in optisol - gs ( supplementary table s1 ) . briefly , corneas were rinsed three times in m199 medium ( gibco , rockville , md , usa ) with 50 g / ml gentamicin ( gibco ) . pieces of endothelium attached to descemet 's membrane were stripped off and stabilized overnight at 37c in 5% co2 in growth medium containing : optimem - i ( gibco ) , 8% fetal bovine serum ( fbs ; hyclone , logan , ut , usa ) , 5 ng / ml human recombinant endothelial growth factor ( peprotech , rocky hill , nj , usa ) , 20 ng / ml human recombinant neural growth factor ( peprotech ) , 100 g / ml bovine pituitary extract ( biomedical technologies , stoughton , ma , usa ) , 20 g / ml l - ascorbic acid ( sigma - aldrich corp . , st . louis , mo , usa ) or 0.5 mm l - ascorbic acid 2-phosphate ( sigma - aldrich corp . ) , 200 mg / l calcium chloride ( life technologies , carlsbad , ca , usa ) , 0.08% chondroitin sulfate ( sigma - aldrich corp . ) , 50 g / ml gentamicin ( gibco ) , and 1 antibiotic / antimycotic solution ( life technologies ) . the next day , the tissue was rinsed in hanks ' balanced salt solution ( gibco ) and incubated in 0.02% edta ( sigma - aldrich corp . ) for 1 hour at 37c . cells were released by passing the tissue 15 to 20 times through a glass pipette and then were resuspended in growth medium . isolated cells and remaining strips of descemet 's membrane from a single cornea were plated in a 3.8-cm tissue culture plate precoated with fnc coating mix ( athena environmental sciences , inc . , baltimore , md , usa ) ; this was considered passage 0 ( p0 ) . medium was changed every other day . for each cornea , the time from plating ( day 0 ) to next passage ( 80%90% confluency ) was recorded . cell passaging was performed by digesting confluent cultures with 0.05% trypsin ( gibco ) for 5 minutes at 37c in 5% co2 , and cell viability was evaluated by trypan blue exclusion assay ( sigma - aldrich corp . ) . briefly , human corneas were washed twice with m199 medium with 50 g / ml gentamicin . after the endothelium and the epithelium were removed , the central stroma was cut with an 8-mm biopsy punch ( sigma - aldrich corp . ) , and rinsed three times in pbs . stromal discs were minced into small pieces and seeded in culture plates . once firmly attached , cells were kept at 37c and 10% co2 , and fed every other day with dulbecco 's modified eagle 's medium supplemented with 10% fbs ( invitrogen ) and 50 g / ml gentamicin ( gibco ) . cultures were imaged in an axio observer a1 phase - contrast microscope ( carl zeiss microscopy , gmbh , oberkochen , germany ) . human corneal endothelial cells were cultured on fnc - coated glass coverslips ( carolina biological supply co. , burlington , nc , usa ) placed in 24-well plates . at approximately 90% confluence , cells were fixed in 4% paraformaldehyde ( electron microscopy sciences , hatfield , pa , usa ) for 10 minutes and rinsed three times with pbs . blocking and permeabilization were performed at room temperature for 30 minutes using 20% normal goat serum and 0.2% triton x-100 in antibody buffer ( 150 mm nacl , 50 mm tris base , 1% bsa , 100 mm l - lysine , 0.04% na azide [ ph = 7.4 ] ) . zo-1-alexa fluor-594-conjugated ( 1:100 ; life technologies ) and anti - human na / k / atpase ( 1:50 ; millipore , billerica , ma , usa ) . after three washes in pbs , coverslips were incubated in secondary antibodies diluted 1:500 in antibody buffer for 2 hours at room temperature or at 4c overnight , rinsed three times with pbs , mounted in vectashield with 4,6-diamidino-2-phenylindole ( dapi ) ( vector laboratories , burlingame , ca , usa ) , and imaged using the upright fluorescent microscope axio imager z1 or the confocal microscope zeiss lsm700 ( carl zeiss microscopy ) . cultured hcecs presenting canonical , fibroblastic , and mixed morphologies were trypsinized and dissociated into single cells , and counted using a neubauer chamber . one hundred thousand cells condition were resuspended in 100 l opti - mem - i without phenol red ( gibco ) and 5% fbs . cells were incubated in the dark at room temperature for 30 minutes with a combination of the following mouse anti - human monoclonal antibodies : cd56-apc , cd90-fitc , cd90-pe , cd90-cy7 , cd166-pe , cd73-fitc , and/or cd109-pe ( all from bd biosciences pharmigen , san jose , ca , usa ) , mouse anti - human car - pe , unconjugated mouse anti - human cd248 ( millipore ) , and human unconjugated na / k atpase ( abcam , cambridge , uk ) . all the primary antibodies were used at a 1:20 dilution . after incubating the cells with anti - cd248 or anti-1 na / k atpase , they were stained with 5% brilliant violet 421 goat anti - mouse igg secondary antibody ( biolegend , san diego , ca , usa ) for 15 minutes in the dark . flow cytometric data was acquired using a bd facs canto flow cytometer and facsdiva software ( bd biosciences , san jose , ca ) . for each run , voltages were adjusted using an unstained hcec control sample and single color positive controls ( anti - mouse beads , stained with one single antibody : abc anti - mouse bead kit ; invitrogen ) . data was stored in a fcs file format , and further analysis was performed using flowjo v x.0.7 ( flowjo llc , ashland , or , usa ) . briefly , a viable hcec population was isolated by gating on the fsc - a versus ssc - a dot plot ( population 1 ) . doublets were excluded by performing two consecutive additional gatings ( fsc - a versus fsc - w , population 2 and ssc - a versus ssc - w , population 3 ) . to quantify the expression of each marker , population three of each sample was used to create a fluorescence histogram . an overlay of the positive histogram and the negative control histogram was created for each fluorophore . three gates including 0.1% , 1% , or 10% of the negative population were selected , and the percentage of positive cells present in these gates was assessed . the threshold of 1% best highlighted the changes of marker expression between canonical , mixed , and fibroblastic cell populations , and therefore was selected for subsequent analysis . given the similarity of unstained canonical and fibroblastic hcecs when analyzed by flow cytometry , either canonical or fibroblastic hcecs were used as negative controls for each run . to compare the expression of cd56 to other markers ( cd109 , cd248 , and car ) , a quadrant dot plot was created , using the negative control population three to determine the limits of the four quadrants . fresh donor corneas stored in optisol - gs ( baush & lomb , rochester , ny , usa ) were minimally processed and the epithelial , stromal , and endothelial layers were isolated . ribonucleic acid extraction from donor corneas and cultured hcecs was performed using the rneasy kit ( hcecs , endothelium , and epithelium ; manufacturer 's protocol ; qiagen , valencia , ca ) or trizol ( stroma ) . purified rna was quantified and analyzed with a nanodrop spectrophotometer ( thermo scientific , wilmington , de , usa ) . per transwell , 20,000 cells were seeded in 24-well plates ( 6.5-mm diameter , 0.4-m pore ; costar , corning , ny , usa ) , previously coated with fnc . transendothelial electrical resistance was measured with an evom2 epithelial volt - ohmmeter ( world precision instruments , sarasota , fl , usa ) for 30 days or until readings reached a steady state , whichever happened first . transendothelial electrical resistance measurements were normalized to the value of the control wells ( growth media without cells ) . a bovine corneal endothelial cell line ( bcec ) was used as a positive control ( atcc crl-2048 , manassas , va , usa ) and fibroblastic hcecs were used as a negative control . ribonucleic acid from at least three biological replicates was independently collected as described above and its quality was assessed with nanodrop ( thermo scientific ) and confirmed with rin ( rna integrity number ) at the university of miami center for genome technology ( miami , fl , usa ) . amplification and processing for hybridization to genechip human gene st 1.0 arrays ( affymetrix , santa clara , ca , usa ) were performed at the university of miami center for genome technology ( miami , fl , usa ) following standard protocols . data were normalized with genespring 12.0 ( agilent , santa clara , ca , usa ) and filtered by intensity . probes between the 20th and 99th percentile range in at least two of three samples per condition were included in the analysis . statistical comparison between conditions was performed using unpaired student 's t - test with benjamini - hochberg correction for multiple testing . final data analysis was conducted using excel software ( microsoft corporation , redmond , wa , usa ) and genego ( thomson reuters , philadelphia , pa , usa ) . statistical analysis was performed using unpaired , 2-tailed student 's t - test ; p less than 0.05 was considered statistically significant . human cadaveric donor corneas not suitable for transplantation and preserved in optisol - gs ( baush & lomb , rochester , ny , usa ) were procured from the lions eye institute for transplant and research ( tampa , fl , usa ) , the national disease research interchange ( ndri ; philadelphia , pa , usa ) , eversight ( ann arbor , mi , usa ) , and the san diego eye bank ( san diego , ca , usa ) . donor confidentiality was maintained by the eye banks and by this laboratory according to the tenets of the declaration of helsinki . the age of donors ranged from 2 to 77 years , with preference for donors under 50-years old and endothelial cell counts above 2000 cells / mm . corneas from donors undergoing chemotherapy at the time of death and with history of diabetes or sepsis were excluded . the time from death to preservation was on average 13 hours and primary cultures of hcecs were initiated within 14 days of preservation in optisol - gs ( supplementary table s1 ) . briefly , corneas were rinsed three times in m199 medium ( gibco , rockville , md , usa ) with 50 g / ml gentamicin ( gibco ) . pieces of endothelium attached to descemet 's membrane were stripped off and stabilized overnight at 37c in 5% co2 in growth medium containing : optimem - i ( gibco ) , 8% fetal bovine serum ( fbs ; hyclone , logan , ut , usa ) , 5 ng / ml human recombinant endothelial growth factor ( peprotech , rocky hill , nj , usa ) , 20 ng / ml human recombinant neural growth factor ( peprotech ) , 100 g / ml bovine pituitary extract ( biomedical technologies , stoughton , ma , usa ) , 20 g / ml l - ascorbic acid ( sigma - aldrich corp . , st . louis , mo , usa ) or 0.5 mm l - ascorbic acid 2-phosphate ( sigma - aldrich corp . ) , 200 mg / l calcium chloride ( life technologies , carlsbad , ca , usa ) , 0.08% chondroitin sulfate ( sigma - aldrich corp . ) , 50 g / ml gentamicin ( gibco ) , and 1 antibiotic / antimycotic solution ( life technologies ) . the next day , the tissue was rinsed in hanks ' balanced salt solution ( gibco ) and incubated in 0.02% edta ( sigma - aldrich corp . ) for 1 hour at 37c . cells were released by passing the tissue 15 to 20 times through a glass pipette and then were resuspended in growth medium . isolated cells and remaining strips of descemet 's membrane from a single cornea were plated in a 3.8-cm tissue culture plate precoated with fnc coating mix ( athena environmental sciences , inc . , baltimore , md , usa ) ; this was considered passage 0 ( p0 ) . medium was changed every other day . for each cornea , the time from plating ( day 0 ) to next passage ( 80%90% confluency ) was recorded . cell passaging was performed by digesting confluent cultures with 0.05% trypsin ( gibco ) for 5 minutes at 37c in 5% co2 , and cell viability was evaluated by trypan blue exclusion assay ( sigma - aldrich corp . ) . briefly , human corneas were washed twice with m199 medium with 50 g / ml gentamicin . after the endothelium and the epithelium were removed , the central stroma was cut with an 8-mm biopsy punch ( sigma - aldrich corp . ) , and rinsed three times in pbs . once firmly attached , cells were kept at 37c and 10% co2 , and fed every other day with dulbecco 's modified eagle 's medium supplemented with 10% fbs ( invitrogen ) and 50 g / ml gentamicin ( gibco ) . cultures were imaged in an axio observer a1 phase - contrast microscope ( carl zeiss microscopy , gmbh , oberkochen , germany ) . human corneal endothelial cells were cultured on fnc - coated glass coverslips ( carolina biological supply co. , burlington , nc , usa ) placed in 24-well plates . at approximately 90% confluence , cells were fixed in 4% paraformaldehyde ( electron microscopy sciences , hatfield , pa , usa ) for 10 minutes and rinsed three times with pbs . blocking and permeabilization were performed at room temperature for 30 minutes using 20% normal goat serum and 0.2% triton x-100 in antibody buffer ( 150 mm nacl , 50 mm tris base , 1% bsa , 100 mm l - lysine , 0.04% na azide [ ph = 7.4 ] ) . zo-1-alexa fluor-594-conjugated ( 1:100 ; life technologies ) and anti - human na / k / atpase ( 1:50 ; millipore , billerica , ma , usa ) . after three washes in pbs , coverslips were incubated in secondary antibodies diluted 1:500 in antibody buffer for 2 hours at room temperature or at 4c overnight , rinsed three times with pbs , mounted in vectashield with 4,6-diamidino-2-phenylindole ( dapi ) ( vector laboratories , burlingame , ca , usa ) , and imaged using the upright fluorescent microscope axio imager z1 or the confocal microscope zeiss lsm700 ( carl zeiss microscopy ) . cultured hcecs presenting canonical , fibroblastic , and mixed morphologies were trypsinized and dissociated into single cells , and counted using a neubauer chamber . one hundred thousand cells condition were resuspended in 100 l opti - mem - i without phenol red ( gibco ) and 5% fbs . cells were incubated in the dark at room temperature for 30 minutes with a combination of the following mouse anti - human monoclonal antibodies : cd56-apc , cd90-fitc , cd90-pe , cd90-cy7 , cd166-pe , cd73-fitc , and/or cd109-pe ( all from bd biosciences pharmigen , san jose , ca , usa ) , mouse anti - human car - pe , unconjugated mouse anti - human cd248 ( millipore ) , and human unconjugated na / k atpase ( abcam , cambridge , uk ) . after incubating the cells with anti - cd248 or anti-1 na / k atpase , they were stained with 5% brilliant violet 421 goat anti - mouse igg secondary antibody ( biolegend , san diego , ca , usa ) for 15 minutes in the dark . flow cytometric data was acquired using a bd facs canto flow cytometer and facsdiva software ( bd biosciences , san jose , ca ) . for each run , voltages were adjusted using an unstained hcec control sample and single color positive controls ( anti - mouse beads , stained with one single antibody : abc anti - mouse bead kit ; invitrogen ) . data was stored in a fcs file format , and further analysis was performed using flowjo v x.0.7 ( flowjo llc , ashland , or , usa ) . briefly , a viable hcec population was isolated by gating on the fsc - a versus ssc - a dot plot ( population 1 ) . doublets were excluded by performing two consecutive additional gatings ( fsc - a versus fsc - w , population 2 and ssc - a versus ssc - w , population 3 ) . to quantify the expression of each marker , population three of each sample was used to create a fluorescence histogram . an overlay of the positive histogram and the negative control histogram was created for each fluorophore . three gates including 0.1% , 1% , or 10% of the negative population were selected , and the percentage of positive cells present in these gates was assessed . the threshold of 1% best highlighted the changes of marker expression between canonical , mixed , and fibroblastic cell populations , and therefore was selected for subsequent analysis . given the similarity of unstained canonical and fibroblastic hcecs when analyzed by flow cytometry , either canonical or fibroblastic hcecs were used as negative controls for each run . to compare the expression of cd56 to other markers ( cd109 , cd248 , and car ) , a quadrant dot plot was created , using the negative control population three to determine the limits of the four quadrants . fresh donor corneas stored in optisol - gs ( baush & lomb , rochester , ny , usa ) were minimally processed and the epithelial , stromal , and endothelial layers were isolated . ribonucleic acid extraction from donor corneas and cultured hcecs was performed using the rneasy kit ( hcecs , endothelium , and epithelium ; manufacturer 's protocol ; qiagen , valencia , ca ) or trizol ( stroma ) . purified rna was quantified and analyzed with a nanodrop spectrophotometer ( thermo scientific , wilmington , de , usa ) . per transwell , 20,000 cells were seeded in 24-well plates ( 6.5-mm diameter , 0.4-m pore ; costar , corning , ny , usa ) , previously coated with fnc . transendothelial electrical resistance was measured with an evom2 epithelial volt - ohmmeter ( world precision instruments , sarasota , fl , usa ) for 30 days or until readings reached a steady state , whichever happened first . transendothelial electrical resistance measurements were normalized to the value of the control wells ( growth media without cells ) . a bovine corneal endothelial cell line ( bcec ) was used as a positive control ( atcc crl-2048 , manassas , va , usa ) and fibroblastic hcecs were used as a negative control . ribonucleic acid from at least three biological replicates was independently collected as described above and its quality was assessed with nanodrop ( thermo scientific ) and confirmed with rin ( rna integrity number ) at the university of miami center for genome technology ( miami , fl , usa ) . amplification and processing for hybridization to genechip human gene st 1.0 arrays ( affymetrix , santa clara , ca , usa ) were performed at the university of miami center for genome technology ( miami , fl , usa ) following standard protocols . data were normalized with genespring 12.0 ( agilent , santa clara , ca , usa ) and filtered by intensity . probes between the 20th and 99th percentile range in at least two of three samples per condition were included in the analysis . statistical comparison between conditions was performed using unpaired student 's t - test with benjamini - hochberg correction for multiple testing . final data analysis was conducted using excel software ( microsoft corporation , redmond , wa , usa ) and genego ( thomson reuters , philadelphia , pa , usa ) . statistical analysis was performed using unpaired , 2-tailed student 's t - test ; p less than 0.05 was considered statistically significant . we first asked whether hcecs in vitro maintain the characteristics observed in vivo , namely cell cell contact inhibition and the canonical cobblestone - like or polygonal morphology . corneal endothelial cells were isolated and cultured from cadaveric donor corneas following a previously published method outlined in figure 1a . cells cultured at high density and for a lower number of passages often formed a monolayer with polygonal canonical typically , the canonical morphology was maintained until passage three or four , similar to previous observations . at later passages , cells often underwent enmt , exhibiting fibroblastic morphology , and losing cell cell contact inhibition ( fig . an exceptional culture from a 15-year - old donor was cultured up to passage 10 without signs of fibroblastic conversion , but at passage 12 , senescence was evident ( fig . 1 g ) as cells became enlarged and proliferation rate dramatically decreased ( not shown ) . overall , hcecs from younger corneas , cultured in vitro , were expanded for 3 or 4 passages , with each cornea yielding a variable number of total cell progeny ( fig . 1h ) that may be adequate to treat several patients . human corneal endothelial cells isolation and culture . g ) bright - field micrographs of cultured hcecs at different passage ( p ) numbers . primary cultures of hcecs often demonstrated the distinctive cobblestone - like morphology until p3 or p4 ( b e ) ; at later passages ( g ) an exceptional culture maintained canonical morphology to p10 , but by p12 showed senescent characteristics including lengthened cells and slowed growth rate . ( h ) cell yields after expansion of hcecs from corneas of young donors for three or four passages . ( i ) scatter plot of the time to confluency in relation to donor age : hcecs cultured from younger donors ( average age : 22 years old ; range , 034 years ; n = 35 ) showed significantly greater proliferation rates ( * * * p < 0.0001 ) compared with older donors ( average age : 50 years old ; range , 3577 years ; n = 20 ) . ( j ) there is a weak correlation between hcec density and in vitro proliferation ( r = 24% ) , but the correlation is statistically significant ( p = 0.0002 ) . ( k ) there was a statistically significant difference between corneal endothelial density measured before enucleation in younger donors ( average endothelial cell density : 3181.6 mm ; range , 25714425 mm ; n = 30 ) compared with older donors ( average endothelial cell density : 2761.5 mm ; range , 19692865 mm ; n = 11 ; p = 0.02 ) . we asked whether the age of the donor influenced culture quality , as has been previously suggested . we looked at the time to reach confluency from passage 0 ( p0 ) to passage 1 ( p1 ) and found that corneas from younger donors ( 2- to 34-years old ) took , on average , 11 days to become confluent , whereas corneas from older donors ( 38- to 77-years old ) took 19 days ( fig . we also found a weak but significant correlation between initial endothelial cell density and time to confluency ( fig . finally , there was a significant difference in initial endothelial cell density between corneas from young donors ( 2- to 34-years old : average endothelial cell density : 3181.6 mm ; range , 25714425 mm ; n = 30 ) and those from older donors ( 38- to 77-years old : average endothelial cell density : 2761.5 mm ; range 19692865 mm ; n = 11 ) . tissue from younger donors had significantly higher endothelial cell counts compared with older donors ( p = 0.02 ; fig . we generally observed that cultures from younger donors demonstrated better attachment and a more uniform morphology . however , cultures from young donors with sepsis or undergoing chemotherapy were not successful , suggesting a direct relationship between hcec culture outcome and donor age and health . there is a paucity of hcec - specific identity markers , and the expression of proteins expressed ubiquitously in tight junction complexes is often cited for identity criteria . for example , we found cultured hcecs expressed such markers including the tight junction protein zo-1 and the channel na / k - atpase . human corneal endothelial cells in culture , morphologically similar to cells represented in figures 1b to 1e , immunostained for zo-1 exhibited typical honeycomb staining at the tight junctions , while na / k - atpase expression was found basolaterally ( fig . 2a ) , but na / k - atpase was also expressed by hcecs with fibroblastic morphology ( fig . we also examined the barrier function of the hcec monolayer using a teer assay , where higher resistance values indicate less permeability at intercellular junctions and therefore , better function . we hypothesized that cells undergoing enmt and demonstrating fibroblastic morphology would have lower barrier function . we found that cultures with the canonical cobblestone - like morphology reached significantly higher teer values than fibroblastic cultures , and cultures with mixed morphology of fibroblastic and hexagonal cells demonstrated intermediate values of teer ( fig . 2c ) . thus , hcecs can be identified by the expression of a combination of markers , and their function can be determined by teer , but the markers used routinely may not have the ability to differentiate canonical hcecs from fibroblastic hcecs or stromal fibroblasts cultured hcecs express characteristic tight - junction - associated markers . ( a ) confocal micrographs of cultured hcecs immunostained for zo-1 and na / k - atpase ; nuclei were counter - stained with dapi . ( b ) bar graph showing no difference between canonical and fibroblastic hcecs in na / k - atpase expression by flow cytometry ( n = 3 ) . histograms of one representative flow cytometry run show no difference in na / k - atpase expression between canonical and fibroblastic cells . ( c ) human corneal endothelial cells function assessed by teer measurements from different hcec cultures . a bovine corneal endothelial cell line ( bcec - line ) was used as positive control ( n = 4 ) . p values resulting from the statistical analysis of teer measured on canonical hcecs compared with mixed 1 , mixed 2 , and fibroblastic hcecs are presented in the table below the graph . top 10 hcec surface markers because the routinely used markers such as zo-1 appear insufficient to distinguish between canonical and fibroblastic hcecs , ( fig . 2 ) , and identifying specific markers for hcecs with the highest barrier function remains a major interest , we next tried to find surface markers to discern between these cell phenotypes . we performed a microarray analysis comparing the transcriptomes of freshly dissected corneal layers ( epithelium , stroma , and endothelium ) and of p0-cultured hcecs , and found that of 28,869 probes , 23,286 ( 81% ) were expressed by at least two of three replicates within at least one condition . ignoring level of expression , many genes were expressed by multiple tissues but some were expressed by subsets or uniquely in single tissues or cells ( fig . principal component analysis revealed four distinct clusters that matched the different tissue samples ( fig . there was very little intersample variability , with pearson 's correlation ( r ) greater than 0.94 ( fig . we found that cultured hcecs were more similar to endothelium than to stroma and epithelium ( fig . 3d ) . thus transcriptomic analysis points to the consistency of cell types in vitro and to the similarity between hcecs in vivo and in vitro . ( a ) venn diagram representing the probes expressed in the microarray dataset in the three corneal layers and freshly cultured hcecs . ( b ) principal component analysis revealed distinct clusters per sample type as labeled ; biological replicates ( dots with the same color ) clustered together . ( c ) pearson correlation ( r ) was higher within biological replicates than across different tissues ( n = 3 ) . ( d ) hierarchical clustering demonstrated that hcecs and endothelium were more closely related than epithelium and stroma . we subsequently focused our analysis on a subset of surface markers with high expression in the endothelium ( p0-hcecs and freshly dissected tissue ) but low expression in stroma , and that did not differ significantly between cultured or fresh endothelium ( table ) . to address whether the expression of such markers in hcecs was affected by fibroblastic enmt , hcec cultures demonstrating canonical , mixed and fibroblastic morphologies ( figs . 5a c ) were assessed for expression of the surface proteins cd56 , car , cd248 , and cd109 by flow cytometry . in a series of independent experiments , we observed a significant difference between canonical and fibroblastic cells in the expression of cd56 , car , cd248 , and cd109 surface markers ( figs . whereas 97% of canonical hcecs were positive for cd56 , 92% for cd248 , and 82% were positive for car , these markers decreased as the cells lost their canonical morphology and became fibroblastic . conversely , cd109 expression was less often detected in canonical hcecs ( 26% on average ) , and increased in expression when the cells underwent enmt to 52% in fibroblastic cell cultures . thus , this series of markers detects shifts in cultured human hcecs from canonical to fibroblastic morphology . ( b ) representative 2d dot plots showing no difference in size and internal complexity between unstained canonical and unstained fibroblastic hcecs . ( c ) representative histogram showing no difference in baseline fluorescence between unstained canonical and fibroblastic hcecs . flow cytometry analysis of surface markers expression differentiates hcec subpopulations in culture . ( a c ) three morphologically distinct hcec cultures , canonical , mixed , and fibroblastic as marked , were carried forward for flow cytometry . a threshold at the top 1% of negative control cells was set to identify positive cells throughout all the independent experiments . quantification of the percentage of positive cells for each marker showed that cd56 ( d ) , cd248 ( e ) , and car ( f ) expressions are low in a fibroblastic culture , while cd109 ( g ) is high . data is representative of three or more independent experiments from separate corneal cultures ( cd56 : n = 10 ; cd109 : n = 6 ; cd248 : n = 3 ; car : n = 4 ; p values : # 0.1 ; * < 0.05 ; * * < 0.001 ; * * * < 0.0001 ) . ( a c ) flow cytometry analysis by dual - color fluorescent dot plot histograms for the canonical ( a ) , mixed ( b ) , and fibroblastic ( c ) hcec cultures show shift in the expression of cd56 , cd248 , car , and cd109 surface markers . each graph is divided into four quadrants determined by the autofluorescence of unstained control cells as in figure 4 , and gated to include 99% of unstained cells in the lower left quadrant ( all negative markers ) . ( d f ) quantification of the percentage of canonical , fibroblastic , and mixed cell populations expressing markers tested in pairs , as marked . ( g ) quantification of flow cytometry experiments showing no difference between canonical and fibroblastic cells in cd166/alcam , cd73 , cd9 , cd90 , and 1na / k atpase expression . ( h ) transendothelial electrical resistance assay using in vitro expanded hcecs whose cd56 expression had been determined by flow cytometry , demonstrating the greater ability of canonical cd56 cells than fibroblastic cd56 to form a barrier . recently other groups reported the expression of cd73 , cd166 , cd9 , and cd90 in corneal endothelial cells . we analyzed by flow cytometry canonical and fibroblastic hcecs in passages two to three and five through eight , respectively , and the high expression of those markers did not vary with morphology ( fig . thus , while these markers may be used to identify hcecs , they may not be adequate to select cell phenotypes based on morphology or function . finally , we examined whether marker expression predicted functional capacity in an in vitro teer assay . we used confluent hcecs that we plated for teer , isolating a subset to be tested by flow cytometry for cd56 expression . cells exhibiting a canonical morphology and a cd56 marker expression demonstrated a superior barrier formation ability measured by teer , compared with morphologically mixed or fibroblastic , cd56 cells ( fig . thus , surface makers together with morphology can be used to characterize a functionally superior hcec culture . we first asked whether hcecs in vitro maintain the characteristics observed in vivo , namely cell cell contact inhibition and the canonical cobblestone - like or polygonal morphology . corneal endothelial cells were isolated and cultured from cadaveric donor corneas following a previously published method outlined in figure 1a . cells cultured at high density and for a lower number of passages often formed a monolayer with polygonal canonical typically , the canonical morphology was maintained until passage three or four , similar to previous observations . at later passages , cells often underwent enmt , exhibiting fibroblastic morphology , and losing cell cell contact inhibition ( fig . an exceptional culture from a 15-year - old donor was cultured up to passage 10 without signs of fibroblastic conversion , but at passage 12 , senescence was evident ( fig . 1 g ) as cells became enlarged and proliferation rate dramatically decreased ( not shown ) . overall , hcecs from younger corneas , cultured in vitro , were expanded for 3 or 4 passages , with each cornea yielding a variable number of total cell progeny ( fig . 1h ) that may be adequate to treat several patients . human corneal endothelial cells isolation and culture . g ) bright - field micrographs of cultured hcecs at different passage ( p ) numbers . primary cultures of hcecs often demonstrated the distinctive cobblestone - like morphology until p3 or p4 ( b e ) ; at later passages ( g ) an exceptional culture maintained canonical morphology to p10 , but by p12 showed senescent characteristics including lengthened cells and slowed growth rate . ( h ) cell yields after expansion of hcecs from corneas of young donors for three or four passages . ( i ) scatter plot of the time to confluency in relation to donor age : hcecs cultured from younger donors ( average age : 22 years old ; range , 034 years ; n = 35 ) showed significantly greater proliferation rates ( * * * p < 0.0001 ) compared with older donors ( average age : 50 years old ; range , 3577 years ; n = 20 ) . ( j ) there is a weak correlation between hcec density and in vitro proliferation ( r = 24% ) , but the correlation is statistically significant ( p = 0.0002 ) . ( k ) there was a statistically significant difference between corneal endothelial density measured before enucleation in younger donors ( average endothelial cell density : 3181.6 mm ; range , 25714425 mm ; n = 30 ) compared with older donors ( average endothelial cell density : 2761.5 mm ; range , 19692865 mm ; n = 11 ; p = 0.02 ) . we asked whether the age of the donor influenced culture quality , as has been previously suggested . we looked at the time to reach confluency from passage 0 ( p0 ) to passage 1 ( p1 ) and found that corneas from younger donors ( 2- to 34-years old ) took , on average , 11 days to become confluent , whereas corneas from older donors ( 38- to 77-years old ) took 19 days ( fig . we also found a weak but significant correlation between initial endothelial cell density and time to confluency ( fig . finally , there was a significant difference in initial endothelial cell density between corneas from young donors ( 2- to 34-years old : average endothelial cell density : 3181.6 mm ; range , 25714425 mm ; n = 30 ) and those from older donors ( 38- to 77-years old : average endothelial cell density : 2761.5 mm ; range 19692865 mm ; n = 11 ) . tissue from younger donors had significantly higher endothelial cell counts compared with older donors ( p = 0.02 ; fig . we generally observed that cultures from younger donors demonstrated better attachment and a more uniform morphology . however , cultures from young donors with sepsis or undergoing chemotherapy were not successful , suggesting a direct relationship between hcec culture outcome and donor age and health . there is a paucity of hcec - specific identity markers , and the expression of proteins expressed ubiquitously in tight junction complexes is often cited for identity criteria . for example , we found cultured hcecs expressed such markers including the tight junction protein zo-1 and the channel na / k - atpase . human corneal endothelial cells in culture , morphologically similar to cells represented in figures 1b to 1e , immunostained for zo-1 exhibited typical honeycomb staining at the tight junctions , while na / k - atpase expression was found basolaterally ( fig . 2a ) , but na / k - atpase was also expressed by hcecs with fibroblastic morphology ( fig . we also examined the barrier function of the hcec monolayer using a teer assay , where higher resistance values indicate less permeability at intercellular junctions and therefore , better function . we hypothesized that cells undergoing enmt and demonstrating fibroblastic morphology would have lower barrier function . we found that cultures with the canonical cobblestone - like morphology reached significantly higher teer values than fibroblastic cultures , and cultures with mixed morphology of fibroblastic and hexagonal cells demonstrated intermediate values of teer ( fig . thus , hcecs can be identified by the expression of a combination of markers , and their function can be determined by teer , but the markers used routinely may not have the ability to differentiate canonical hcecs from fibroblastic hcecs or stromal fibroblasts ( a ) confocal micrographs of cultured hcecs immunostained for zo-1 and na / k - atpase ; nuclei were counter - stained with dapi . ( b ) bar graph showing no difference between canonical and fibroblastic hcecs in na / k - atpase expression by flow cytometry ( n = 3 ) . histograms of one representative flow cytometry run show no difference in na / k - atpase expression between canonical and fibroblastic cells . ( c ) human corneal endothelial cells function assessed by teer measurements from different hcec cultures . a bovine corneal endothelial cell line ( bcec - line ) p values resulting from the statistical analysis of teer measured on canonical hcecs compared with mixed 1 , mixed 2 , and fibroblastic hcecs are presented in the table below the graph . because the routinely used markers such as zo-1 appear insufficient to distinguish between canonical and fibroblastic hcecs , ( fig . 2 ) , and identifying specific markers for hcecs with the highest barrier function remains a major interest , we next tried to find surface markers to discern between these cell phenotypes . we performed a microarray analysis comparing the transcriptomes of freshly dissected corneal layers ( epithelium , stroma , and endothelium ) and of p0-cultured hcecs , and found that of 28,869 probes , 23,286 ( 81% ) were expressed by at least two of three replicates within at least one condition . ignoring level of expression , many genes were expressed by multiple tissues but some were expressed by subsets or uniquely in single tissues or cells ( fig . principal component analysis revealed four distinct clusters that matched the different tissue samples ( fig . there was very little intersample variability , with pearson 's correlation ( r ) greater than 0.94 ( fig . we found that cultured hcecs were more similar to endothelium than to stroma and epithelium ( fig . 3d ) . thus transcriptomic analysis points to the consistency of cell types in vitro and to the similarity between hcecs in vivo and in vitro . ( a ) venn diagram representing the probes expressed in the microarray dataset in the three corneal layers and freshly cultured hcecs . ( b ) principal component analysis revealed distinct clusters per sample type as labeled ; biological replicates ( dots with the same color ) clustered together . ( c ) pearson correlation ( r ) was higher within biological replicates than across different tissues ( n = 3 ) . ( d ) hierarchical clustering demonstrated that hcecs and endothelium were more closely related than epithelium and stroma . we subsequently focused our analysis on a subset of surface markers with high expression in the endothelium ( p0-hcecs and freshly dissected tissue ) but low expression in stroma , and that did not differ significantly between cultured or fresh endothelium ( table ) . to address whether the expression of such markers in hcecs was affected by fibroblastic enmt , hcec cultures demonstrating canonical , mixed and fibroblastic morphologies ( figs . 5a c ) were assessed for expression of the surface proteins cd56 , car , cd248 , and cd109 by flow cytometry . in a series of independent experiments , we observed a significant difference between canonical and fibroblastic cells in the expression of cd56 , car , cd248 , and cd109 surface markers ( figs . whereas 97% of canonical hcecs were positive for cd56 , 92% for cd248 , and 82% were positive for car , these markers decreased as the cells lost their canonical morphology and became fibroblastic . conversely , cd109 expression was less often detected in canonical hcecs ( 26% on average ) , and increased in expression when the cells underwent enmt to 52% in fibroblastic cell cultures . thus , this series of markers detects shifts in cultured human hcecs from canonical to fibroblastic morphology . ( b ) representative 2d dot plots showing no difference in size and internal complexity between unstained canonical and unstained fibroblastic hcecs . ( c ) representative histogram showing no difference in baseline fluorescence between unstained canonical and fibroblastic hcecs . ( a c ) three morphologically distinct hcec cultures , canonical , mixed , and fibroblastic as marked , were carried forward for flow cytometry . a threshold at the top 1% of negative control cells was set to identify positive cells throughout all the independent experiments . quantification of the percentage of positive cells for each marker showed that cd56 ( d ) , cd248 ( e ) , and car ( f ) expressions are low in a fibroblastic culture , while cd109 ( g ) is high . data is representative of three or more independent experiments from separate corneal cultures ( cd56 : n = 10 ; cd109 : n = 6 ; cd248 : n = 3 ; car : n = 4 ; p values : # 0.1 ; * < 0.05 ; * * < 0.001 ; * * * < 0.0001 ) . ( a c ) flow cytometry analysis by dual - color fluorescent dot plot histograms for the canonical ( a ) , mixed ( b ) , and fibroblastic ( c ) hcec cultures show shift in the expression of cd56 , cd248 , car , and cd109 surface markers . each graph is divided into four quadrants determined by the autofluorescence of unstained control cells as in figure 4 , and gated to include 99% of unstained cells in the lower left quadrant ( all negative markers ) . ( d f ) quantification of the percentage of canonical , fibroblastic , and mixed cell populations expressing markers tested in pairs , as marked . ( g ) quantification of flow cytometry experiments showing no difference between canonical and fibroblastic cells in cd166/alcam , cd73 , cd9 , cd90 , and 1na / k atpase expression . ( h ) transendothelial electrical resistance assay using in vitro expanded hcecs whose cd56 expression had been determined by flow cytometry , demonstrating the greater ability of canonical cd56 cells than fibroblastic cd56 to form a barrier . recently other groups reported the expression of cd73 , cd166 , cd9 , and cd90 in corneal endothelial cells . we analyzed by flow cytometry canonical and fibroblastic hcecs in passages two to three and five through eight , respectively , and the high expression of those markers did not vary with morphology ( fig . thus , while these markers may be used to identify hcecs , they may not be adequate to select cell phenotypes based on morphology or function . finally , we examined whether marker expression predicted functional capacity in an in vitro teer assay . we used confluent hcecs that we plated for teer , isolating a subset to be tested by flow cytometry for cd56 expression . cells exhibiting a canonical morphology and a cd56 marker expression demonstrated a superior barrier formation ability measured by teer , compared with morphologically mixed or fibroblastic , cd56 cells ( fig . thus , surface makers together with morphology can be used to characterize a functionally superior hcec culture . corneal endothelial insufficiency is currently treated by different variants of corneal transplant , replacing either the whole cornea or the endothelial layer . these are limited by tissue availability , access to highly trained specialists , surgical complications , immune rejection , and cost . here we describe steps toward corneal endothelial cell therapy based on in vitro culture , expansion and identification of high quality , functional hcecs . a successful culture starts with good quality donor tissue : the origin and qualities of the donor cornea including donor age , health status , and use of chemotherapy or other toxic substances play a large part in proliferative potential , morphology , and function . here , we also documented the positive correlation between younger donor age and proliferation and total cell yield . with increasing numbers of passages in culture , enmt often occurs and the hcecs lose their canonical cobblestone morphology and functional efficacy in barrier formation , in vitro and in vivo . molecular and cellular mechanisms are not yet completely understood , but inflammatory molecules such as il-1 have been implicated in initiating a cascade of events leading to the activation of pi-3 kinase and fibroblast growth factor 2 . to date , medium optimization , addition of growth factors and optimization of growth substrates have increased cell yields of hcec cultures , but may not prevent fibroblastic transformation . given the loss of function of fibroblastic hcecs , the ability to identify and select functional canonical cells in a mixed culture is critical . our data confirms in canonical cultures the expression of zo-1 and na / k / atpase , typical markers of the endothelial monolayer . however , these markers are expressed by canonical hcecs only while confluent and forming tight junctions , and their expression decreases once the cells are removed from a monolayer . this underlines the necessity for identifying other markers for canonical hcecs in suspension . to pursue this , we used microarray analysis of corneal epithelial , stromal , and endothelial tissue , as well as in vitro cultured hcecs to isolate candidate surface markers present either exclusively in the stroma , or in both the native endothelium and the in vitro cultured hcecs . we further tested the expression levels of nine of these candidate markers by flow cytometry in canonical , mixed , and fibroblastic in vitro cultured hcecs . we found four surface - expressed proteins that differ significantly between canonical and fibroblastic hcecs . cd56 , or neural cell adhesion molecule ( ncam ) , is a surface glycoprotein that exists in multiple isoforms and is expressed by multiple cell types . it plays an important role in cell adhesion and cell interactions , migration , and embryogenesis . neural cell adhesion molecule expression has been demonstrated in the embryonic chick cornea , with decrease in expression and localization toward the posterior regions of the cornea after birth . neural cell adhesion molecule has also been localized to the mouse retina and human adult corneal endothelium , consistent with hcecs ' embryologic origin from neural crest cells . neural cell adhesion molecule is expressed by hcecs produced in vitro , however , as the cells undergo enmt , its expression decreases . importantly , we found that cd56 expression in cultures also correlated with higher functional barrier capacity in teer assays . cd248 , or endosialin , belongs to the family of c - type lectin transmembrane receptors that may play a role in cell cell adhesion , host defense , and tumor neoangiogenesis , but their role in hcec biology is unknown . a third canonical marker identified here , coxsackie adenovirus receptor ( car ) , is part of the junctional adhesion molecules ( jams ) family known to participate in a variety of cellular processes , such as leukocyte - platelet - vascular endothelium interactions , and tight - junction formation in epithelial and endothelial cells . the presence of car in the corneal endothelium was previously described , but its biological significance remains unknown . finally , cd109 , a cell surface antigen involved in the tgf- pathway , has been studied in white blood cells , vascular endothelial cells , keratinocytes , and activated platelets . its expression increases in some malignancies , an interesting observation consistent with our data demonstrating increased expression as hcecs undergo enmt . for all of these new markers , other potential candidates have been identified from microarray analyses and tested via flow cytometry to differentiate canonical hcecs from hcecs undergoing enmt , including cd166 ( alcam ) , expressed in corneal epithelial limbal stem cells but also in corneal stromal stem cells , cd90 , expressed in corneal stromal fibroblasts , but also in canonical corneal endothelial cells , and cd73 and cd9 . for these last two , no difference between corneal layers was detected by microarray , and further testing by flow cytometry remained negative in our experiments . given our testing of both gene and protein expression , and use of exclusively human corneal tissue and primary corneal endothelial cells , these data raise questions as to whether cd9 and cd73 will be useful markers to differentiate canonical from fibroblastic hcecs . together , the specific surface markers identified here and their correlation to cell function represent a novel set of criteria for the selection of in vitro expanded hcecs that we would hypothesize are most likely to be functionally competent to replace damaged corneal endothelial cells , and thus represent a step toward hcec therapy . along with hcec culture and cell characterization , effort directed toward delivering in vitro cultured hcecs to the patient 's endothelial surface ongoing efforts should demonstrate the feasibility of hcec in vitro expansion , selection of cultured hcecs with highest function , and development of cell delivery approaches . recent animal studies using corneal endothelial cells revealed that the cells integrate in vivo to the host endothelium , and are able to restore corneal transparency after injury , but many questions such as dosage and stability remain unanswered . our donor tissue selection criteria , together with the exhaustive characterization provided in this study , are essential for obtaining high quality , functional cultured hcecs , bringing us a step closer to the clinic with a human corneal endothelial cell therapy .

purposehuman corneal endothelial cell ( hcec ) density decreases with age , surgical complications , or disease , leading to vision impairment . such endothelial dysfunction is an indication for corneal transplantation , although there is a worldwide shortage of transplant - grade tissue . to overcome the current poor donor availability , here we isolate , expand , and characterize hcecs in vitro as a step toward cell therapy.methodshuman corneal endothelial cells were isolated from cadaveric corneas and expanded in vitro . cell identity was evaluated based on morphology and immunocytochemistry , and gene expression analysis and flow cytometry were used to identify novel hcec - specific markers . the functional ability of hcec to form barriers was assessed by transendothelial electrical resistance ( teer ) assays.resultscultured hcecs demonstrated canonical morphology for up to four passages and later underwent endothelial - to - mesenchymal transition ( enmt ) . quality of donor tissue influenced cell measures in culture including proliferation rate . cultured hcecs expressed identity markers , and microarray analysis revealed novel endothelial - specific markers that were validated by flow cytometry . finally , canonical hcecs expressed higher levels of cd56 , which correlated with higher teer than fibroblastic hcecs.conclusionsin vitro expansion of hcecs from cadaveric donor corneas yields functional cells identifiable by morphology and a panel of novel markers . markers described correlated with function in culture , suggesting a basis for cell therapy for corneal endothelial dysfunction .

Materials and Methods Donor Tissue Human Corneal Cell Culture Immunohistochemistry Flow Cytometry RNA Isolation Transendothelial Electrical Resistance (TEER) Microarray Analysis Statistical Analysis Results Isolation and In Vitro Expansion of HCECs Identity and Function of HCECs In Vitro Canonical HCECs Express a Subset of Specific Markers That Define Their Identity Discussion Supplementary Material

human cadaveric donor corneas not suitable for transplantation and preserved in optisol - gs ( baush & lomb , rochester , ny , usa ) were procured from the lions eye institute for transplant and research ( tampa , fl , usa ) , the national disease research interchange ( ndri ; philadelphia , pa , usa ) , eversight ( ann arbor , mi , usa ) , and the san diego eye bank ( san diego , ca , usa ) . once firmly attached , cells were kept at 37c and 10% co2 , and fed every other day with dulbecco 's modified eagle 's medium supplemented with 10% fbs ( invitrogen ) and 50 g / ml gentamicin ( gibco ) . human corneal endothelial cells were cultured on fnc - coated glass coverslips ( carolina biological supply co. , burlington , nc , usa ) placed in 24-well plates . cultured hcecs presenting canonical , fibroblastic , and mixed morphologies were trypsinized and dissociated into single cells , and counted using a neubauer chamber . three gates including 0.1% , 1% , or 10% of the negative population were selected , and the percentage of positive cells present in these gates was assessed . given the similarity of unstained canonical and fibroblastic hcecs when analyzed by flow cytometry , either canonical or fibroblastic hcecs were used as negative controls for each run . fresh donor corneas stored in optisol - gs ( baush & lomb , rochester , ny , usa ) were minimally processed and the epithelial , stromal , and endothelial layers were isolated . ribonucleic acid extraction from donor corneas and cultured hcecs was performed using the rneasy kit ( hcecs , endothelium , and epithelium ; manufacturer 's protocol ; qiagen , valencia , ca ) or trizol ( stroma ) . transendothelial electrical resistance measurements were normalized to the value of the control wells ( growth media without cells ) . a bovine corneal endothelial cell line ( bcec ) was used as a positive control ( atcc crl-2048 , manassas , va , usa ) and fibroblastic hcecs were used as a negative control . human cadaveric donor corneas not suitable for transplantation and preserved in optisol - gs ( baush & lomb , rochester , ny , usa ) were procured from the lions eye institute for transplant and research ( tampa , fl , usa ) , the national disease research interchange ( ndri ; philadelphia , pa , usa ) , eversight ( ann arbor , mi , usa ) , and the san diego eye bank ( san diego , ca , usa ) . cell passaging was performed by digesting confluent cultures with 0.05% trypsin ( gibco ) for 5 minutes at 37c in 5% co2 , and cell viability was evaluated by trypan blue exclusion assay ( sigma - aldrich corp . ) human corneal endothelial cells were cultured on fnc - coated glass coverslips ( carolina biological supply co. , burlington , nc , usa ) placed in 24-well plates . data was stored in a fcs file format , and further analysis was performed using flowjo v x.0.7 ( flowjo llc , ashland , or , usa ) . three gates including 0.1% , 1% , or 10% of the negative population were selected , and the percentage of positive cells present in these gates was assessed . given the similarity of unstained canonical and fibroblastic hcecs when analyzed by flow cytometry , either canonical or fibroblastic hcecs were used as negative controls for each run . fresh donor corneas stored in optisol - gs ( baush & lomb , rochester , ny , usa ) were minimally processed and the epithelial , stromal , and endothelial layers were isolated . ribonucleic acid extraction from donor corneas and cultured hcecs was performed using the rneasy kit ( hcecs , endothelium , and epithelium ; manufacturer 's protocol ; qiagen , valencia , ca ) or trizol ( stroma ) . a bovine corneal endothelial cell line ( bcec ) was used as a positive control ( atcc crl-2048 , manassas , va , usa ) and fibroblastic hcecs were used as a negative control . we first asked whether hcecs in vitro maintain the characteristics observed in vivo , namely cell cell contact inhibition and the canonical cobblestone - like or polygonal morphology . corneal endothelial cells were isolated and cultured from cadaveric donor corneas following a previously published method outlined in figure 1a . overall , hcecs from younger corneas , cultured in vitro , were expanded for 3 or 4 passages , with each cornea yielding a variable number of total cell progeny ( fig . ( h ) cell yields after expansion of hcecs from corneas of young donors for three or four passages . ( j ) there is a weak correlation between hcec density and in vitro proliferation ( r = 24% ) , but the correlation is statistically significant ( p = 0.0002 ) . finally , there was a significant difference in initial endothelial cell density between corneas from young donors ( 2- to 34-years old : average endothelial cell density : 3181.6 mm ; range , 25714425 mm ; n = 30 ) and those from older donors ( 38- to 77-years old : average endothelial cell density : 2761.5 mm ; range 19692865 mm ; n = 11 ) . there is a paucity of hcec - specific identity markers , and the expression of proteins expressed ubiquitously in tight junction complexes is often cited for identity criteria . human corneal endothelial cells in culture , morphologically similar to cells represented in figures 1b to 1e , immunostained for zo-1 exhibited typical honeycomb staining at the tight junctions , while na / k - atpase expression was found basolaterally ( fig . we found that cultures with the canonical cobblestone - like morphology reached significantly higher teer values than fibroblastic cultures , and cultures with mixed morphology of fibroblastic and hexagonal cells demonstrated intermediate values of teer ( fig . thus , hcecs can be identified by the expression of a combination of markers , and their function can be determined by teer , but the markers used routinely may not have the ability to differentiate canonical hcecs from fibroblastic hcecs or stromal fibroblasts cultured hcecs express characteristic tight - junction - associated markers . ( b ) bar graph showing no difference between canonical and fibroblastic hcecs in na / k - atpase expression by flow cytometry ( n = 3 ) . ( c ) human corneal endothelial cells function assessed by teer measurements from different hcec cultures . p values resulting from the statistical analysis of teer measured on canonical hcecs compared with mixed 1 , mixed 2 , and fibroblastic hcecs are presented in the table below the graph . we performed a microarray analysis comparing the transcriptomes of freshly dissected corneal layers ( epithelium , stroma , and endothelium ) and of p0-cultured hcecs , and found that of 28,869 probes , 23,286 ( 81% ) were expressed by at least two of three replicates within at least one condition . 5a c ) were assessed for expression of the surface proteins cd56 , car , cd248 , and cd109 by flow cytometry . in a series of independent experiments , we observed a significant difference between canonical and fibroblastic cells in the expression of cd56 , car , cd248 , and cd109 surface markers ( figs . whereas 97% of canonical hcecs were positive for cd56 , 92% for cd248 , and 82% were positive for car , these markers decreased as the cells lost their canonical morphology and became fibroblastic . conversely , cd109 expression was less often detected in canonical hcecs ( 26% on average ) , and increased in expression when the cells underwent enmt to 52% in fibroblastic cell cultures . flow cytometry analysis of surface markers expression differentiates hcec subpopulations in culture . ( a c ) three morphologically distinct hcec cultures , canonical , mixed , and fibroblastic as marked , were carried forward for flow cytometry . ( a c ) flow cytometry analysis by dual - color fluorescent dot plot histograms for the canonical ( a ) , mixed ( b ) , and fibroblastic ( c ) hcec cultures show shift in the expression of cd56 , cd248 , car , and cd109 surface markers . ( g ) quantification of flow cytometry experiments showing no difference between canonical and fibroblastic cells in cd166/alcam , cd73 , cd9 , cd90 , and 1na / k atpase expression . ( h ) transendothelial electrical resistance assay using in vitro expanded hcecs whose cd56 expression had been determined by flow cytometry , demonstrating the greater ability of canonical cd56 cells than fibroblastic cd56 to form a barrier . recently other groups reported the expression of cd73 , cd166 , cd9 , and cd90 in corneal endothelial cells . we analyzed by flow cytometry canonical and fibroblastic hcecs in passages two to three and five through eight , respectively , and the high expression of those markers did not vary with morphology ( fig . thus , while these markers may be used to identify hcecs , they may not be adequate to select cell phenotypes based on morphology or function . we used confluent hcecs that we plated for teer , isolating a subset to be tested by flow cytometry for cd56 expression . cells exhibiting a canonical morphology and a cd56 marker expression demonstrated a superior barrier formation ability measured by teer , compared with morphologically mixed or fibroblastic , cd56 cells ( fig . corneal endothelial cells were isolated and cultured from cadaveric donor corneas following a previously published method outlined in figure 1a . ( h ) cell yields after expansion of hcecs from corneas of young donors for three or four passages . ( j ) there is a weak correlation between hcec density and in vitro proliferation ( r = 24% ) , but the correlation is statistically significant ( p = 0.0002 ) . ( k ) there was a statistically significant difference between corneal endothelial density measured before enucleation in younger donors ( average endothelial cell density : 3181.6 mm ; range , 25714425 mm ; n = 30 ) compared with older donors ( average endothelial cell density : 2761.5 mm ; range , 19692865 mm ; n = 11 ; p = 0.02 ) . however , cultures from young donors with sepsis or undergoing chemotherapy were not successful , suggesting a direct relationship between hcec culture outcome and donor age and health . there is a paucity of hcec - specific identity markers , and the expression of proteins expressed ubiquitously in tight junction complexes is often cited for identity criteria . human corneal endothelial cells in culture , morphologically similar to cells represented in figures 1b to 1e , immunostained for zo-1 exhibited typical honeycomb staining at the tight junctions , while na / k - atpase expression was found basolaterally ( fig . we found that cultures with the canonical cobblestone - like morphology reached significantly higher teer values than fibroblastic cultures , and cultures with mixed morphology of fibroblastic and hexagonal cells demonstrated intermediate values of teer ( fig . thus , hcecs can be identified by the expression of a combination of markers , and their function can be determined by teer , but the markers used routinely may not have the ability to differentiate canonical hcecs from fibroblastic hcecs or stromal fibroblasts ( a ) confocal micrographs of cultured hcecs immunostained for zo-1 and na / k - atpase ; nuclei were counter - stained with dapi . ( b ) bar graph showing no difference between canonical and fibroblastic hcecs in na / k - atpase expression by flow cytometry ( n = 3 ) . ( c ) human corneal endothelial cells function assessed by teer measurements from different hcec cultures . a bovine corneal endothelial cell line ( bcec - line ) p values resulting from the statistical analysis of teer measured on canonical hcecs compared with mixed 1 , mixed 2 , and fibroblastic hcecs are presented in the table below the graph . we performed a microarray analysis comparing the transcriptomes of freshly dissected corneal layers ( epithelium , stroma , and endothelium ) and of p0-cultured hcecs , and found that of 28,869 probes , 23,286 ( 81% ) were expressed by at least two of three replicates within at least one condition . 5a c ) were assessed for expression of the surface proteins cd56 , car , cd248 , and cd109 by flow cytometry . in a series of independent experiments , we observed a significant difference between canonical and fibroblastic cells in the expression of cd56 , car , cd248 , and cd109 surface markers ( figs . whereas 97% of canonical hcecs were positive for cd56 , 92% for cd248 , and 82% were positive for car , these markers decreased as the cells lost their canonical morphology and became fibroblastic . conversely , cd109 expression was less often detected in canonical hcecs ( 26% on average ) , and increased in expression when the cells underwent enmt to 52% in fibroblastic cell cultures . ( a c ) three morphologically distinct hcec cultures , canonical , mixed , and fibroblastic as marked , were carried forward for flow cytometry . ( a c ) flow cytometry analysis by dual - color fluorescent dot plot histograms for the canonical ( a ) , mixed ( b ) , and fibroblastic ( c ) hcec cultures show shift in the expression of cd56 , cd248 , car , and cd109 surface markers . ( h ) transendothelial electrical resistance assay using in vitro expanded hcecs whose cd56 expression had been determined by flow cytometry , demonstrating the greater ability of canonical cd56 cells than fibroblastic cd56 to form a barrier . recently other groups reported the expression of cd73 , cd166 , cd9 , and cd90 in corneal endothelial cells . we analyzed by flow cytometry canonical and fibroblastic hcecs in passages two to three and five through eight , respectively , and the high expression of those markers did not vary with morphology ( fig . thus , while these markers may be used to identify hcecs , they may not be adequate to select cell phenotypes based on morphology or function . finally , we examined whether marker expression predicted functional capacity in an in vitro teer assay . cells exhibiting a canonical morphology and a cd56 marker expression demonstrated a superior barrier formation ability measured by teer , compared with morphologically mixed or fibroblastic , cd56 cells ( fig . these are limited by tissue availability , access to highly trained specialists , surgical complications , immune rejection , and cost . here we describe steps toward corneal endothelial cell therapy based on in vitro culture , expansion and identification of high quality , functional hcecs . a successful culture starts with good quality donor tissue : the origin and qualities of the donor cornea including donor age , health status , and use of chemotherapy or other toxic substances play a large part in proliferative potential , morphology , and function . with increasing numbers of passages in culture , enmt often occurs and the hcecs lose their canonical cobblestone morphology and functional efficacy in barrier formation , in vitro and in vivo . to pursue this , we used microarray analysis of corneal epithelial , stromal , and endothelial tissue , as well as in vitro cultured hcecs to isolate candidate surface markers present either exclusively in the stroma , or in both the native endothelium and the in vitro cultured hcecs . we further tested the expression levels of nine of these candidate markers by flow cytometry in canonical , mixed , and fibroblastic in vitro cultured hcecs . cd56 , or neural cell adhesion molecule ( ncam ) , is a surface glycoprotein that exists in multiple isoforms and is expressed by multiple cell types . importantly , we found that cd56 expression in cultures also correlated with higher functional barrier capacity in teer assays . cd248 , or endosialin , belongs to the family of c - type lectin transmembrane receptors that may play a role in cell cell adhesion , host defense , and tumor neoangiogenesis , but their role in hcec biology is unknown . finally , cd109 , a cell surface antigen involved in the tgf- pathway , has been studied in white blood cells , vascular endothelial cells , keratinocytes , and activated platelets . for all of these new markers , other potential candidates have been identified from microarray analyses and tested via flow cytometry to differentiate canonical hcecs from hcecs undergoing enmt , including cd166 ( alcam ) , expressed in corneal epithelial limbal stem cells but also in corneal stromal stem cells , cd90 , expressed in corneal stromal fibroblasts , but also in canonical corneal endothelial cells , and cd73 and cd9 . for these last two , no difference between corneal layers was detected by microarray , and further testing by flow cytometry remained negative in our experiments . given our testing of both gene and protein expression , and use of exclusively human corneal tissue and primary corneal endothelial cells , these data raise questions as to whether cd9 and cd73 will be useful markers to differentiate canonical from fibroblastic hcecs . together , the specific surface markers identified here and their correlation to cell function represent a novel set of criteria for the selection of in vitro expanded hcecs that we would hypothesize are most likely to be functionally competent to replace damaged corneal endothelial cells , and thus represent a step toward hcec therapy . along with hcec culture and cell characterization , effort directed toward delivering in vitro cultured hcecs to the patient 's endothelial surface ongoing efforts should demonstrate the feasibility of hcec in vitro expansion , selection of cultured hcecs with highest function , and development of cell delivery approaches . recent animal studies using corneal endothelial cells revealed that the cells integrate in vivo to the host endothelium , and are able to restore corneal transparency after injury , but many questions such as dosage and stability remain unanswered . our donor tissue selection criteria , together with the exhaustive characterization provided in this study , are essential for obtaining high quality , functional cultured hcecs , bringing us a step closer to the clinic with a human corneal endothelial cell therapy .

significant unmet needs exist in the treatment of major depressive disorder , such as suboptimal efficacy and residual cognitive dysfunction.a paradigm shift from the traditional monoamine therapeutics to approaches integrating glutamatergic function has occurred recently in antidepressant research , and has been especially fueled by the surprising rapid and sustained antidepressant effect of ketamine.we review the evidence that glutamate neurotransmission can be modulated indirectly by the 5-ht system through the 5-ht1a , 5-ht1b , 5-ht3 , and 5-ht7 receptors , and discuss the therapeutic potential of a multimodal approach , combining one or more 5-ht receptor mechanisms with 5-ht reuptake inhibition.we review the available information for the two multimodal compounds vortioxetine and vilazodone , which are examples of this approach . significant unmet needs exist in the treatment of major depressive disorder , such as suboptimal efficacy and residual cognitive dysfunction . a paradigm shift from the traditional monoamine therapeutics to approaches integrating glutamatergic function has occurred recently in antidepressant research , and has been especially fueled by the surprising rapid and sustained antidepressant effect of ketamine . we review the evidence that glutamate neurotransmission can be modulated indirectly by the 5-ht system through the 5-ht1a , 5-ht1b , 5-ht3 , and 5-ht7 receptors , and discuss the therapeutic potential of a multimodal approach , combining one or more 5-ht receptor mechanisms with 5-ht reuptake inhibition . we review the available information for the two multimodal compounds vortioxetine and vilazodone , which are examples of this approach . over the past 50 years , pharmacological treatments for major depressive disorder ( mdd ) have evolved from the older tricyclic antidepressants and monoamine oxidase inhibitors , to selective serotonin ( 5-ht ) reuptake inhibitors ( ssri ) and serotonin and norepinephrine ( ne ) reuptake inhibitors ( snris ) . in recent years , antidepressant combination therapies with multifunctional pharmacologic mechanisms have been used to enhance therapeutic outcomes . some combinations include an ssri plus the 5-ht1a receptor and adrenergic receptor antagonist pindolol , or ssris augmented with atypical antipsychotics . despite these therapeutic evolutions , significant unmet needs still exist in treating depression , including improving suboptimal treatment response and remission rates , and cognitive impairments in domains such as memory , attention , executive function , and speed of processing . moreover , some cognitive disturbances may predict the development of mood disorders , and furthermore may persist beyond remission . since cognitive dysfunction in depression contributes significantly to disability in some patients , the glutamate system is the major excitatory neurotransmitter system in the brain and is essential for cognitive processing . in depressed patients , neurochemical assessments have found increased basal glutamate levels in serum or plasma , though changes in its levels in cerebrospinal fluid and brain tissue are somewhat inconsistent . recent studies using magnetic resonance spectroscopy ( mrs ) in depressed patients have generally found reductions in glx , a combined measure of glutamate and glutamine , possibly suggesting that the total glutamatergic pool available for synaptic and metabolic activities is reduced in depression . however , studies that have directly measured glutamate using mrs have also found inconsistent results , with some groups finding increases , decreases , or no change in glutamate concentrations . there is also evidence from studies of post - mortem brain tissue in depressed patients or suicide victims for altered expression of n - methyl - d - aspartate ( nmda ) and -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid ( ampa ) receptors . given the complexity of glutamatergic neurotransmission and the diversity of these results , it is difficult to come to a definitive conclusion on the role of glutamate in the etiology of major depression at this time . in the future , information on functional single nucleotide polymorphisms related to the glutamate system may provide another valuable method of examining glutamate 's role in this disease . nonetheless , interest in the role of glutamate in depression is quickly accreting , primarily due to the observation that the noncompetitive nmda receptor antagonist ketamine engenders a fast and relatively long - lasting antidepressant effect . this observation has prompted a new focus in antidepressant development toward integrating glutamatergic function , leading to the suggestion of a wide range of glutamate targets for the treatment of depression . 5-ht neurotransmission is regulated both by the serotonin transporter ( sert ) , which has been a target of antidepressants for the past 30 years , and by modulation via 5-ht receptor subtypes , some of which ( such as the 5-ht1a receptor ) may be independent therapeutic targets for the treatment of depression . a substantial body of data shows that , in addition to modulating 5-ht neurotransmission , multiple 5-ht receptor subtypes can also modulate glutamate neurotransmission . this may be reflected in results from a recent preclinical study , which found that ketamine 's fast antidepressant activity was abolished by 5-ht depletion , suggesting that these effects may be serotonin - dependent . thus , there may be an opportunity to integrate monoamine and glutamate strategies for treating depression . a new class of multimodal antidepressants has emerged , which , in addition to inhibiting the sert , also modulate 5-ht receptors , and may represent an example of this integrative strategy . in this review , we summarize the current knowledge of putative glutamatergic antidepressants , 5-ht receptor - mediated glutamate modulation , and current evidence that multimodal serotonergic antidepressants with indirectly modulating roles on glutamate transmission are active in treating lowered mood and impaired cognition . the glutamate receptors are divided into two major families : ionotropic and metabotropic glutamate receptors ( mglurs ) . the metabotropic family consists of group i receptors ( mglur1 and mglur5 ) , which potentiate both presynaptic glutamate release and postsynaptic nmda currents , and group ii ( mglur2 and mglur3 ) and group iii receptors ( mglur4 , mglur6 , mglur7 , and mglur8 ) , which in general suppress glutamate function . glutamate receptors are widely expressed in the brain , and some of them have been implicated in the treatment of depression . preclinical and clinical compounds acting via these targets and showing potential antidepressant activity are listed in table 1.table 1examples of glutamatergic compounds with antidepressant or antidepressant - like propertiescompound examplesmechanism of actiondevelopment stageeffectsreferencesketaminenmda antagonistclinical userapid ( 4 h ) antidepressant effect ; sustained for up to 1 week 35,149 memantinenmda antagonistclinical useno effect 37 lamotrigineinhibition of glutamate releaseclinical useantidepressant properties in unipolar patients 46,47 riluzoleincrease in glutamate uptakeclinical useantidepressant efficacy in treatment - resistant and bipolar depression 48,49 traxoprodilnr2b antagonistclinical developmentantidepressant effect in treatment - resistant depression after a single infusion , sustained up to 1 week 36 aniracetamampa potentiatorclinical developmentmemory - enhancing effects , antidepressant - like behavioral effects 43,44,150 ly392098ampa potentiatorpreclinicalantidepressant - like effects in the tail suspension and forced - swim tests 151 mpepmglur5 antagonistpreclinicalantidepressant - like effects in the mouse tail - suspension and rat forced swim tests 50 ly341495mglur2/3 antagonistpreclinicalantidepressant - like effects ; enhanced spatial memory 52,152 mgs0039mglur2/3 antagonistpreclinicalantidepressant - like effects 51 glyx13nmda receptor glycine site partial agonistpreclinicalantidepressant - like effects 153 examples of glutamatergic compounds with antidepressant or antidepressant - like properties over - activation of extrasynaptic nmda receptors is one of several hypothesized glutamate - related pathophysiologies for depression . in support of this idea , the noncompetitive nmda receptor antagonist ketamine at a single i.v . dosing shows rapid ( 4 h ) antidepressant effect that is sustained for up to 7 days in therapy - resistant depressed patients . this rate of onset is extremely fast compared to the 2 - 3 weeks that approved antidepressants require . a single infusion of a subtype selective nmda nr2b antagonist traxoprodil has shown a robust separation from placebo in treatment - resistant depression ( 60% vs 20% response ) with sustained effects up to 1 week . however memantine , a use - dependent nmda receptor antagonist , has not demonstrated the same efficacy as ketamine , though it was not tested in the same paradigm as ketamine . part of the mechanism for the antidepressant effect of ketamine may involve disinhibition of pyramidal cell firing as a result of the antagonism of nmda receptors located on interneurons . however , it remains to be seen whether the nmda receptor blockade alone mediates this fast antidepressant activity . in support of a role for ampa receptors in treating depression , preclinical studies suggest that ketamine exerts its antidepressant - like effect through ampa receptors , and that fast action is accompanied by rapid neuronal and synaptic adaptation . it is widely believed that neuroadaptive changes represent a key event during antidepressant treatment , and may play a role in the delayed onset of efficacy in traditional antidepressants . thus , ketamine 's rapid effects on neuroadaptation may be a key mechanism in its antidepressant effects , and may converge with the general actions of antidepressant treatments suggested in the past decades . furthermore , the ampa receptor potentiator aniracetam has shown an antidepressant - like profile . lamotrigine , a modulator of glutamate release via its action on sodium and calcium channels , is approved for relapse prevention in bipolar disorder in the united states , and may have antidepressant properties in unipolar patients . additionally , it may accelerate the rate of onset in combination with traditional antidepressants . riluzole , which acts to rebalance glutamate levels by enhancing glutamate transport in astrocytes , has shown efficacy in treatment - resistant and bipolar depression . further examples of targets in the glutamate system with antidepressant - like implications include mglur2/3 and mglur5 antagonists or negative allosteric modulators . thus , although there is evidence that drugs that negatively modulate some aspects of glutamate neurotransmission have antidepressant - like effects , there is also evidence that increasing other aspects of glutamate signaling can have antidepressant - like effects . it remains to be seen which variables are the true mediators of these effects . in comparison , the prominent role of glutamatergic neurotransmission in cognitive function is better understood . antagonism of nmda receptors as well as other experimental manipulations that reduce aspects of glutamatergic neurotransmission , such as antagonism at ampa or mglu5 receptors , are known to consistently impair function across a range of cognitive domains . accordingly , the glutamatergic neurotransmitter system has become a common target in developing cognition - enhancing drugs , with the broad theme that increasing synaptic glutamate neurotransmission , for example using positive allosteric modulators at ampa ( ampakines ) , mglur5 ( cdppb ) , or nmda receptors ( d - cycloserine ) , improves cognitive function in rodent models . however , improving mood and cognition by directly modulating glutamatergic neurotransmission may be difficult , as excessive glutamatergic activation can lead to excitotoxic effects and cognitive impairment . furthermore , the near - ubiquitous expression of glutamatergic receptors in the brain may hamper the specificity of drug development . thus , a strategy to indirectly modulate glutamatergic neurotransmission in selected brain regions may be more advantageous . a recent preclinical report demonstrated that 5-ht depletion abolished ketamine 's antidepressant - like activity , suggesting that 5-ht plays an important role in its action . these data make it reasonable to explore a strategy in which 5-ht receptor modulation can be used to alter glutamate neurotransmission in a manner that may improve both mood and cognitive function . here we discuss four 5-ht receptors known to be involved in the action of multimodal antidepressants that have been approved or are in the approval process , and which have the potential to modulate the glutamate system based on their localization and function . the 5-ht1a receptor is an inhibitory autoreceptor or heteroceptor located on serotonergic and other neurons , whose activation typically results in suppression of neuronal activity . the main function of presynaptic autoreceptors localized in the midbrain raphe nuclei is to self - regulate the function of the serotonergic system . desensitization of these autoreceptors is believed to play an important role in the onset of action of sert inhibitors . the antidepressant potential of 5-ht1a receptor agonism or partial agonism has been studied in both preclinical and clinical settings . as postsynaptic heteroreceptors , 5-ht1a is localized in the hippocampus , septum , amygdala , and corticolimbic areas . based on immunocytochemical studies , the 5-ht1a receptor is expressed in both pyramidal cells and gabaergic interneurons in the cortex and hippocampus . unlike presynaptic 5-ht1a receptors , which mainly act through inhibition of adenylate cyclase , postsynaptic 5-ht1a receptors exert their inhibitory action through g protein - coupled inwardly rectifying k channels . due to the inhibitory nature of gabaergic interneurons , stimulation of 5-ht1a receptors located on interneurons can paradoxically increase cortical pyramidal cell firing , although higher doses can suppress it , probably due to the action of 5-ht1a receptors on the pyramidal cells . similarly , 5-ht1a receptor stimulation resulted in inhibition of gabaergic interneurons in the hippocampus . thus , based on the localization of 5-ht1a receptors on both gaba and glutamate neurons ( figure 1 ) , their activation may lead to either an increase or a decrease in glutamate neurotransmission depending on which subpopulations of 5-ht1a receptors are activated.figure 1a schematic diagram of the hypothesized modulatory role of 5-ht receptors on glutamatergic neurotransmission . a glutamatergic pyramidal neuron and several gaba interneurons expressing the 5-ht3 , 5-ht1a , the multimodal compounds vortioxetine and vilazodone and their possible sites of action are also shown . note that 5-ht1a , 5-ht1b , and 5-ht7 receptors may be localized on different neuronal populations . symbols used : vla , vilazodone ; vor , vortioxetine . a schematic diagram of the hypothesized modulatory role of 5-ht receptors on glutamatergic neurotransmission . a glutamatergic pyramidal neuron and several gaba interneurons expressing the 5-ht3 , 5-ht1a , 5-ht7 , and 5-ht1b receptors on either dendrites or axon terminals are shown . the multimodal compounds vortioxetine and vilazodone and their possible sites of action are also shown . note that 5-ht1a , 5-ht1b , and 5-ht7 receptors may be localized on different neuronal populations . symbols used : vla , vilazodone ; vor , vortioxetine . based on the above interaction between effects mediated through the 5-ht1a receptor and glutamatergic neurons , agonists of the 5-ht1a receptor are predicted to have a memory - modulating role , and this has been demonstrated in various preclinical studies . the 5-ht1a receptor full agonist flesinoxan impairs working memory in a delayed conditional discrimination task in normal rats . mixed results have been shown in a passive avoidance test in mice , in which pretreatment with flesinoxan either decreased or increased memory function , depending on when it was administered . in contrast , a memory - enhancing profile was consistently observed with 5-ht1a agonism in animals with learning and memory deficits . for example , the 5-ht1a receptor agonist 8-oh - dpat reversed learning deficits induced by scopolamine and mk-801 in an autoshaping learning task . interestingly , a postsynaptic - selective 5-ht1a receptor agonist f15599 was reported to improve working and reference memory in rats with phencyclidine - induced memory deficits . last , 5-ht1a receptor agonists , such as tandospirone , seem also to be able to alleviate the memory deficits induced by subchronic phencyclidine treatment . thus , based on the localization and function of 5-ht1a heteroreceptors , 5-ht1a receptor stimulation has the potential to enhance or suppress glutamatergic neurotransmission , and thus may also have biphasic effects on mood or cognitive function . like the 5-ht1a receptors , 5-ht1b receptors are distributed as autoreceptors or heteroreceptors throughout the brain , in areas such as the ventral pallidum , globus pallidus , substantia nigra , dorsal subiculum cerebral cortex , and the hippocampus . unlike the 5-ht1a autoreceptors , which are localized in somatodendritic regions of 5-ht neurons , 5-ht1b receptors are localized either presynaptically at nerve terminals or postsynaptically on dendrites . postsynaptic 5-ht1b receptors are co - localized with nmda or ampa receptors on dentrites , and are thus well - positioned to modulate glutamate transmission . recently , cai etal demonstrated that 5-ht1b receptor agonism increases hippocampal excitatory field potentials through a cam kinase - dependent pathway . in the dorsal subiculum , however , 5-ht1b receptors are localized on ca1 pyramidal axon terminals as inhibitory heteroceptors , and activation of these receptors attenuates glutamate transmission in the hippocampus due to its negative coupling to adenylate cyclase . it has been shown that the 5-ht1b receptor agonist cp-94253 can modulate 5-ht synthesis in the flinders sensitive line rat , an animal model of depression . in intracerebral microdialysis studies , stimulation of 5-ht1b receptors by ru 24969 potentiated the antidepressant - like effects of ssris and imipramine . additionally , 5-ht1b receptor stimulation with the selective agonist cp-94253 in mice displayed an antidepressant - like profile in the forced swim test . intrahippocampal microinjection of the 5-ht1b receptor agonist cp-93129 impairs spatial learning performance in the radial maze task . on the other hand , the 5-ht1b receptor antagonist sb-224289 enhanced memory consolidation during learning in an associative autoshaping learning task , and reversed the cognitive deficits induced by either the cholinergic inhibitor scopolamine or the nmda receptor antagonist mk-801 . in an aversive contextual learning task in mice , thus , 5-ht1b receptors may be able to positively or negatively modulate glutamate transmission and may be linked to the pathophysiology of depression . due to the somewhat contrasting antidepressant - like properties of 5-ht1b receptor agonism and memory deficit - reducing effect of 5-ht1b receptor antagonism , a balance of stimulation versus blockade of this receptor may be needed . based on this idea , a partial agonist for the 5-ht1b receptor may be a reasonable approach , although at the time of writing , the authors are not aware of any empirical investigations of the effects of 5-ht1b partial agonism on mood and cognitive function . among 5-ht receptors , the 5-ht3 receptor is the only known excitatory ion channel , and is expressed throughout the brain , including the following regions : ( 1 ) hippocampus ; ( 2 ) amygdala ; and ( 3 ) entorhinal , frontal , and cingulate cortices . immunohistochemical studies show that 5-ht3 receptors are localized in postsynaptic dendrites , especially of gabaergic interneurons in cortical and hippocampal regions . these receptors function as a mechanism of 5-ht - mediated excitation of gaba neurons . in freely moving rats , the 5-ht3 receptor antagonist ondansetron significantly suppressed the firing rate of ca1 hippocampal gabaergic interneurons and concomitantly increased the firing rate of glutamatergic pyramidal cells by disinhibition . consistent with the above , activation of 5-ht3 receptors can suppress both the spontaneous firing and nmda - evoked responses of the pyramidal neurons in the rat medial prefrontal cortex . thus , 5-ht3 receptor antagonism enhances glutamate transmission by reducing gaba - mediated inhibition , as illustrated in figure 1 . this mechanism may explain previous reports that 5-ht3 receptor antagonism by ondansetron enhances long - term potentiation ( ltp ) and hippocampal and cortical theta rhythms . for example , the 5-ht3 receptor antagonist itasetron showed memory - enhancing effects in a multiple - choice avoidance behavioral task , and ondansetron blocks scopolamine - induced deficits in learning . in addition to the previously mentioned effects on cognition , 5-ht3 receptor antagonists have antidepressant - like effects . newer antagonists also show antidepressant - like activities in the forced swim test and in olfactory bulbectomized rats . in conclusion , 5-ht3 receptor antagonism shows antidepressant - like activity and increased cognitive function in preclinical studies , possibly through facilitation of glutamate neurotransmission by reducing the activity of inhibitory gaba neurons . the 5-ht7 receptor is a g - protein - coupled receptor ( gpcr ) with positive coupling to adenylate cyclase , and is highly expressed in the brain , including the thalamus , hypothalamus , hippocampus , and cortex . in midbrain slices of rat brain containing the dorsal and median raphe nuclei , the mixed 5-ht receptor agonist 5-carboxamido - tryptamine inhibited glutamate release , and this was reversed by the 5-ht7 receptor antagonist sb-258719 . thus , 5-ht7 receptors in the axon terminals of the glutamatergic cortico - raphe neurons may serve as heteroreceptors that inhibit glutamate release . the 5-ht7 receptor is also expressed on the cell bodies of pyramidal neurons . in normal animals , activation of the 5-ht7 receptor leads to increased firing of glutamatergic neurons in the cortex and hippocampus . however , these effects on glutamatergic neurotransmission may be accompanied by increased inhibitory gabaergic transmission , likely due to expression in both pyramidal neurons and gabaergic interneurons . these concomitant effects were demonstrated in the hippocampus with an increase in the frequencies of both spontaneous inhibitory postsynaptic currents recorded in pyramidal neurons and spontaneous excitatory postsynaptic currents recorded in interneurons . based on these data , 5-ht7 receptor activation has mixed effects on glutamatergic neurotransmission , but the overall effect in normal rodents appears to be excitatory . importantly , this relationship may be altered in disease states , as 5-ht7 receptor activation in 6-hydroxydopamine - lesioned animals led to a net inhibition , rather than excitation , of pyramidal cell firing in the same study . based on these results , 5-ht7 receptor antagonism may result in either increases or decreases in glutamatergic neurotransmission within the context of depression . although the effects of 5-ht7 receptor modulation on glutamatergic neurotransmission are currently somewhat unclear , clear antidepressant - like activities of 5-ht7 antagonism have been reported in a number of preclinical studies . treatment with the 5-ht7 receptor antagonist sb-269970 reduced immobility in the forced swim and tail suspension tests , and there was a further synergistic effect on extracellular 5-ht release in the frontal cortex when sb-269970 was combined with the ssri citalopram . therefore , the results from preclinical studies suggest that 5-ht7 receptor antagonism might be a novel strategy for treating depression . additionally , memory - enhancing effects of 5-ht7 antagonists have been shown in preclinical models and have been reviewed elsewhere . in cases where learning or memory was disrupted by nmda antagonists such as phencyclidine or mk-801 , interestingly , combined 5-ht7 receptor antagonism and sert inhibition produced a synergistic effect in a preclinical test of executive function . these data support a modulatory role of 5-ht7 receptors on glutamate transmission as mentioned above . there are currently two multimodal compounds with clinically documented antidepressant activity : vilazodone , which is approved for clinical use in the u.s . , and vortioxetine , which is undergoing regulatory review . given the complexity of the serotonergic modulation of glutamate , it is not possible to predict the net effect that multimodal serotonergic compounds will have on glutamate neurotransmission . thus , the need for empirical data on the effects of these compounds on glutamate neurotransmission is paramount . vilazodone is a recently approved antidepressant with high affinities for the sert ( ic50 0.5 nm ) and 5-ht1a receptor ( ec50 0.2 nm ) ( table 2 ) . vilazodone is a partial agonist at the 5-ht1a receptor , but with a relatively high intrinsic activity69% of the magnitude of the full 5-ht1a receptor agonist 8-oh - dpat . in preclinical studies , vilazodone seems to outperform the ssris paroxetine and fluoxetine , as measured by 5-ht release and ultrasonic vocalization . however , the fact that antidepressant - like effects are observed at moderate but not higher doses in the rat and mouse forced swim test may suggest that its 5-ht1a receptor partial agonism may inhibit the expression of rodent antidepressant - like behaviors . vilazodone 's potential to interact with glutamate neurotransmission the antidepressant efficacy of vilazodone was seen only in some of the clinical trials , partly due to the need to balance the higher dose ( 40 mg ) needed versus the high rate of gastrointestinal side effect , and thus its efficacy and safety profiles in comparison to current antidepressants require further clinical evaluation . table 2clinical compounds with serotonin ( 5-ht ) transporter ( sert ) inhibition plus activity at one or more 5-ht receptors linked to glutamatergic modulationvilazodonevortioxetinetargettype of activityhuman ic50 ( nm)human ki ( nm)rat ki ( nm)5-ht3 antagonist3.71.15-ht7 antagonist192005-ht1b partial agonist33165-ht1a agonist0.2 ( 69%)15 ( full)230sertinhibitor0.51.68.6references129112 , 134 , 135the in vitro pharmacological activities were from either binding or functional measurements . clinical compounds with serotonin ( 5-ht ) transporter ( sert ) inhibition plus activity at one or more 5-ht receptors linked to glutamatergic modulation the in vitro pharmacological activities were from either binding or functional measurements . numbers in parentheses denote agonist efficacy . vortioxetine is an investigational multimodal antidepressant that acts as a 5-ht3 , 5-ht7 , and 5-ht1d receptor antagonist ; 5-ht1b receptor partial agonist ; 5-ht1a receptor agonist ; and sert inhibitor in vitro ( table 2 ) . its pharmacological profile indicates that vortioxetine has the potential to modulate glutamate transmission through all of the four 5-ht receptor pathways discussed above ( figure 1 ) . multiple reports of preclinical studies have shown the antidepressant - like activities of vortioxetine . further , in clinical studies , its efficacy as an antidepressant has been demonstrated in several studies to date , although statistically significant separation from placebo has not been observed in every clinical trial . recently , it was reported that vortioxetine enhanced time - dependent contextual fear memory and object recognition memory in rats . additionally , 5-ht depletion - induced memory deficits were dose - dependently reversed by vortioxetine treatment , while escitalopram and duloxetine were inactive . these data strongly suggest that the receptor activities of vortioxetine contribute to its cognition - improving properties in rats . in further support of the relevance of the receptor mechanism , this study reported improved memory performance in rats by a selective 5-ht1a receptor agonist and a 5-ht3 receptor antagonist . furthermore , a recent clinical study in elderly depressed patients showed a beneficial effect of vortioxetine compared to placebo in cognitive tests of processing speed , verbal learning , and memory . it should be noted that vortioxetine has a 10-fold lower in vitro affinity for rat 5-ht7 ( ki = 200 nm ) compared with human 5-ht7 receptors ( ki = 19 nm ) , and a 15-fold lower affinity at rat 5-ht1a ( ki = 230 nm ) compared with human 5-ht1a receptors ( ki = 15 nm ) ( table 2 ) . thus , the contribution of the 5-ht7 and 5-ht1a receptors in the clinic may be underestimated by evaluation of preclinical models . based on the current preclinical understanding of the mechanisms and the preclinical and clinical results , we hypothesize that vortioxetine 's multimodal profile including 5-ht3 and 5-ht7 antagonism , 5-ht1b partial agonism , and 5-ht1a agonism could result in enhanced glutamate transmission and contribute to its antidepressant and cognitive enhancing properties ( figure 1 ) . however , the way in which vortioxetine modulates glutamate transmission remains to be empirically determined . pharmacological treatments for major depressive disorder have evolved from monoamine - based therapies to integration of glutamatergic mechanisms . data from current clinical and preclinical compounds targeting nmda , ampa , and mglur receptors and glutamate transport present new opportunities for the treatment of depression . the serotonergic system can modulate glutamate transmission through 5-ht3 , 5-ht1a , 5-ht7 , and 5-ht1b receptors . these 5-ht receptor targets present opportunities for integrating glutamatergic modulation into monoamine - based therapies , without the direct use of glutamatergic compounds . the multimodal compounds vilazodone and vortioxetine are examples of this approach with diverse mechanisms , to indirectly modulate glutamate transmission by respectively targeting the 5-ht1a receptor , or 5-ht3 , 5-ht1a , 5-ht7 , and 5-ht1b receptors along with the sert . clinical results with these multimodal compounds will provide valuable insights into whether exploiting serotonergic modulation of glutamate transmission is an effective strategy in treating depression . the work by both authors was performed as full - time employees of lundbeck at the time of the study . vortioxetine is currently under development by h. lundbeck a / s and the takeda pharmaceutical company , ltd .

monoamine - based treatments for depression have evolved greatly over the past several years , but shortcomings such as suboptimal efficacy , treatment lag , and residual cognitive dysfunction are still significant . preclinical and clinical studies using compounds directly targeting glutamatergic neurotransmission present new opportunities for antidepressant treatment , with ketamine having a surprisingly rapid and sustained antidepressant effect that is presumably mediated through glutamate - dependent mechanisms . while direct modulation of glutamate transmission for antidepressant and cognition - enhancing actions may be hampered by nonspecific effects , indirect modulation through the serotonin ( 5-ht ) system may be a viable alternative approach . based on localization and function , 5-ht can modulate glutamate neurotransmission at least through the 5-ht1a , 5-ht1b , 5-ht3 , and 5-ht7 receptors , which presents a rational pharmacological opportunity for modulating glutamatergic transmission without the direct use of glutamatergic compounds . combining one or more of these glutamate - modulating 5-ht targets with 5-ht transporter inhibition may offer new therapeutic opportunities . the multimodal compounds vortioxetine and vilazodone are examples of this approach with diverse mechanisms , and their different clinical effects will provide valuable insights into serotonergic modulation of glutamate transmission for the potential treatment of depression and associated cognitive dysfunction .

Clinical Implications Introduction Antidepressant Effects by Modulation of Glutamate Transmission Modulation of Glutamate Transmission by 5-HT Receptors Multimodal Antidepressants Conclusions Disclosures

significant unmet needs exist in the treatment of major depressive disorder , such as suboptimal efficacy and residual cognitive dysfunction.a paradigm shift from the traditional monoamine therapeutics to approaches integrating glutamatergic function has occurred recently in antidepressant research , and has been especially fueled by the surprising rapid and sustained antidepressant effect of ketamine.we review the evidence that glutamate neurotransmission can be modulated indirectly by the 5-ht system through the 5-ht1a , 5-ht1b , 5-ht3 , and 5-ht7 receptors , and discuss the therapeutic potential of a multimodal approach , combining one or more 5-ht receptor mechanisms with 5-ht reuptake inhibition.we review the available information for the two multimodal compounds vortioxetine and vilazodone , which are examples of this approach . significant unmet needs exist in the treatment of major depressive disorder , such as suboptimal efficacy and residual cognitive dysfunction . a paradigm shift from the traditional monoamine therapeutics to approaches integrating glutamatergic function has occurred recently in antidepressant research , and has been especially fueled by the surprising rapid and sustained antidepressant effect of ketamine . we review the evidence that glutamate neurotransmission can be modulated indirectly by the 5-ht system through the 5-ht1a , 5-ht1b , 5-ht3 , and 5-ht7 receptors , and discuss the therapeutic potential of a multimodal approach , combining one or more 5-ht receptor mechanisms with 5-ht reuptake inhibition . we review the available information for the two multimodal compounds vortioxetine and vilazodone , which are examples of this approach . over the past 50 years , pharmacological treatments for major depressive disorder ( mdd ) have evolved from the older tricyclic antidepressants and monoamine oxidase inhibitors , to selective serotonin ( 5-ht ) reuptake inhibitors ( ssri ) and serotonin and norepinephrine ( ne ) reuptake inhibitors ( snris ) . despite these therapeutic evolutions , significant unmet needs still exist in treating depression , including improving suboptimal treatment response and remission rates , and cognitive impairments in domains such as memory , attention , executive function , and speed of processing . recent studies using magnetic resonance spectroscopy ( mrs ) in depressed patients have generally found reductions in glx , a combined measure of glutamate and glutamine , possibly suggesting that the total glutamatergic pool available for synaptic and metabolic activities is reduced in depression . given the complexity of glutamatergic neurotransmission and the diversity of these results , it is difficult to come to a definitive conclusion on the role of glutamate in the etiology of major depression at this time . nonetheless , interest in the role of glutamate in depression is quickly accreting , primarily due to the observation that the noncompetitive nmda receptor antagonist ketamine engenders a fast and relatively long - lasting antidepressant effect . this observation has prompted a new focus in antidepressant development toward integrating glutamatergic function , leading to the suggestion of a wide range of glutamate targets for the treatment of depression . 5-ht neurotransmission is regulated both by the serotonin transporter ( sert ) , which has been a target of antidepressants for the past 30 years , and by modulation via 5-ht receptor subtypes , some of which ( such as the 5-ht1a receptor ) may be independent therapeutic targets for the treatment of depression . a substantial body of data shows that , in addition to modulating 5-ht neurotransmission , multiple 5-ht receptor subtypes can also modulate glutamate neurotransmission . this may be reflected in results from a recent preclinical study , which found that ketamine 's fast antidepressant activity was abolished by 5-ht depletion , suggesting that these effects may be serotonin - dependent . a new class of multimodal antidepressants has emerged , which , in addition to inhibiting the sert , also modulate 5-ht receptors , and may represent an example of this integrative strategy . in this review , we summarize the current knowledge of putative glutamatergic antidepressants , 5-ht receptor - mediated glutamate modulation , and current evidence that multimodal serotonergic antidepressants with indirectly modulating roles on glutamate transmission are active in treating lowered mood and impaired cognition . the metabotropic family consists of group i receptors ( mglur1 and mglur5 ) , which potentiate both presynaptic glutamate release and postsynaptic nmda currents , and group ii ( mglur2 and mglur3 ) and group iii receptors ( mglur4 , mglur6 , mglur7 , and mglur8 ) , which in general suppress glutamate function . glutamate receptors are widely expressed in the brain , and some of them have been implicated in the treatment of depression . preclinical and clinical compounds acting via these targets and showing potential antidepressant activity are listed in table 1.table 1examples of glutamatergic compounds with antidepressant or antidepressant - like propertiescompound examplesmechanism of actiondevelopment stageeffectsreferencesketaminenmda antagonistclinical userapid ( 4 h ) antidepressant effect ; sustained for up to 1 week 35,149 memantinenmda antagonistclinical useno effect 37 lamotrigineinhibition of glutamate releaseclinical useantidepressant properties in unipolar patients 46,47 riluzoleincrease in glutamate uptakeclinical useantidepressant efficacy in treatment - resistant and bipolar depression 48,49 traxoprodilnr2b antagonistclinical developmentantidepressant effect in treatment - resistant depression after a single infusion , sustained up to 1 week 36 aniracetamampa potentiatorclinical developmentmemory - enhancing effects , antidepressant - like behavioral effects 43,44,150 ly392098ampa potentiatorpreclinicalantidepressant - like effects in the tail suspension and forced - swim tests 151 mpepmglur5 antagonistpreclinicalantidepressant - like effects in the mouse tail - suspension and rat forced swim tests 50 ly341495mglur2/3 antagonistpreclinicalantidepressant - like effects ; enhanced spatial memory 52,152 mgs0039mglur2/3 antagonistpreclinicalantidepressant - like effects 51 glyx13nmda receptor glycine site partial agonistpreclinicalantidepressant - like effects 153 examples of glutamatergic compounds with antidepressant or antidepressant - like properties over - activation of extrasynaptic nmda receptors is one of several hypothesized glutamate - related pathophysiologies for depression . dosing shows rapid ( 4 h ) antidepressant effect that is sustained for up to 7 days in therapy - resistant depressed patients . part of the mechanism for the antidepressant effect of ketamine may involve disinhibition of pyramidal cell firing as a result of the antagonism of nmda receptors located on interneurons . in support of a role for ampa receptors in treating depression , preclinical studies suggest that ketamine exerts its antidepressant - like effect through ampa receptors , and that fast action is accompanied by rapid neuronal and synaptic adaptation . it is widely believed that neuroadaptive changes represent a key event during antidepressant treatment , and may play a role in the delayed onset of efficacy in traditional antidepressants . thus , ketamine 's rapid effects on neuroadaptation may be a key mechanism in its antidepressant effects , and may converge with the general actions of antidepressant treatments suggested in the past decades . lamotrigine , a modulator of glutamate release via its action on sodium and calcium channels , is approved for relapse prevention in bipolar disorder in the united states , and may have antidepressant properties in unipolar patients . thus , although there is evidence that drugs that negatively modulate some aspects of glutamate neurotransmission have antidepressant - like effects , there is also evidence that increasing other aspects of glutamate signaling can have antidepressant - like effects . in comparison , the prominent role of glutamatergic neurotransmission in cognitive function is better understood . antagonism of nmda receptors as well as other experimental manipulations that reduce aspects of glutamatergic neurotransmission , such as antagonism at ampa or mglu5 receptors , are known to consistently impair function across a range of cognitive domains . accordingly , the glutamatergic neurotransmitter system has become a common target in developing cognition - enhancing drugs , with the broad theme that increasing synaptic glutamate neurotransmission , for example using positive allosteric modulators at ampa ( ampakines ) , mglur5 ( cdppb ) , or nmda receptors ( d - cycloserine ) , improves cognitive function in rodent models . however , improving mood and cognition by directly modulating glutamatergic neurotransmission may be difficult , as excessive glutamatergic activation can lead to excitotoxic effects and cognitive impairment . thus , a strategy to indirectly modulate glutamatergic neurotransmission in selected brain regions may be more advantageous . here we discuss four 5-ht receptors known to be involved in the action of multimodal antidepressants that have been approved or are in the approval process , and which have the potential to modulate the glutamate system based on their localization and function . the antidepressant potential of 5-ht1a receptor agonism or partial agonism has been studied in both preclinical and clinical settings . based on immunocytochemical studies , the 5-ht1a receptor is expressed in both pyramidal cells and gabaergic interneurons in the cortex and hippocampus . unlike presynaptic 5-ht1a receptors , which mainly act through inhibition of adenylate cyclase , postsynaptic 5-ht1a receptors exert their inhibitory action through g protein - coupled inwardly rectifying k channels . thus , based on the localization of 5-ht1a receptors on both gaba and glutamate neurons ( figure 1 ) , their activation may lead to either an increase or a decrease in glutamate neurotransmission depending on which subpopulations of 5-ht1a receptors are activated.figure 1a schematic diagram of the hypothesized modulatory role of 5-ht receptors on glutamatergic neurotransmission . a glutamatergic pyramidal neuron and several gaba interneurons expressing the 5-ht3 , 5-ht1a , the multimodal compounds vortioxetine and vilazodone and their possible sites of action are also shown . note that 5-ht1a , 5-ht1b , and 5-ht7 receptors may be localized on different neuronal populations . a glutamatergic pyramidal neuron and several gaba interneurons expressing the 5-ht3 , 5-ht1a , 5-ht7 , and 5-ht1b receptors on either dendrites or axon terminals are shown . the multimodal compounds vortioxetine and vilazodone and their possible sites of action are also shown . note that 5-ht1a , 5-ht1b , and 5-ht7 receptors may be localized on different neuronal populations . based on the above interaction between effects mediated through the 5-ht1a receptor and glutamatergic neurons , agonists of the 5-ht1a receptor are predicted to have a memory - modulating role , and this has been demonstrated in various preclinical studies . thus , based on the localization and function of 5-ht1a heteroreceptors , 5-ht1a receptor stimulation has the potential to enhance or suppress glutamatergic neurotransmission , and thus may also have biphasic effects on mood or cognitive function . like the 5-ht1a receptors , 5-ht1b receptors are distributed as autoreceptors or heteroreceptors throughout the brain , in areas such as the ventral pallidum , globus pallidus , substantia nigra , dorsal subiculum cerebral cortex , and the hippocampus . unlike the 5-ht1a autoreceptors , which are localized in somatodendritic regions of 5-ht neurons , 5-ht1b receptors are localized either presynaptically at nerve terminals or postsynaptically on dendrites . postsynaptic 5-ht1b receptors are co - localized with nmda or ampa receptors on dentrites , and are thus well - positioned to modulate glutamate transmission . in the dorsal subiculum , however , 5-ht1b receptors are localized on ca1 pyramidal axon terminals as inhibitory heteroceptors , and activation of these receptors attenuates glutamate transmission in the hippocampus due to its negative coupling to adenylate cyclase . it has been shown that the 5-ht1b receptor agonist cp-94253 can modulate 5-ht synthesis in the flinders sensitive line rat , an animal model of depression . in an aversive contextual learning task in mice , thus , 5-ht1b receptors may be able to positively or negatively modulate glutamate transmission and may be linked to the pathophysiology of depression . due to the somewhat contrasting antidepressant - like properties of 5-ht1b receptor agonism and memory deficit - reducing effect of 5-ht1b receptor antagonism , a balance of stimulation versus blockade of this receptor may be needed . based on this idea , a partial agonist for the 5-ht1b receptor may be a reasonable approach , although at the time of writing , the authors are not aware of any empirical investigations of the effects of 5-ht1b partial agonism on mood and cognitive function . among 5-ht receptors , the 5-ht3 receptor is the only known excitatory ion channel , and is expressed throughout the brain , including the following regions : ( 1 ) hippocampus ; ( 2 ) amygdala ; and ( 3 ) entorhinal , frontal , and cingulate cortices . thus , 5-ht3 receptor antagonism enhances glutamate transmission by reducing gaba - mediated inhibition , as illustrated in figure 1 . in conclusion , 5-ht3 receptor antagonism shows antidepressant - like activity and increased cognitive function in preclinical studies , possibly through facilitation of glutamate neurotransmission by reducing the activity of inhibitory gaba neurons . however , these effects on glutamatergic neurotransmission may be accompanied by increased inhibitory gabaergic transmission , likely due to expression in both pyramidal neurons and gabaergic interneurons . based on these data , 5-ht7 receptor activation has mixed effects on glutamatergic neurotransmission , but the overall effect in normal rodents appears to be excitatory . based on these results , 5-ht7 receptor antagonism may result in either increases or decreases in glutamatergic neurotransmission within the context of depression . these data support a modulatory role of 5-ht7 receptors on glutamate transmission as mentioned above . there are currently two multimodal compounds with clinically documented antidepressant activity : vilazodone , which is approved for clinical use in the u.s . , and vortioxetine , which is undergoing regulatory review . given the complexity of the serotonergic modulation of glutamate , it is not possible to predict the net effect that multimodal serotonergic compounds will have on glutamate neurotransmission . thus , the need for empirical data on the effects of these compounds on glutamate neurotransmission is paramount . vilazodone is a partial agonist at the 5-ht1a receptor , but with a relatively high intrinsic activity69% of the magnitude of the full 5-ht1a receptor agonist 8-oh - dpat . vilazodone 's potential to interact with glutamate neurotransmission the antidepressant efficacy of vilazodone was seen only in some of the clinical trials , partly due to the need to balance the higher dose ( 40 mg ) needed versus the high rate of gastrointestinal side effect , and thus its efficacy and safety profiles in comparison to current antidepressants require further clinical evaluation . table 2clinical compounds with serotonin ( 5-ht ) transporter ( sert ) inhibition plus activity at one or more 5-ht receptors linked to glutamatergic modulationvilazodonevortioxetinetargettype of activityhuman ic50 ( nm)human ki ( nm)rat ki ( nm)5-ht3 antagonist3.71.15-ht7 antagonist192005-ht1b partial agonist33165-ht1a agonist0.2 ( 69%)15 ( full)230sertinhibitor0.51.68.6references129112 , 134 , 135the in vitro pharmacological activities were from either binding or functional measurements . clinical compounds with serotonin ( 5-ht ) transporter ( sert ) inhibition plus activity at one or more 5-ht receptors linked to glutamatergic modulation the in vitro pharmacological activities were from either binding or functional measurements . vortioxetine is an investigational multimodal antidepressant that acts as a 5-ht3 , 5-ht7 , and 5-ht1d receptor antagonist ; 5-ht1b receptor partial agonist ; 5-ht1a receptor agonist ; and sert inhibitor in vitro ( table 2 ) . its pharmacological profile indicates that vortioxetine has the potential to modulate glutamate transmission through all of the four 5-ht receptor pathways discussed above ( figure 1 ) . additionally , 5-ht depletion - induced memory deficits were dose - dependently reversed by vortioxetine treatment , while escitalopram and duloxetine were inactive . it should be noted that vortioxetine has a 10-fold lower in vitro affinity for rat 5-ht7 ( ki = 200 nm ) compared with human 5-ht7 receptors ( ki = 19 nm ) , and a 15-fold lower affinity at rat 5-ht1a ( ki = 230 nm ) compared with human 5-ht1a receptors ( ki = 15 nm ) ( table 2 ) . based on the current preclinical understanding of the mechanisms and the preclinical and clinical results , we hypothesize that vortioxetine 's multimodal profile including 5-ht3 and 5-ht7 antagonism , 5-ht1b partial agonism , and 5-ht1a agonism could result in enhanced glutamate transmission and contribute to its antidepressant and cognitive enhancing properties ( figure 1 ) . pharmacological treatments for major depressive disorder have evolved from monoamine - based therapies to integration of glutamatergic mechanisms . data from current clinical and preclinical compounds targeting nmda , ampa , and mglur receptors and glutamate transport present new opportunities for the treatment of depression . the serotonergic system can modulate glutamate transmission through 5-ht3 , 5-ht1a , 5-ht7 , and 5-ht1b receptors . these 5-ht receptor targets present opportunities for integrating glutamatergic modulation into monoamine - based therapies , without the direct use of glutamatergic compounds . the multimodal compounds vilazodone and vortioxetine are examples of this approach with diverse mechanisms , to indirectly modulate glutamate transmission by respectively targeting the 5-ht1a receptor , or 5-ht3 , 5-ht1a , 5-ht7 , and 5-ht1b receptors along with the sert . clinical results with these multimodal compounds will provide valuable insights into whether exploiting serotonergic modulation of glutamate transmission is an effective strategy in treating depression .

biologically functional proteins and/or protein domains / regions that appear to exist as an ensemble of reversible conformers with only little or no well defined secondary / tertiary structures , and are being recognized to contain amino acid sequences that fail to automatically fold into their fully compact functional conformations under physiological conditions , have grown exponentially in last decade or so [ 110 ] . these are often known as intrinsically disordered ( i d ) proteins , which possess protein surfaces with largely unstructured and dynamic conformations [ 110 ] . one common characteristic of many i d protein regions is high number of charged amino acids and low hydrophobicity , which acts to destabilize an ordered conformation . importance of i d regions / domains in cell signaling and regulation can be easily judged by the fact that these i d containing regions / domains are reported to be much higher in eukaryotic genomes when compared with prokaryotes [ 1215 ] . the abundance of such i d protein regions / domains in eukaryotes could be due to the fact that their flexible and dynamic conformation promotes recognition of target molecules or functional binding partners , by creating large interaction surfaces suitable for macromolecular interactions [ 911 ] . there are reports showing that transcription factors with modular structures commonly possess one or more of i d regions / domains , and it is believed that nature has created such flexibility for specific functions that may require large structural flexibility under physiological conditions [ 12 , 16 ] . in spite of having common characteristics of i d nature , these regions / domains often do not share sequence homology with other members and are quite variable in size compared to other similar domains within the transcription factors [ 1720 ] . for example , steroid receptors , which possess an i d activation domain located in their n - terminal domain , are quite variable in size and sequence homology . due to unstructured nature of these i d domains it has been quite difficult to study their three - dimensional structures , and only in the recent years , we have begun to understand their structural basis [ 2123 ] . however , compared to proteins with globular structures , still not much is known about their three - dimensional structures . as we have begun to understand their physical and functional characteristics , it is now well accepted that in order to function optimally , these i d regions need to acquire well defined conformations under physiological conditions [ 2426 ] . to fully understand how precisely a transcription factor transmits the signal to regulate the expression of its specific target gene(s ) , it is pivotal to gain structural and functional information about i d regions , particularly those within the activation domain . it is likely that conformational flexibility of i d region allows it to adopt protein surfaces such that an efficient interaction can be established with other target binding partners that can result in i d sequences to achieve ordered conformation(s ) to carry out their functions [ 17 , 2832 ] . are the conformational transitions taking place during folding / unfolding of these i d regions , highly dynamic process ? how do internal and external factors influence their structural dynamics in a particular cellular environment ? these and several other fundamental questions warrant an answer to understand this complex yet extremely important phenomenon with far reaching biological consequences . therefore it is important to address the underlying structural and functional correlations that govern this critical , yet not fully understood process . some studies have shown that transcription factors remodel chromatin structure in an extremely dynamic situation such that they have the capacity to rapidly form and reform multiprotein complexes involving critical coregulatory proteins including those from the fundamental initiation complex machinery . thus , the role of their i d region / domain(s ) with flexible conformations becomes much more important , and in fact this could provide a mechanism for inclusion or exclusion of specific protein complexes that may ultimately influence the final outcome responsible for regulation of target gene either through activation or repression [ 3336 ] . it is now well accepted fact that transcriptional regulation is a highly complex and dynamic process that allows relatively small number of transcription factors to generate a huge variety of gene expression through various permutations and combinations of their interactions with target binding partners [ 3745 ] . thus , the notion that i d domains / regions of transcription factors must have significantly ordered conformation in their normal cellular milieu under physiological conditions pose a paradox that must be solved before we can fully understand their role in gene regulation . it is important to note that under physiological conditions , there are several cellular events including molecular crowding , both due to the presence of small molecules and/or macromolecules that could influence the structure formation in such i d proteins . since transcription factors function through interactions with a network of gene assembly at the level of protein expression , these kinds of conditional folding become much more relevant for them . in the recent years , data from both experimental and computational approaches are supporting this theory , and are helping us in gaining knowledge about the functional structures of i d proteins / peptides [ 5 , 6 ] . in this paper , we have discussed various ways by which these i d regions / domains of the transcription factors could acquire functionally ordered conformation(s ) , essential for their optimal functions under physiological conditions . for years , we have been relying on the theory that according to thermodynamic hypothesis of anfinsen , the amino acid sequences can provide all information needed to determine the fold of a protein , and only one collapsed folded state is possible for a specific sequence . until recently , no data challenged this concept ; however , more and more we learn about characteristics of protein folding process , it becomes clearer that it is not possible to predict with certainty the folded form of a protein from its primary sequence , and recent progresses made with the class of proteins that are i d or contain i d regions seem to challenge this hypothesis [ 17 ] . in our opinion , the biggest drawback with such a theory is that it does not consider the highly dynamic and mobile nature of protein conformation that gives such a profound flexibility to protein to adopt a number of conformations in a given cellular environment in a rapid manner . i d proteins can be divided into two classes ( at least ) ; those that can adopt a unique native conformation when subjected to stabilizing conditions ( i.e. , the addition of natural ligands such as proteins and dna or stabilizing osmolytes ) , and those that apparently have no stable native state [ 7 , 17 ] . the i d regions of many transcription factors fall into the category of those i d proteins that can fold into unique structures , suggesting that if functional properties of the transcription factors are coupled to folding / unfolding in the i d region , thermodynamic analysis of the equilibrium should provide a quantitative characterization of their function [ 7 , 12 , 34 ] . in this situation , the change in free energy should be reflected to favor folding process due to the factors responsible for folding . in fact , we and others have discovered several ways to make the i d portion of several transcription factors to fold into a functionally active form that can facilitate transcriptional activity of related protein [ 4753 ] . thus , the knowledge we have gained so far supports the idea that the conditional binding / folding of the i d regions of the transcription factors may be an important requirement for its role in gene regulation . since transcription factors work in a very selective manner to regulate specific sets of genes , conformational flexibility of i d region / domain may set up appropriate assembly of coregulatory proteins in an efficient and selective manner to regulate the target gene . it is no secret that i d stretches are quite common in proteins with essential basic cellular functions ( including many transcription factors ) , and thus may be recognized as a separate functional and structural entity based upon the basis of structure and function within the protein classes [ 13 ] . however , it may be premature to do so , unless more structural and functional characterization of such proteins becomes available . due to their differential structural and functional characteristics from those of ordered proteins , i d proteins require special experimental and computational tools for their characterization [ 1 , 3 ] . in a number of signaling proteins , sites of posttranslational modifications ( such as site - specific phosphorylation ) are located within i d region / domain [ 6 , 54 ] . one of the main reasons for such propensity is to facilitate extensive formation of hydrogen binding between the backbones and/or side chains that can occur through disorder - order transition within the i d region . the structural flexibility of i d proteins helps them to more easily and specifically adapt to protein : protein and protein : dna interaction cascades and possibly in gene regulation including alternative splicing . knowledge of these factors and the kind of conformations adopted by the i d regions / domains within the transcription factors will lead to an understanding of the role of order / disorder transition in the transcription process . details of some of the possible events that might lead to functionally folded conformations in the i d regions / domains under physiological conditions are discussed in the sections below in detail . organic osmolytes are found widely in nature to protect cellular proteins against harsh conditions such as the effects of dehydrating conditions , other hypertonic states , or the build - up of potentially denaturing metabolites , and are known to interact with the peptide backbone of proteins [ 5669 ] . osmolytes are synthesized by microorganisms , plants , and animals in response to environmental stress to protect proteins against denaturation [ 5669 ] . the free energy of these interactions corresponds to the propensity of protein to either fold or unfold due to presence of osmolytes [ 6466 ] . in spite of small energy magnitude of such interactions , peptide bonds are by far the most numerous structural component of a protein [ 6264 ] . it is the balance between osmolyte - backbone interactions and amino acid side chain - solvent interactions that determines the outcome on protein folding . in most cases , i d regions do not contain sufficient hydrophobic residues to fold spontaneously , thus addition of an osmolyte shifts the balance to a favorable negative free energy for folding . the unfavorable interaction of the osmolyte with the peptide backbone causes the preferential exclusion of the osmolyte from the protein - water interface , and it dominates over any favorable interaction of the osmolyte with the side chains of amino acids of the protein . it is the balance between osmolyte - backbone interactions and amino acid side chain - solvent interactions that determines the outcome on folding . the quantity of osmolyte required depends on both its inherent solvophobic interaction with peptide backbone and the free energy balance provided by the sum of all backbone - osmolyte interactions and the sum of all amino acid side chain - solvent interactions [ 68 , 69 ] . it has recently been shown that the effects of differing osmolytes are additive , so that under physiological conditions , cellular profolding molecules ( such as osmolytes ) may reach even higher summative concentrations [ 6466 ] . certain plants , animals , and microorganisms have adapted to environmental stresses that change the intracellular water activity by producing small organic osmolyte molecules [ 61 , 62 ] . indeed , in some organisms and in mammalian cells , certain class of osmolytes arise to counteract the effects of high intracellular concentrations of urea or other denaturing conditions on the biological activity of relevant proteins [ 61 , 62 ] . osmolytes are known to perform vital functions in many different tissues in the human body , particularly kidney and brain . without presence of relatively large quantities of osmolytes for example , urea tends to decrease the kcat and increase the km of enzymatic reactions , while the counteracting osmolyte tmao tends to have the opposite effects , that is , increasing kcat and decreasing km . furthermore , it has been shown that urea and osmolyte , trimethylamine - n - oxide have opposite effects alone or in combination . it is therefore logical to believe that many other osmolytes could have similar effects depending upon the environmental conditions and cellular effects . studies undertaken in last several years from various laboratories on osmolytes suggest that osmolyte - induced structures are in fact native - like with functional activities under physiological conditions [ 6164 ] . osmolytes are natural substances , used by many organisms to enhance proper protein folding [ 6164 ] . human kidney , for example , contains several osmolytes , and it has been calculated that osmolyte concentrations in whole tissues often reaches quite high relative to cell water content , suggesting that in certain cells / tissues , their concentrations are almost surely much higher [ 61 , 62 ] . it is well accepted fact that when a protein folds into a cooperative manner , it should result in a native - like functional species , and the consensus is that when cooperative folding in the presence of an osmolyte occurs , it is to the native folded structure [ 61 , 62 ] . osmolytes can force i d protein to fold into native - like functional species with significant secondary and tertiary structural contents in it . we have used several osmolytes to cooperatively fold an i d activation region ( af1 ) located in the n - terminal domain of the glucocorticoid receptor . we have shown that when af1 is incubated in increasing concentrations of natural organic osmolytes representative of three classes : certain amino acids ( proline ) , methylamines ( sarcosine ) , and polyols ( sorbitol ) , the i d af1 peptide folds into functionally active conformation(s ) that selectively binds several critical coregulatory proteins , and subsequent transcriptional transactivation activity . a study has shown that oral administration of an osmolyte , trehalose can inhibit polyglutamine - mediated protein aggregation in cerebrum of transgenic mouse model of huntington disease and increased life span . it has been suggested that these beneficial effects of trehalose are due to stabilizing the partially unfolded polyglutamine - containing huntingtin protein . this protein aggregation / misfolding process constitutes a hallmark of neurodegenerative pathologies , including alzheimer 's , huntington 's , and parkinson 's diseases , and if osmolytes can provide a unifying mechanism of action , this may have far reaching consequences in developing better therapeutic tools for the management of such diseases . such effects of osmolytes on protein folding pathways have become important to study . under physiological conditions , the cellular compositions of osmolytes may vary significantly ; therefore , different protein folding pathways utilized in the cell may depend upon the cellular environment within it . understanding the role of osmolytes in cell regulation will not only allow to predict the action of osmolytes on macromolecular interactions in stressed and crowded environments typical of cellular conditions , but will also provide insights on how osmolytes may be involved in pathologies or in their prevention . it is well established that to regulate transcription , transcription factors act on specific genes by binding to regulatory element sites in the dna , generally located upstream from the relevant transcription start site , and termed as response element . once bound to its specific response element through high affinity and specificity for the relatively short dna sequences contained therein , the dna bound transcription factor collects a variety of other coregulators that modify chromatin structure and/or interact with the proteins from the primary transcription initiation complex to regulate transcription from the relevant promoter [ 73 , 74 ] . thus , both protein - dna and protein : protein recognition are central processes in transcription factors function , and several reports indicate that these interactions are often accompanied by conformational changes leading to folding of the i d region(s ) in a protein molecule [ 21 , 73 , 74 ] . there are reports that dna binding stabilizes the overall global fold of protein in a manner that is consistent with folding - coupled target recognition as a mechanism to control site - specific recombination , and protein flexibility is involved in such induced - fit recognition particularly in i d dna binding proteins . it is an established fact that transient interactions between transcription factors and site - specific dna sequences are common and fundamental to many cellular processes , and protein flexibility is found to play a major role in protein : dna binding where conformational flexibility of protein acts to maximize efficiency of protein : dna binding . for transcription factors , protein first binds dna nonspecifically ( with low affinity ) in a partially or fully unfolded state and undergoes folding of i d sequences when it finds specific dna site to which it binds tightly with high affinity . an important biological implication of this binding / folding phenomenon is that in early events protein backbone mobility may play an important role in a specific binding with target molecule ; whereas later events may lead to specific signals being passed to the target gene(s ) from the complex of proteins , which emerges only after appropriate conformational changes take place . based on these observations , it is logical to hypothesize that site - specific nucleotide sequence of the regulatory element sites affects not only the overall affinity of the transcription factor for its regulatory element site , but also influences its overall conformation such that the i d region(s ) of these proteins can acquire much needed ordered conformation(s ) . as a result of such events i d surfaces on the protein molecule can be modified to accommodate various critical ancillary factors [ 73 , 74 ] . since transcriptional regulation for a specific gene depends upon the interactions of these coregulatory proteins , the exact dna sequences of the available sites in the regulatory region of the dna of the gene could help determine gene regulation [ 73 , 74 ] . biophysical studies ( using circular dichroism and fluorescence emissions ) carried out by us have shown that stoichiometric binding to a consensus response element of the glucocorticoid receptor ( an intracellular transcription factor , belonging to the nuclear hormone receptors superfamily ) results in a considerable amount of binding energy being devoted to intramolecular rearrangement in its n - terminal domain where a powerful i d transactivation domain is located . similar studies from other groups using the progesterone receptor have also been reported that its site - specific dna binding results in additional structure in its i d n - terminal domain . together , these results suggest that one of the reasons why sequence specific dna binding has such a profound effect on function of the transcription factors in general and the steroid receptors in particular may be so that their i d sequences may acquire an ordered conformation(s ) . since many transcription factors possess i d activation domain that is responsible for their transcriptional transactivation activity , and this activation domain provides a platform for interaction with other coregulatory proteins , dna binding induced conformational alterations in transcription factors is of immense importance in regulating the expression of target genes [ 5 , 12 , 17 ] . of course , conformational changes in other parts of the molecule can not be ruled out . for example , in case of the steroid receptors , dna - binding induced structural changes in the n - terminal i d domain may be influenced by other intramolecular cross communications such as interactions between n- and c - terminal domains , and/or due to binding of specific ligands . though these studies certainly provide a reasonable explanation of why such a specific protein : dna interaction takes place in a promoter region involving transcription factor , resolution of these models will require further future experiments . of course , availability of three - dimensional structure of such i d containing region bound to dna through their dna binding domain will provide much needed information . since gene regulation is an essential function in all organisms and provides the ability to respond to signals that reflect intra- and extra - cellular environmental conditions , understanding the role of protein : dna interactions involved in the regulation of gene expression has been a major challenge . in the recent years , a broad range of techniques have been used to explore the molecular and energetic basis of dna recognition , assembly , and allosteric changes within regulatory proteins that involves transcription factors . it is a well established fact that there are a number of proteins often known as coregulatory proteins that make physical and functional interactions with dna - bound transcription factors and participate in their transcriptional activation function . these coregulators act as coactivators or corepressors depending upon the up- or down - regulation of the target gene by specific transcription factor . of course , addition of several additional cofactors can not be ruled out that may be involved either directly or indirectly ; some of them are ubiquitous , while others cell - specific . in fact , for many transcription factors , it has been reported that their effects on transcriptional activity may be cell- and promoter - specific and potential explanation for these effects can be attributed to the formation of the assembly of transcription factor with other coregulatory proteins in a particular cellular setup . thus , specific combination of transcription factor and coactivators / corepressors results in the specific control of particular genes . but the obvious questions then come to mind : how is the choice of coregulator interaction with specific transcription factor made ? some of the explanation for this can be provided from the fact that differing surfaces of the transcription factor are important for regulation of various genes . there are several reports showing that protein : protein interactions may result in induced - fit alterations in the structure formation in i d region of the transcription factors . in figure 1 , we have illustrated a model of binding / folding for i d domains / regions under physiological conditions . many i d regions are known to undergo to more ordered conformational transition after interacting with their protein binding targets . for example , i d kinase - inducible transcriptional - activation domain ( kid ) of creb folds into a more ordered conformation on binding to its target peptide in cbp . i d activation domain of c - myc ( another transcription factor known to regulate the transcription of genes involved in normal cell growth , differentiation , and apoptosis ) selectively binds to proteins from the basal transcription factors , and undergoes induction of protein conformation in the i d domain of c - myc during this interaction with the target factor . similar studies have been shown involving the activation domain and its target protein for other transcription factors [ 3032 ] . we and others have shown that when an i d regulatory region of steroid receptor binds to its coregulatory protein from basal transcription machinery , structure is formed in the i d activation domain of the steroid receptor [ 27 , 29 ] . dyson and wright have suggested that the binding of i d regions to their targets is often regulated by covalent modifications , which leads to simple biological switches . applied to the i d region of transcription factors , this induced - fit model of folding hypothesizes that these i d regions do not adopt fully ordered conformation(s ) until they bind to one or more of their key target partner proteins . it appears that many transcription factors need to be more flexible in order to be efficient in carrying out their functions . because specific region(s ) of these transcription factors act by interacting with specific binding partner proteins , it is likely that their flexible structure helps them create a favorable surface for these interactions [ 5 , 12 ] . in the recent years , much attention has been focused on the role of protein : protein interactions in gene regulation by transcription factors , although a systematic analysis of all possible interactions and underlying mechanisms is still lacking . due to varied expression patterns , many cell types contain an assortment of different factors that can interact with a single transcription factor . it seems likely that the precise protein assembly , based on relative affinities combined with the allosteric effects , largely define the final transcriptional potential of each transcription factor in a given cell [ 5 , 17 ] . thus , coregulatory proteins may influence or modulate the activity of a transcription factor through multiple mechanisms [ 5 , 7476 ] . in order to fully understand the mechanisms of gene regulation by transcription as we learn more and more about the role of binding / folding events in gene regulation by transcription factors , it becomes clearer that highly flexible and dynamic nature of i d regions / domains is an inherent advantage in these molecules that exploits its protein surfaces for critical interactions with various coregulatory proteins in order to achieve desired targets in an efficient and highly specific manner . under physiological conditions , the ultimate composition of the assembly and kind of induced folding in the i d regions / domain may dictate the final outcome of the signals to be passed by specific transcription factor to the target gene . as we start to understand more about folded functional conformations of these i d stretches and the sequence of events that lead to such folding , we should have answers to many questions that regulate the expression of gene . in addition to protein : protein interactions , for some transcription factors , rnas are also known to function as cofactors , therefore id - rich transcription factors may offer a platform of rna binding . it is also important to note that structural flexibility is a common phenomenon in several protein : rna recognition processes , and these interactions often involve conformational changes in the structure of the rna , protein , or both . no doubt the elucidation of the human genome has provided us an incredible opportunity to find out an immense amount of structural information that may be contained within the human genome , yet our efforts must be devoted to understand how the expression of this genetic information is regulated and how the interactions between the vast array of expressed proteins are controlled . with large data generated from various research groups on protein : protein interactions involving transcription factor and its relationship with target gene regulations , it has become possible to visualize a global view of biological networks . dynamic macromolecular interactions are key elements in the regulation of many biological systems , particularly in gene regulations by transcription factors . in addition to other factors , the dynamic nature of such protein : protein interactions in the recent years , many observations led us to believe that direct protein : protein interaction may be an essential step in realizing properly folded and functionally active structure in their i d region . though limited , data indicate that the i d sequences may be adopting functionally folded conformation(s ) under physiological conditions through these interactions . however , it remains to be determined what kind of functional ordered conformation(s ) these i d domains adopt , and whether there are multiple folded conformations generated depending upon the nature of binding partner(s ) involved . the resulting structurally modified conformation(s ) of these i d regions may further be providing protein surfaces to attract other target molecules , essential for functions . available knowledge so far on how these i d domains of transcription factors adopt ordered conformation(s ) under physiological conditions supports this notion . however , more studies are needed to determine precise mechanisms through which these i d regions / domains acquire a well defined structure . it is now recognized that the conformational flexibility of i d domains / regions is a general feature of most transcription factor proteins , we must take into account their structural features in a network sense as recently described . since several small molecules as potential drug targets have been found to act by blocking specific protein : protein interactions , and that i d regions of transcription factors are known to form the platform for many such protein : protein interactions , a better structural and functional understanding of i d proteins will be a potent tool for drug designing . the utilization of various drug delivery systems such as nanotechnology - based products is anticipated to revolutionize treatments for diseases in future . thus , rapidly growing field of i d proteins ' structural analyses combined with their functional behavior will allow cross - disciplinary researchers the opportunity to design and develop multifunctional approaches to develop better therapeutic tools that will generate novel ideas and help accelerate critical advances in the field of biomedical research .

a number of proteins with intrinsically disordered ( i d ) regions / domains are reported to be found disproportionately higher in transcription factors . available evidences suggest that presence of i d region / domain within a transcription factor plays an important role in its biological functions . these i d sequences provide large flexible surfaces that can allow them to make more efficient physical and functional interactions with their target partners . since transcription factors regulate expression of target genes by interacting with specific coregulatory proteins , these i d regions / domains can be used as a platform for such large macromolecular interactions , and may represent a mechanism for regulation of cellular processes . the precise structural basis for the function of these i d regions / domains of the transcription factors remains to be determined . in the recent years there has been growing evidence suggesting that an induced fit - like process leads to imposition of folded functional structure in these i d domains on which large multiprotein complexes are built . these multiprotein complexes may eventually dictate the final outcome of the gene regulation by the transcription factors .

1. Introduction 2. Factors Responsible for Bringing Ordered Conformation(s) in the ID Region/Domain of the Transcription Factors 3. Osmolyte-Induced Folding of Intrinsically Disordered Region/Domain of the Transcription Factors 4. Role of Site-Specific DNA Binding in the Induced Folding of Intrinsically Disordered Region/Domain of the Transcription Factors 5. Role of Protein:Protein Interactions in Giving ID Region a Functionally Folded Conformation 6. Summary and Perspectives

these are often known as intrinsically disordered ( i d ) proteins , which possess protein surfaces with largely unstructured and dynamic conformations [ 110 ] . importance of i d regions / domains in cell signaling and regulation can be easily judged by the fact that these i d containing regions / domains are reported to be much higher in eukaryotic genomes when compared with prokaryotes [ 1215 ] . the abundance of such i d protein regions / domains in eukaryotes could be due to the fact that their flexible and dynamic conformation promotes recognition of target molecules or functional binding partners , by creating large interaction surfaces suitable for macromolecular interactions [ 911 ] . there are reports showing that transcription factors with modular structures commonly possess one or more of i d regions / domains , and it is believed that nature has created such flexibility for specific functions that may require large structural flexibility under physiological conditions [ 12 , 16 ] . in spite of having common characteristics of i d nature , these regions / domains often do not share sequence homology with other members and are quite variable in size compared to other similar domains within the transcription factors [ 1720 ] . due to unstructured nature of these i d domains it has been quite difficult to study their three - dimensional structures , and only in the recent years , we have begun to understand their structural basis [ 2123 ] . as we have begun to understand their physical and functional characteristics , it is now well accepted that in order to function optimally , these i d regions need to acquire well defined conformations under physiological conditions [ 2426 ] . to fully understand how precisely a transcription factor transmits the signal to regulate the expression of its specific target gene(s ) , it is pivotal to gain structural and functional information about i d regions , particularly those within the activation domain . it is likely that conformational flexibility of i d region allows it to adopt protein surfaces such that an efficient interaction can be established with other target binding partners that can result in i d sequences to achieve ordered conformation(s ) to carry out their functions [ 17 , 2832 ] . are the conformational transitions taking place during folding / unfolding of these i d regions , highly dynamic process ? some studies have shown that transcription factors remodel chromatin structure in an extremely dynamic situation such that they have the capacity to rapidly form and reform multiprotein complexes involving critical coregulatory proteins including those from the fundamental initiation complex machinery . thus , the role of their i d region / domain(s ) with flexible conformations becomes much more important , and in fact this could provide a mechanism for inclusion or exclusion of specific protein complexes that may ultimately influence the final outcome responsible for regulation of target gene either through activation or repression [ 3336 ] . it is now well accepted fact that transcriptional regulation is a highly complex and dynamic process that allows relatively small number of transcription factors to generate a huge variety of gene expression through various permutations and combinations of their interactions with target binding partners [ 3745 ] . thus , the notion that i d domains / regions of transcription factors must have significantly ordered conformation in their normal cellular milieu under physiological conditions pose a paradox that must be solved before we can fully understand their role in gene regulation . it is important to note that under physiological conditions , there are several cellular events including molecular crowding , both due to the presence of small molecules and/or macromolecules that could influence the structure formation in such i d proteins . since transcription factors function through interactions with a network of gene assembly at the level of protein expression , these kinds of conditional folding become much more relevant for them . in the recent years , data from both experimental and computational approaches are supporting this theory , and are helping us in gaining knowledge about the functional structures of i d proteins / peptides [ 5 , 6 ] . in this paper , we have discussed various ways by which these i d regions / domains of the transcription factors could acquire functionally ordered conformation(s ) , essential for their optimal functions under physiological conditions . until recently , no data challenged this concept ; however , more and more we learn about characteristics of protein folding process , it becomes clearer that it is not possible to predict with certainty the folded form of a protein from its primary sequence , and recent progresses made with the class of proteins that are i d or contain i d regions seem to challenge this hypothesis [ 17 ] . i d proteins can be divided into two classes ( at least ) ; those that can adopt a unique native conformation when subjected to stabilizing conditions ( i.e. the i d regions of many transcription factors fall into the category of those i d proteins that can fold into unique structures , suggesting that if functional properties of the transcription factors are coupled to folding / unfolding in the i d region , thermodynamic analysis of the equilibrium should provide a quantitative characterization of their function [ 7 , 12 , 34 ] . in fact , we and others have discovered several ways to make the i d portion of several transcription factors to fold into a functionally active form that can facilitate transcriptional activity of related protein [ 4753 ] . thus , the knowledge we have gained so far supports the idea that the conditional binding / folding of the i d regions of the transcription factors may be an important requirement for its role in gene regulation . since transcription factors work in a very selective manner to regulate specific sets of genes , conformational flexibility of i d region / domain may set up appropriate assembly of coregulatory proteins in an efficient and selective manner to regulate the target gene . it is no secret that i d stretches are quite common in proteins with essential basic cellular functions ( including many transcription factors ) , and thus may be recognized as a separate functional and structural entity based upon the basis of structure and function within the protein classes [ 13 ] . due to their differential structural and functional characteristics from those of ordered proteins , i d proteins require special experimental and computational tools for their characterization [ 1 , 3 ] . in a number of signaling proteins , sites of posttranslational modifications ( such as site - specific phosphorylation ) are located within i d region / domain [ 6 , 54 ] . one of the main reasons for such propensity is to facilitate extensive formation of hydrogen binding between the backbones and/or side chains that can occur through disorder - order transition within the i d region . the structural flexibility of i d proteins helps them to more easily and specifically adapt to protein : protein and protein : dna interaction cascades and possibly in gene regulation including alternative splicing . knowledge of these factors and the kind of conformations adopted by the i d regions / domains within the transcription factors will lead to an understanding of the role of order / disorder transition in the transcription process . details of some of the possible events that might lead to functionally folded conformations in the i d regions / domains under physiological conditions are discussed in the sections below in detail . the unfavorable interaction of the osmolyte with the peptide backbone causes the preferential exclusion of the osmolyte from the protein - water interface , and it dominates over any favorable interaction of the osmolyte with the side chains of amino acids of the protein . studies undertaken in last several years from various laboratories on osmolytes suggest that osmolyte - induced structures are in fact native - like with functional activities under physiological conditions [ 6164 ] . human kidney , for example , contains several osmolytes , and it has been calculated that osmolyte concentrations in whole tissues often reaches quite high relative to cell water content , suggesting that in certain cells / tissues , their concentrations are almost surely much higher [ 61 , 62 ] . it is well accepted fact that when a protein folds into a cooperative manner , it should result in a native - like functional species , and the consensus is that when cooperative folding in the presence of an osmolyte occurs , it is to the native folded structure [ 61 , 62 ] . we have used several osmolytes to cooperatively fold an i d activation region ( af1 ) located in the n - terminal domain of the glucocorticoid receptor . we have shown that when af1 is incubated in increasing concentrations of natural organic osmolytes representative of three classes : certain amino acids ( proline ) , methylamines ( sarcosine ) , and polyols ( sorbitol ) , the i d af1 peptide folds into functionally active conformation(s ) that selectively binds several critical coregulatory proteins , and subsequent transcriptional transactivation activity . this protein aggregation / misfolding process constitutes a hallmark of neurodegenerative pathologies , including alzheimer 's , huntington 's , and parkinson 's diseases , and if osmolytes can provide a unifying mechanism of action , this may have far reaching consequences in developing better therapeutic tools for the management of such diseases . understanding the role of osmolytes in cell regulation will not only allow to predict the action of osmolytes on macromolecular interactions in stressed and crowded environments typical of cellular conditions , but will also provide insights on how osmolytes may be involved in pathologies or in their prevention . it is well established that to regulate transcription , transcription factors act on specific genes by binding to regulatory element sites in the dna , generally located upstream from the relevant transcription start site , and termed as response element . once bound to its specific response element through high affinity and specificity for the relatively short dna sequences contained therein , the dna bound transcription factor collects a variety of other coregulators that modify chromatin structure and/or interact with the proteins from the primary transcription initiation complex to regulate transcription from the relevant promoter [ 73 , 74 ] . thus , both protein - dna and protein : protein recognition are central processes in transcription factors function , and several reports indicate that these interactions are often accompanied by conformational changes leading to folding of the i d region(s ) in a protein molecule [ 21 , 73 , 74 ] . there are reports that dna binding stabilizes the overall global fold of protein in a manner that is consistent with folding - coupled target recognition as a mechanism to control site - specific recombination , and protein flexibility is involved in such induced - fit recognition particularly in i d dna binding proteins . it is an established fact that transient interactions between transcription factors and site - specific dna sequences are common and fundamental to many cellular processes , and protein flexibility is found to play a major role in protein : dna binding where conformational flexibility of protein acts to maximize efficiency of protein : dna binding . for transcription factors , protein first binds dna nonspecifically ( with low affinity ) in a partially or fully unfolded state and undergoes folding of i d sequences when it finds specific dna site to which it binds tightly with high affinity . an important biological implication of this binding / folding phenomenon is that in early events protein backbone mobility may play an important role in a specific binding with target molecule ; whereas later events may lead to specific signals being passed to the target gene(s ) from the complex of proteins , which emerges only after appropriate conformational changes take place . based on these observations , it is logical to hypothesize that site - specific nucleotide sequence of the regulatory element sites affects not only the overall affinity of the transcription factor for its regulatory element site , but also influences its overall conformation such that the i d region(s ) of these proteins can acquire much needed ordered conformation(s ) . as a result of such events i d surfaces on the protein molecule can be modified to accommodate various critical ancillary factors [ 73 , 74 ] . since transcriptional regulation for a specific gene depends upon the interactions of these coregulatory proteins , the exact dna sequences of the available sites in the regulatory region of the dna of the gene could help determine gene regulation [ 73 , 74 ] . biophysical studies ( using circular dichroism and fluorescence emissions ) carried out by us have shown that stoichiometric binding to a consensus response element of the glucocorticoid receptor ( an intracellular transcription factor , belonging to the nuclear hormone receptors superfamily ) results in a considerable amount of binding energy being devoted to intramolecular rearrangement in its n - terminal domain where a powerful i d transactivation domain is located . similar studies from other groups using the progesterone receptor have also been reported that its site - specific dna binding results in additional structure in its i d n - terminal domain . together , these results suggest that one of the reasons why sequence specific dna binding has such a profound effect on function of the transcription factors in general and the steroid receptors in particular may be so that their i d sequences may acquire an ordered conformation(s ) . since many transcription factors possess i d activation domain that is responsible for their transcriptional transactivation activity , and this activation domain provides a platform for interaction with other coregulatory proteins , dna binding induced conformational alterations in transcription factors is of immense importance in regulating the expression of target genes [ 5 , 12 , 17 ] . for example , in case of the steroid receptors , dna - binding induced structural changes in the n - terminal i d domain may be influenced by other intramolecular cross communications such as interactions between n- and c - terminal domains , and/or due to binding of specific ligands . though these studies certainly provide a reasonable explanation of why such a specific protein : dna interaction takes place in a promoter region involving transcription factor , resolution of these models will require further future experiments . since gene regulation is an essential function in all organisms and provides the ability to respond to signals that reflect intra- and extra - cellular environmental conditions , understanding the role of protein : dna interactions involved in the regulation of gene expression has been a major challenge . in the recent years , a broad range of techniques have been used to explore the molecular and energetic basis of dna recognition , assembly , and allosteric changes within regulatory proteins that involves transcription factors . it is a well established fact that there are a number of proteins often known as coregulatory proteins that make physical and functional interactions with dna - bound transcription factors and participate in their transcriptional activation function . these coregulators act as coactivators or corepressors depending upon the up- or down - regulation of the target gene by specific transcription factor . in fact , for many transcription factors , it has been reported that their effects on transcriptional activity may be cell- and promoter - specific and potential explanation for these effects can be attributed to the formation of the assembly of transcription factor with other coregulatory proteins in a particular cellular setup . thus , specific combination of transcription factor and coactivators / corepressors results in the specific control of particular genes . but the obvious questions then come to mind : how is the choice of coregulator interaction with specific transcription factor made ? some of the explanation for this can be provided from the fact that differing surfaces of the transcription factor are important for regulation of various genes . there are several reports showing that protein : protein interactions may result in induced - fit alterations in the structure formation in i d region of the transcription factors . many i d regions are known to undergo to more ordered conformational transition after interacting with their protein binding targets . i d activation domain of c - myc ( another transcription factor known to regulate the transcription of genes involved in normal cell growth , differentiation , and apoptosis ) selectively binds to proteins from the basal transcription factors , and undergoes induction of protein conformation in the i d domain of c - myc during this interaction with the target factor . we and others have shown that when an i d regulatory region of steroid receptor binds to its coregulatory protein from basal transcription machinery , structure is formed in the i d activation domain of the steroid receptor [ 27 , 29 ] . dyson and wright have suggested that the binding of i d regions to their targets is often regulated by covalent modifications , which leads to simple biological switches . applied to the i d region of transcription factors , this induced - fit model of folding hypothesizes that these i d regions do not adopt fully ordered conformation(s ) until they bind to one or more of their key target partner proteins . because specific region(s ) of these transcription factors act by interacting with specific binding partner proteins , it is likely that their flexible structure helps them create a favorable surface for these interactions [ 5 , 12 ] . in the recent years , much attention has been focused on the role of protein : protein interactions in gene regulation by transcription factors , although a systematic analysis of all possible interactions and underlying mechanisms is still lacking . it seems likely that the precise protein assembly , based on relative affinities combined with the allosteric effects , largely define the final transcriptional potential of each transcription factor in a given cell [ 5 , 17 ] . thus , coregulatory proteins may influence or modulate the activity of a transcription factor through multiple mechanisms [ 5 , 7476 ] . in order to fully understand the mechanisms of gene regulation by transcription as we learn more and more about the role of binding / folding events in gene regulation by transcription factors , it becomes clearer that highly flexible and dynamic nature of i d regions / domains is an inherent advantage in these molecules that exploits its protein surfaces for critical interactions with various coregulatory proteins in order to achieve desired targets in an efficient and highly specific manner . under physiological conditions , the ultimate composition of the assembly and kind of induced folding in the i d regions / domain may dictate the final outcome of the signals to be passed by specific transcription factor to the target gene . as we start to understand more about folded functional conformations of these i d stretches and the sequence of events that lead to such folding , we should have answers to many questions that regulate the expression of gene . in addition to protein : protein interactions , for some transcription factors , rnas are also known to function as cofactors , therefore id - rich transcription factors may offer a platform of rna binding . it is also important to note that structural flexibility is a common phenomenon in several protein : rna recognition processes , and these interactions often involve conformational changes in the structure of the rna , protein , or both . dynamic macromolecular interactions are key elements in the regulation of many biological systems , particularly in gene regulations by transcription factors . in addition to other factors , the dynamic nature of such protein : protein interactions in the recent years , many observations led us to believe that direct protein : protein interaction may be an essential step in realizing properly folded and functionally active structure in their i d region . though limited , data indicate that the i d sequences may be adopting functionally folded conformation(s ) under physiological conditions through these interactions . however , it remains to be determined what kind of functional ordered conformation(s ) these i d domains adopt , and whether there are multiple folded conformations generated depending upon the nature of binding partner(s ) involved . the resulting structurally modified conformation(s ) of these i d regions may further be providing protein surfaces to attract other target molecules , essential for functions . available knowledge so far on how these i d domains of transcription factors adopt ordered conformation(s ) under physiological conditions supports this notion . however , more studies are needed to determine precise mechanisms through which these i d regions / domains acquire a well defined structure . it is now recognized that the conformational flexibility of i d domains / regions is a general feature of most transcription factor proteins , we must take into account their structural features in a network sense as recently described . since several small molecules as potential drug targets have been found to act by blocking specific protein : protein interactions , and that i d regions of transcription factors are known to form the platform for many such protein : protein interactions , a better structural and functional understanding of i d proteins will be a potent tool for drug designing . thus , rapidly growing field of i d proteins ' structural analyses combined with their functional behavior will allow cross - disciplinary researchers the opportunity to design and develop multifunctional approaches to develop better therapeutic tools that will generate novel ideas and help accelerate critical advances in the field of biomedical research .

traumatic spinal cord injury ( tsci ) causes permanent disability characterized by paralysis and loss of sensitivity as well as multiple metabolic and systemic alterations associated with the dysfunction of the autonomic nervous system . until now , there is no effective treatment for both acute and chronic sci , despite several strategies that have been carried out to promote regeneration and improve function . within these strategies , due to its organized structure , the use of peripheral nerve acts as a physical guide via which axons are encouraged to grow . they are also distally connected and act as a neuroprotector for the preserved spinal cord [ 26 ] . furthermore , due to the action of the schwann cells and macrophages stemming from the ppn , the regeneration and axonal remyelination are supported , since they are capable of secreting growth factors such as brain - derived neurotrophic factor ( bdnf ) , nerve growth factor ( ngf ) , neurotrophin-3 ( nt-3 ) , and granulocyte - macrophage colony - stimulating factor ( gm - csf ) which promote neuronal survival [ 713 ] . another strategy employed is the use of adult bone marrow stromal cells ( bmscs ) since they have the capacity for self - renewal and differentiation . it has been demonstrated that the use of bmscs in tsci assists in modulating the central nervous system ( cns ) environment to promote its repair and secreting anti - inflammatory and antiapoptotic molecules and growth factors , which promote axonal growth , immunomodulation , angiogenesis , remyelination , and protection from cell death caused by apoptosis . they have been shown to have the capacity to differentiate into different neural linages both in vitro and in vivo , including neurons , astrocytes , oligodendrocytes , schwann cells , and microglia [ 15 , 16 ] . furthermore , they promote axonal regrowth , remyelination , and the improvement of locomotor function , since they are capable of secreting growth factors such as bdnf , nt-3 , vegf , and bfgf [ 1722 ] . as well as those previously mentioned , there are multiple strategies that have been observed to promote axonal regrowth and the reworking of the central nervous system ( cns ) in mammals that have suffered a tsci . however , none of these alone has been able to reestablish total functionality of the injured spinal cord ( sc ) . as such , it is feasible to believe that the use of two of these in conjunction would produce greater functionality . the objective of this study was to evaluate the morphological and functional effect of transplanting bmscs and ppn into chronic paraplegic rat model that has undergone complete spinal cord transection . our hypothesis was that the combination of ppn and bmscs would give better results compared to those obtained from untreated rats or rats treated with individual transplants . this study was authorized by the research and ethics committee of the hospital de especialidades , centro medico nacional siglo xxi , instituto mexicano del seguro social ( imss ) ; use , handling , and care of animals were carried out following the official mexican standard ( norma oficial mexicana ) nom-062-zoo-1999 , which is supported on international standards . a total of 84 fischer 344 rats were used , aged between 810 weeks and weighing between 200 and 220 g. for the tsci and transplant procedures , 39 females were used having been divided into four random groups ( immunofluorescence and histology : control group : 7 animals ; fibrin glue group : 8 animals ; ppn group : 12 animals ; ppn + bmscs : 12 animals ; electron microscopy : 3 animals per group ) , 25 males were used as sciatic nerve donors , and 20 males were used to obtain bmscs . in order to produce the spinal injury , the rats were anaesthetized by the intraperitoneal injection of a mixture of ketamine ( 70 mg / kg ) and xylazine ( 10 mg / kg ) . a laminectomy was carried out at t9 before a sagittal section was made to the dural sac on the dorsal side . a complete transection of the exposed spinal cord was carried out immediately using microsurgery scissors ; finally , the surgical wound was stitched using layered closure . twenty - one days before the transplant , the peripheral nerve donor rats were anaesthetized before undergoing a complete transverse section of the sciatic nerve in the upper part of the thigh ; the caudal stump of the sectioned nerve was fixed to the surrounding muscle with a 5 - 0 nylon suture . on the day of the transplant , the rat was anaesthetized to extract a segment of sciatic nerve distal to the cut of approximately 2 cm in length . the nerve fragment was placed in chilled isotonic saline solution until the time of transplantation . the bone marrow was obtained from both femurs and tibias using a 200 l micropipette and deposited in a 15 ml conical tube with culture medium ( dulbecco 's modified eagle ( dmem ) gibco ) . following this , the cells were then separated using a ficoll ( sigma ) ( 3 ml ) gradient centrifuged at 2000 rpm at 24c for 30 minutes . the total number of nucleated cells obtained was quantified and 9 10 cells were seeded into a 75 cm culture flask ( in 5 ml of dmem with 20% fetal bovine serum ( fbs ) , from gibco ) , 1 ml of l - glutamine ( gibco ) , 5 ml of hepes ( sigma ) , and 1 ml of penicillin - streptomycin ( gibco ) ; they were then placed in a water - jacketed incubator at 37c with 5% co2 until the cells formed a fibroblast monolayer . finally , the bmscs were reseeded onto the fibroblast layer and maintained for two weeks until transplantation time . the cells were centrifuged at 1500 rpm for five minutes and they were incubated with primary antibodies ( cd117 millipore ; cd13 santa cruz biotechnology inc ; cd34 santa cruz biotechnology inc , all at a dilution of 1 : 100 ) in darkness for 20 minutes at 4c . they were washed twice with facs buffer before being centrifuged again at 1500 rpm for five minutes . the cells marked with cd117 were incubated in darkness for two hours with the secondary antibody ( alexa 488 or 586 , molecular probes invitrogen 1 : 200 ) ; they were then fixed in 4% paraformaldehyde for 1 hour before finally being quantified and analyzed with flow cytometry using the cellquest pro ( bd biosciences ) program . 80.45% of the cells were positive for cd13 ( marker for subpopulation of mesenchymal stem cells ) ; 11.49% were positive for cd117 ( marker for subpopulation of mesenchymal stem cells ) ; and 10.69% of the cellular population was positive for cd34 ( specific control marker for hematopoietic stem cells ) . as such , the majority of cells transplanted were adult mesenchymal stem cells . four weeks after the spinal cord transection , the rats were assigned to one of four experimental groups . in group 1 ( control , n = 7 ) , the surgical wound was reopened enough to expose the dural sac . in group 2 ( positive control n = 8) , the dural sac was reopened and the scar was carefully removed from the spinal cord and the end of the medullary stumps , leaving a cavity of approximately 6 mm of amputated length ; the cavity was filled with fibrin glue ( baxter ) . in group 3 ( n = 12 ) , the same procedure was carried out as described for group 2 , except , in this case , 3 or 4 segments of the sciatic peripheral nerve , each of approximately 6 mm in length , were transplanted lengthways into the medullary cavity ; the implants were fixed with fibrin glue ( baxter ) . in group 4 ( n = 12 ) , in addition to the procedures detailed for group 3 , bmscs were transplanted via four injections , two in the proximal medullary stump and two in the distal medullary stump , in the center of each hemicord ; each injection contained 3 10 cells in 5 l of hank 's solution ( sigma ) . hindlimb locomotion was evaluated using the basso , beattie , bresnahan ( bbb ) locomotor rating scale . the scale of the bbb evaluates the movements of the hindlimb of the animals ; the scale oscillates between 021 points , where 0 is no movement and 21 is a normal movement of the hindlimbs . the animals were evaluated 24 hours after the transplant and every two weeks during the following 8 weeks . eight weeks after transplantation , the animals were anaesthetized with sodium pentobarbital ( 40 mg / kg i.p ) before being perfused by intracardiac injection with 4% paraformaldehyde . a 2 cm long segment was obtained from the spinal cord with the injury zone in the center . the samples were placed in a 30% sucrose solution in pbs for 24 hours ; 12 m thick sagittal frozen serial sections were then cut on a leica cm1510s cryostat . following this , the sections were incubated for 48 hours at 4c with the primary antibodies ( protein basic myelin ( pbm ) d18 ; neuritin fl-142 , and gap-43 h-100 , santa cruz biotechnology inc . ) . the sections were later washed with pbs and incubated for 2 hours with the secondary antibody ( alexa 488 anti - rabbit or anti - mouse , molecular probes invitrogen ) ; they were washed with pbs and stained using propidium iodide ( red nuclei ) for 1 minute . finally , they were covered with vectashield ( vector labs ) in order to be analyzed with a fluorescence microscope ( carl zeiss ) . for each specimen , 6 photos were taken from the epicenter ( transplant area ) and the zones both proximal and distal to it . with the image - pro plus 5.1 ( media cybernetics ) program , the intensity of green channel pixels corresponding to the alexa 488 per area was quantified . a histogram , with previously calibrated intensity and spatial scale , was obtained from the intensity values contained within the image 's bitmap to establish the intensity values such as the integrated intensity of each image . the optical density was determined in relation to the control group and expressed in pixels / mm . the kluver - barrera staining method was performed on two specimens of each group ; the sections were hydrated before being put into 95% alcohol ; they were then placed in a luxol fast blue solution overnight at 37c . following this , the excess colorant was removed using 95% alcohol and they were rinsed with distilled water ; they were then washed with lithium carbonate and again rinsed with 70% alcohol and with distilled water . the sections were immediately placed in a purple cresol solution for 6 minutes before being returned to the 70% alcohol and dehydrated with absolute alcohol . finally , the samples were observed using a nikon ( eclipse e600 ) microscope and the morphological changes were observed . the specimens were fixed with a karnovsky solution ( 2.5% glutaraldehyde and 2% paraformaldehyde in a sorensen solution ) during 4 hours at 4c . following this , the excess osmium tetroxide was removed by twice washing with distilled water for two minutes each time . they were dehydrated in alcohols of increasing concentration ( 50% , 70% , 95% , and 100% ) to reach propylene oxide . following dehydration , the tissue was included in aldarite synthetic resin and was left to polymerize for 24 hours at 60 or 70c . once the blocks were carried out , semifine cuts were made to locate the zone to be evaluated before fine cuts were made that would be fixed to copper grids and stained with uranyl acetate and lead . the sections were analyzed with a zeiss 906 transmission electron microscope and , for each group , 10 images were taken from the transition zone only between the ppn transplant and both the proximal and distal surrounding spinal cords in which only morphological changes were observed . the graph prism 5.0 statistics program was used for descriptive analysis ; measures of central tendency and statistical dispersion were used alongside tables and graphs . for statistical inference , a nonparametric anova analysis test was used with kruskal - wallis ranges to determine the differences between groups , followed by a mann - whitney u test to identify the groups between which there was a difference . the hind extremity evaluation using the bbb rating scale showed severe motor function deterioration in the initial evaluation following the different experimental procedures . scores improved marginally as time passed , especially for the ppn and bmscs groups which reached an average rating close to 4 in contrast to the control group which maintained a rating close to 1 ( figure 2 ) . in the qualitative evaluation of the histology images , a greater quantity of gap-43 positive axons ( figure 3 ) and neuritin was found in the transplant group compared to the control group , in the zones both rostral and caudal to the injury ( figure 4 ) . furthermore , the ppn + bmscs group axons were thicker than those observed in the ppn group ( figure 3 ) . finally , the presence of growth cones marked with neuritin was only present in the ppn and ppn + bmscs groups ( figure 4 ) . a greater number of pbm - positive axons were also observed in the ppn and ppn + bmscs groups in the zones rostral and caudal to the transplant ( figure 5 ) . the gap-43 fluorescence intensity was significantly greater in the groups that received a treatment ( ppn + bmscs , fg , and ppn ) versus the control group ( p < 0.05 ) in both the rostral and caudal zones ( figures 6(a ) and 6(b ) ) ; additionally , in the caudal zone ( figure 6(b ) ) , the fluorescence intensity was significantly greater in the ppn + bmscs and ppn groups versus fg ( p < 0.05 ) . the neuritin fluorescence intensity ( figures 6(c ) and 6(d ) ) and pbm ( figures 6(e ) and 6(f ) ) in both the rostral and caudal zones was significantly greater in the ppn + bmscs and ppn groups versus the fg and control groups ( p < 0.05 ) . in the ppn and ppn + bmscs groups , it was observed that the axon myelin found had a well - defined and better - preserved structure ; furthermore , there were a greater number of myelinated axons in contrast to control and fg groups ( figure 7 ) . finally , in the ppn + bmscs group , there were several thin axons rounded by a thin sheath of myelin and beside to a schwann cell , which we consider the new axons ( figure 7 ) . in both treatment groups , it was observed that the myelinated axons were ensheathed by the schwann cells ( figure 7 ) ; on the other hand , in the ppn + bmscs group , the bmscs were found along with the axons and the schwann cells ( figure 8) . from the samples analyzed with luxol fast blue , it was observed that in the groups receiving ppn and ppn + bmscs transplant , the surrounding spinal cord structure was adequately preserved and there was good acceptance between the transplanted ppn and the preserved sc ( figure 9 ) . functional recovery of the spinal cord following a traumatic injury with complete paralysis and secondary loss of sphincter control has been achieved using diverse therapeutic strategies in animal models . it is with that objective , therefore , that proposals for new alternatives using both single and combined treatment alternatives continue to be made . it would appear that better results are achieved using combined treatments compared to single treatments . using an acute model of complete transection , guzen et al . demonstrated that axonal regrowth and improved locomotor function took place following ppn transplantation ; they also demonstrated that axonal regeneration and locomotor improvement increased when the ppn was combined with fgf-2 , making it more effective than the use of ppn alone . using an acute model of complete transection , koda et al . also demonstrated that axonal regrowth and improved locomotor function occurred following the transplantation of bmscs ; however , the combination of bmscs + bdnf showed greater effectiveness since it promoted a greater number of regenerated axons and increased locomotor function . in this study , the therapy proposed to encourage axonal regrowth , remyelination , and functional recovery was the combined use of ppn and bmsc ; considering that separately , each one of them has been shown to have a beneficial effect following the tsci as previously mentioned . the evaluation of axonal regrowth using the gap-43 protein , known to be related to axonic fiber growth following a tsci , showed a greater number of protein - positive fibers in the group transplanted with ppn than in control group . coinciding with yuan et al . in an axotomy model , gap-43 positive fibers were found following the ppn transplant . following the ppn transplantation in an acute model of complete transection , guzen et al furthermore , this study also observed that the ppn + bmscs group had a greater number of gap-43 positive fibers compared to the ppn group and they were also thicker than those of the ppn group . to date , no publications on the complete transection model are known to associate the use of bmscs and the expression of gap-43 . however , kov et al . observed the expression of gap-43 following the transplantation of bmscs in a compression model . finding gap-43 positive fibers in another study using a contusion model , kamada et al the expression of gap-43 might be mainly due to the ppn since it expresses different substances to support its regeneration such as trophic factors , adhesion molecules , and extracellular matrix molecules ; these factors provide a stimulating environment to facilitate the growth of cns axons and support locomotor function . however , since this expression is greater in the combined group , this could also be due to the contribution of the bmscs given that the use of bmscs in tsci helps modulate the cns environment to promote self - repair , secreting trophic substances that promote axonal growth . as well as observing the presence of gap-43 , this study only found neuritin immunoreactivity in the transplant groups ppn and ppn + bmscs , indicative of the presence of growth cones ; however , there was no significant difference between the transplant groups . there are no publications on complete section models in respect to this marker ; however , sarah busch and colleagues have associated the presence of growth cones with the regrowth of sensory axons in a spinal cord compression model . myelination is essential to maintaining optimum function of the cns since it promotes the conduction of nerve impulses and provides metabolic and trophic support ; so , when a tsci occurs , the destruction of myelin affects motor and sensory function in the aforementioned manner . structural affectation in different tsci models can be identified by evaluating the level of myelination . pbm has been used to evaluate the level of myelination in different experimental models . in this study , a greater expression of pbm was observed in the transplant groups , especially in the distal segment of the spinal cord and mainly in the ppn + bmscs group . furthermore , in the electron microscope study , it was demonstrated that the ppn + bmscs group had a greater quantity of myelinated axons which were more robust and which had ramifications . there are no studies known to evaluate the effect of ppn and bmscs transplant on pbm in a chronic complete transection model . however , in an acute complete transection model , chen et al . demonstrated that they obtained a greater number of myelinated axons after transplanting bmscs and the myelin had a uniform and more dense structure . this property can be explained by the capability of the bmscs to differentiate into myelinating cells . this is in line with the studies carried out by akiyama et al . who used a model of radiation injury , in which the myelinating cells were destroyed after transplantation of bmscs , demonstrating that they promoted the myelination of bare axons which improved the nerve impulse . additionally , cells with a morphology that was different from that of the nervous tissue were observed near the myelinated axons ; these cells could correspond to differentiated bmscs , which can differentiate into myelinating cells as mentioned above , and , together with the schwann cells resulting from the ppn , they help bring about a better myelination of regenerated axons as was demonstrated by dam - hieu et al . in a model of hemicordotomy , in which the axons were myelinated by the actions of the schwann cells following the transplantation of ppn . finally , and as a result of the beneficial mechanisms already mentioned in relation to both the ppn and the bmscs , it was observed that , in the ppn and ppn + bmscs transplant groups , the surrounding spinal cord structure was adequately preserved and that there was good acceptance with the ppn , which can be categorized as neuroprotection . , in which they observed that the use of ppn acted like a shock absorber for substances produced by the secondary injury mechanisms , protecting the sc surrounding the injury zone , expressed as improved medullary tissue preservation . on the other hand , feng et al . showed that , following the use of ppn in a model of contusion , there was greater neuronal preservation , which is due to the fact that the ppn promotes a microenvironment in which the schwann cells secrete growth factors such as bdnf , ngf , and nt3 which improve neuronal survival . it has been seen that the neuroprotective capacity of the bmscs comes from the secretion of different substances , which can promote immunomodulation , repair , remyelination , axonal regrowth , and improved function . using a model of compression , quertainmont et al . demonstrated that , in transplanting bmscs , their neuroprotective effects came from the secretion of molecules such as the ciliary neurotrophic factor ( cnt - f ) , the monocyte chemoattracting protein-1 ( mcp-1 ) , and the granulocyte - macrophage colony - stimulating factor ( gm - csf ) which support the survival and differentiation of oligodendrocyte precursor cells , promoting the clearance of myelin debris and protecting the neurons and glial cells from apoptosis . they also observed an increase in the secretion of anti - inflammatory cytokines such as ifn- and il-10 as well as the secretion of growth factors such as bdnf and ngf , which help protect the neurons during toxic events , promote regrowth , and repair and reorganize the neuronal connections and which also stimulate neurogenesis and protect tissue , decreasing scar formation . also observed were the antiangiogenic effects of the secretion of the vascular endothelial growth factor ( vegf ) . despite the fact that there were significant locomotion differences between transplanted and nontransplanted animals , the improvement was modest . on the other hand , although there was a tendency in favor of combined transplantation ( ppn + bmscs ) , the difference versus ppn group was not significant . in both cases , it can be due to the short term functional follow - up , because it has been seen that improvement begins at the third month after implemented treatment . poor functional improvement was observed in models of complete transection that received no additional treatment , obtaining an average bbb rating scale score of 4 points . following transplantation of ppn in an acute model of complete transection , guzen et al . obtained an average score of 5 on the bbb rating scale eight weeks after treatment . following the transplantation of bmscs in another acute injury study , chen et al . observed an average of 8 points on the same scale following eight weeks of treatment . it is possible that there is additional functional improvement with long term follow - up as reported by vaquero and zurita in a severe contusion model in which the experimental animals achieved a 13 point score with bmscs transplantation after a one - year follow - up . the transplant of ppn + bmscs in a chronic complete spinal cord transection model showed significantly great myelination in the preserved spinal cord rostral and caudal to the transplant area compared to ppn . however , although axonal regrowth and functionality were not significant , the ppn + bmscs group showed high levels . the transplanted groups showed significantly greater axonal regrowth , myelination , and functionality than the control groups . the use of new combinations that potentially increase locomotor function in the same injury model used in this study is also proposed .

functional recovery following spinal cord injury ( sci ) is limited by poor axonal and cellular regeneration as well as the failure to replace damaged myelin . employed separately , both the transplantation of the predegenerated peripheral nerve ( ppn ) and the transplantation of bone marrow stromal cells ( bmscs ) have been shown to promote the regrowth and remyelination of the damaged central axons in sci models of hemisection , transection , and contusion injury . with the aim to test the effects of the combined transplantation of ppn and bmsc on regrowth , remyelination , and locomotor function in an adult rat model of spinal cord ( sc ) transection , 39 fischer 344 rats underwent sc transection at t9 level . four weeks later they were randomly assigned to traumatic spinal cord injury ( tsci ) without treatment , tsci + fibrin glue ( fg ) , tsci + fg + ppn , and tsci + fg + ppn + bmscs . eight weeks after , transplantation was carried out on immunofluorescence and electron microscope studies . the results showed greater axonal regrowth and remyelination in experimental groups tsci + fg + ppn and tsci + fg + ppn + bmscs analyzed with gap-43 , neuritin , and myelin basic protein . it is concluded that the combined treatment of ppn and bmscs is a favorable strategy for axonal regrowth and remyelination in a chronic sc transection model .

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

traumatic spinal cord injury ( tsci ) causes permanent disability characterized by paralysis and loss of sensitivity as well as multiple metabolic and systemic alterations associated with the dysfunction of the autonomic nervous system . until now , there is no effective treatment for both acute and chronic sci , despite several strategies that have been carried out to promote regeneration and improve function . furthermore , due to the action of the schwann cells and macrophages stemming from the ppn , the regeneration and axonal remyelination are supported , since they are capable of secreting growth factors such as brain - derived neurotrophic factor ( bdnf ) , nerve growth factor ( ngf ) , neurotrophin-3 ( nt-3 ) , and granulocyte - macrophage colony - stimulating factor ( gm - csf ) which promote neuronal survival [ 713 ] . another strategy employed is the use of adult bone marrow stromal cells ( bmscs ) since they have the capacity for self - renewal and differentiation . it has been demonstrated that the use of bmscs in tsci assists in modulating the central nervous system ( cns ) environment to promote its repair and secreting anti - inflammatory and antiapoptotic molecules and growth factors , which promote axonal growth , immunomodulation , angiogenesis , remyelination , and protection from cell death caused by apoptosis . they have been shown to have the capacity to differentiate into different neural linages both in vitro and in vivo , including neurons , astrocytes , oligodendrocytes , schwann cells , and microglia [ 15 , 16 ] . furthermore , they promote axonal regrowth , remyelination , and the improvement of locomotor function , since they are capable of secreting growth factors such as bdnf , nt-3 , vegf , and bfgf [ 1722 ] . as well as those previously mentioned , there are multiple strategies that have been observed to promote axonal regrowth and the reworking of the central nervous system ( cns ) in mammals that have suffered a tsci . however , none of these alone has been able to reestablish total functionality of the injured spinal cord ( sc ) . as such , it is feasible to believe that the use of two of these in conjunction would produce greater functionality . the objective of this study was to evaluate the morphological and functional effect of transplanting bmscs and ppn into chronic paraplegic rat model that has undergone complete spinal cord transection . our hypothesis was that the combination of ppn and bmscs would give better results compared to those obtained from untreated rats or rats treated with individual transplants . this study was authorized by the research and ethics committee of the hospital de especialidades , centro medico nacional siglo xxi , instituto mexicano del seguro social ( imss ) ; use , handling , and care of animals were carried out following the official mexican standard ( norma oficial mexicana ) nom-062-zoo-1999 , which is supported on international standards . a total of 84 fischer 344 rats were used , aged between 810 weeks and weighing between 200 and 220 g. for the tsci and transplant procedures , 39 females were used having been divided into four random groups ( immunofluorescence and histology : control group : 7 animals ; fibrin glue group : 8 animals ; ppn group : 12 animals ; ppn + bmscs : 12 animals ; electron microscopy : 3 animals per group ) , 25 males were used as sciatic nerve donors , and 20 males were used to obtain bmscs . a laminectomy was carried out at t9 before a sagittal section was made to the dural sac on the dorsal side . a complete transection of the exposed spinal cord was carried out immediately using microsurgery scissors ; finally , the surgical wound was stitched using layered closure . twenty - one days before the transplant , the peripheral nerve donor rats were anaesthetized before undergoing a complete transverse section of the sciatic nerve in the upper part of the thigh ; the caudal stump of the sectioned nerve was fixed to the surrounding muscle with a 5 - 0 nylon suture . the bone marrow was obtained from both femurs and tibias using a 200 l micropipette and deposited in a 15 ml conical tube with culture medium ( dulbecco 's modified eagle ( dmem ) gibco ) . the total number of nucleated cells obtained was quantified and 9 10 cells were seeded into a 75 cm culture flask ( in 5 ml of dmem with 20% fetal bovine serum ( fbs ) , from gibco ) , 1 ml of l - glutamine ( gibco ) , 5 ml of hepes ( sigma ) , and 1 ml of penicillin - streptomycin ( gibco ) ; they were then placed in a water - jacketed incubator at 37c with 5% co2 until the cells formed a fibroblast monolayer . the cells marked with cd117 were incubated in darkness for two hours with the secondary antibody ( alexa 488 or 586 , molecular probes invitrogen 1 : 200 ) ; they were then fixed in 4% paraformaldehyde for 1 hour before finally being quantified and analyzed with flow cytometry using the cellquest pro ( bd biosciences ) program . four weeks after the spinal cord transection , the rats were assigned to one of four experimental groups . in group 2 ( positive control n = 8) , the dural sac was reopened and the scar was carefully removed from the spinal cord and the end of the medullary stumps , leaving a cavity of approximately 6 mm of amputated length ; the cavity was filled with fibrin glue ( baxter ) . in group 3 ( n = 12 ) , the same procedure was carried out as described for group 2 , except , in this case , 3 or 4 segments of the sciatic peripheral nerve , each of approximately 6 mm in length , were transplanted lengthways into the medullary cavity ; the implants were fixed with fibrin glue ( baxter ) . the scale of the bbb evaluates the movements of the hindlimb of the animals ; the scale oscillates between 021 points , where 0 is no movement and 21 is a normal movement of the hindlimbs . eight weeks after transplantation , the animals were anaesthetized with sodium pentobarbital ( 40 mg / kg i.p ) before being perfused by intracardiac injection with 4% paraformaldehyde . a 2 cm long segment was obtained from the spinal cord with the injury zone in the center . following this , the sections were incubated for 48 hours at 4c with the primary antibodies ( protein basic myelin ( pbm ) d18 ; neuritin fl-142 , and gap-43 h-100 , santa cruz biotechnology inc . ) the sections were later washed with pbs and incubated for 2 hours with the secondary antibody ( alexa 488 anti - rabbit or anti - mouse , molecular probes invitrogen ) ; they were washed with pbs and stained using propidium iodide ( red nuclei ) for 1 minute . finally , they were covered with vectashield ( vector labs ) in order to be analyzed with a fluorescence microscope ( carl zeiss ) . for each specimen , 6 photos were taken from the epicenter ( transplant area ) and the zones both proximal and distal to it . with the image - pro plus 5.1 ( media cybernetics ) program , the intensity of green channel pixels corresponding to the alexa 488 per area was quantified . the kluver - barrera staining method was performed on two specimens of each group ; the sections were hydrated before being put into 95% alcohol ; they were then placed in a luxol fast blue solution overnight at 37c . they were dehydrated in alcohols of increasing concentration ( 50% , 70% , 95% , and 100% ) to reach propylene oxide . the sections were analyzed with a zeiss 906 transmission electron microscope and , for each group , 10 images were taken from the transition zone only between the ppn transplant and both the proximal and distal surrounding spinal cords in which only morphological changes were observed . scores improved marginally as time passed , especially for the ppn and bmscs groups which reached an average rating close to 4 in contrast to the control group which maintained a rating close to 1 ( figure 2 ) . in the qualitative evaluation of the histology images , a greater quantity of gap-43 positive axons ( figure 3 ) and neuritin was found in the transplant group compared to the control group , in the zones both rostral and caudal to the injury ( figure 4 ) . furthermore , the ppn + bmscs group axons were thicker than those observed in the ppn group ( figure 3 ) . finally , the presence of growth cones marked with neuritin was only present in the ppn and ppn + bmscs groups ( figure 4 ) . a greater number of pbm - positive axons were also observed in the ppn and ppn + bmscs groups in the zones rostral and caudal to the transplant ( figure 5 ) . the gap-43 fluorescence intensity was significantly greater in the groups that received a treatment ( ppn + bmscs , fg , and ppn ) versus the control group ( p < 0.05 ) in both the rostral and caudal zones ( figures 6(a ) and 6(b ) ) ; additionally , in the caudal zone ( figure 6(b ) ) , the fluorescence intensity was significantly greater in the ppn + bmscs and ppn groups versus fg ( p < 0.05 ) . the neuritin fluorescence intensity ( figures 6(c ) and 6(d ) ) and pbm ( figures 6(e ) and 6(f ) ) in both the rostral and caudal zones was significantly greater in the ppn + bmscs and ppn groups versus the fg and control groups ( p < 0.05 ) . in the ppn and ppn + bmscs groups , it was observed that the axon myelin found had a well - defined and better - preserved structure ; furthermore , there were a greater number of myelinated axons in contrast to control and fg groups ( figure 7 ) . finally , in the ppn + bmscs group , there were several thin axons rounded by a thin sheath of myelin and beside to a schwann cell , which we consider the new axons ( figure 7 ) . in both treatment groups , it was observed that the myelinated axons were ensheathed by the schwann cells ( figure 7 ) ; on the other hand , in the ppn + bmscs group , the bmscs were found along with the axons and the schwann cells ( figure 8) . from the samples analyzed with luxol fast blue , it was observed that in the groups receiving ppn and ppn + bmscs transplant , the surrounding spinal cord structure was adequately preserved and there was good acceptance between the transplanted ppn and the preserved sc ( figure 9 ) . functional recovery of the spinal cord following a traumatic injury with complete paralysis and secondary loss of sphincter control has been achieved using diverse therapeutic strategies in animal models . it is with that objective , therefore , that proposals for new alternatives using both single and combined treatment alternatives continue to be made . using an acute model of complete transection , guzen et al . demonstrated that axonal regrowth and improved locomotor function took place following ppn transplantation ; they also demonstrated that axonal regeneration and locomotor improvement increased when the ppn was combined with fgf-2 , making it more effective than the use of ppn alone . using an acute model of complete transection , koda et al . also demonstrated that axonal regrowth and improved locomotor function occurred following the transplantation of bmscs ; however , the combination of bmscs + bdnf showed greater effectiveness since it promoted a greater number of regenerated axons and increased locomotor function . in this study , the therapy proposed to encourage axonal regrowth , remyelination , and functional recovery was the combined use of ppn and bmsc ; considering that separately , each one of them has been shown to have a beneficial effect following the tsci as previously mentioned . following the ppn transplantation in an acute model of complete transection , guzen et al furthermore , this study also observed that the ppn + bmscs group had a greater number of gap-43 positive fibers compared to the ppn group and they were also thicker than those of the ppn group . to date , no publications on the complete transection model are known to associate the use of bmscs and the expression of gap-43 . observed the expression of gap-43 following the transplantation of bmscs in a compression model . finding gap-43 positive fibers in another study using a contusion model , kamada et al the expression of gap-43 might be mainly due to the ppn since it expresses different substances to support its regeneration such as trophic factors , adhesion molecules , and extracellular matrix molecules ; these factors provide a stimulating environment to facilitate the growth of cns axons and support locomotor function . however , since this expression is greater in the combined group , this could also be due to the contribution of the bmscs given that the use of bmscs in tsci helps modulate the cns environment to promote self - repair , secreting trophic substances that promote axonal growth . as well as observing the presence of gap-43 , this study only found neuritin immunoreactivity in the transplant groups ppn and ppn + bmscs , indicative of the presence of growth cones ; however , there was no significant difference between the transplant groups . there are no publications on complete section models in respect to this marker ; however , sarah busch and colleagues have associated the presence of growth cones with the regrowth of sensory axons in a spinal cord compression model . myelination is essential to maintaining optimum function of the cns since it promotes the conduction of nerve impulses and provides metabolic and trophic support ; so , when a tsci occurs , the destruction of myelin affects motor and sensory function in the aforementioned manner . in this study , a greater expression of pbm was observed in the transplant groups , especially in the distal segment of the spinal cord and mainly in the ppn + bmscs group . furthermore , in the electron microscope study , it was demonstrated that the ppn + bmscs group had a greater quantity of myelinated axons which were more robust and which had ramifications . there are no studies known to evaluate the effect of ppn and bmscs transplant on pbm in a chronic complete transection model . however , in an acute complete transection model , chen et al . this is in line with the studies carried out by akiyama et al . who used a model of radiation injury , in which the myelinating cells were destroyed after transplantation of bmscs , demonstrating that they promoted the myelination of bare axons which improved the nerve impulse . additionally , cells with a morphology that was different from that of the nervous tissue were observed near the myelinated axons ; these cells could correspond to differentiated bmscs , which can differentiate into myelinating cells as mentioned above , and , together with the schwann cells resulting from the ppn , they help bring about a better myelination of regenerated axons as was demonstrated by dam - hieu et al . in a model of hemicordotomy , in which the axons were myelinated by the actions of the schwann cells following the transplantation of ppn . finally , and as a result of the beneficial mechanisms already mentioned in relation to both the ppn and the bmscs , it was observed that , in the ppn and ppn + bmscs transplant groups , the surrounding spinal cord structure was adequately preserved and that there was good acceptance with the ppn , which can be categorized as neuroprotection . , in which they observed that the use of ppn acted like a shock absorber for substances produced by the secondary injury mechanisms , protecting the sc surrounding the injury zone , expressed as improved medullary tissue preservation . showed that , following the use of ppn in a model of contusion , there was greater neuronal preservation , which is due to the fact that the ppn promotes a microenvironment in which the schwann cells secrete growth factors such as bdnf , ngf , and nt3 which improve neuronal survival . it has been seen that the neuroprotective capacity of the bmscs comes from the secretion of different substances , which can promote immunomodulation , repair , remyelination , axonal regrowth , and improved function . demonstrated that , in transplanting bmscs , their neuroprotective effects came from the secretion of molecules such as the ciliary neurotrophic factor ( cnt - f ) , the monocyte chemoattracting protein-1 ( mcp-1 ) , and the granulocyte - macrophage colony - stimulating factor ( gm - csf ) which support the survival and differentiation of oligodendrocyte precursor cells , promoting the clearance of myelin debris and protecting the neurons and glial cells from apoptosis . they also observed an increase in the secretion of anti - inflammatory cytokines such as ifn- and il-10 as well as the secretion of growth factors such as bdnf and ngf , which help protect the neurons during toxic events , promote regrowth , and repair and reorganize the neuronal connections and which also stimulate neurogenesis and protect tissue , decreasing scar formation . also observed were the antiangiogenic effects of the secretion of the vascular endothelial growth factor ( vegf ) . on the other hand , although there was a tendency in favor of combined transplantation ( ppn + bmscs ) , the difference versus ppn group was not significant . poor functional improvement was observed in models of complete transection that received no additional treatment , obtaining an average bbb rating scale score of 4 points . following transplantation of ppn in an acute model of complete transection , guzen et al . obtained an average score of 5 on the bbb rating scale eight weeks after treatment . following the transplantation of bmscs in another acute injury study , chen et al . it is possible that there is additional functional improvement with long term follow - up as reported by vaquero and zurita in a severe contusion model in which the experimental animals achieved a 13 point score with bmscs transplantation after a one - year follow - up . the transplant of ppn + bmscs in a chronic complete spinal cord transection model showed significantly great myelination in the preserved spinal cord rostral and caudal to the transplant area compared to ppn . however , although axonal regrowth and functionality were not significant , the ppn + bmscs group showed high levels . the transplanted groups showed significantly greater axonal regrowth , myelination , and functionality than the control groups . the use of new combinations that potentially increase locomotor function in the same injury model used in this study is also proposed .

non - hodgkin s lymphomas ( nhls ) comprise a heterogeneous group of malignancies of the lymphoid tissues . in the us , the great majority ( 80%85% ) present with a b - cell phenotype , while 15%20% of nhls are derived from t - cell subtypes and rarely from natural killer ( nk ) cells . because of the high heterogeneity of diseases included in the nhl category , the clinical presentation varies greatly according to the subtype and area of involvement , ranging from indolent to highly aggressive forms . in 2013 , 69,740 new cases of nhl were estimated , with 19,020 deaths , making nhl the seventh - leading type of cancer in the us and accounting for 4% of new cases and 3% of cancer - related deaths . the incidence of nhl is currently rising , with a 3.7% yearly percentage increase recorded between 1975 and 1991 and a 0.3% yearly increase from 1992 to 2007 . this has been at least in part attributed to the strict relationship between the occurrence of nhl and a competent immune system , with diseases or treatments that weaken the immune system often favoring the development of nhl . indeed , patients affected with human immunodeficiency virus ( hiv ) , inherited immunodeficiency syndromes , or autoimmune diseases , or treated with high - dose chemotherapy , as well as recipients of organ - transplant or hematopoietic stem cells , have a higher risk of developing nhl . based on similar mechanisms , infections also play an important role in the development of some lymphomas by impairing multiple immune functions or inducing chronic inflammatory responses . barr virus ( ebv ) is strongly associated with the occurrence of burkitt s lymphoma , nasal nk - t lymphomas , and posttransplant lymphoproliferative diseases ( ptlds ) , helicobacter pylori is a risk factor for gastric mucosal associated lymphomas , hepatitis c virus is associated with splenic marginal - zone lymphomas , borrelia burgdorferi with cutaneous mucosa - associated lymphoid - tissue lymphomas , and chlamydophila psittaci with ocular adrenal lymphomas.1 all these conditions are associated , to different extents , with a defect of the b - cell , t - cell , or nk - cell compartments that play a central role in patrolling the body and preventing the proliferation of transformed cell clones . for lymphomas associated with conditions that directly affect the immune system ( hiv , primary immunodeficiency , chemotherapy ) , a failure in immunosurveillance leads to the development of nhl , while in the case of infection - associated lymphomas , dysfunctional immunosurveillance needs to be associated with chronic antigen exposure and the presence of oncogenic viruses . in this case , genetics and defective deoxyribonucleic acid - damage responses play a relevant role in the pathogenesis of the diseases.2 the median age at diagnosis for nhl is 66 years , with more than 9% of patients over the age of 85 years . importantly , the patient s comorbidities related to this older age often restrict the applicability of standard chemotherapy regimens.3 the response rates of patients with nhl to conventional chemotherapy are generally greater than 50% . however , despite the numerous drugs and combinations available , a significant fraction of nhl patients eventually relapse due to incomplete eradication of tumor cells . numerous regimens have been studied as options for salvage therapy and for aggressive nhl , but despite the inclusion of high - dose chemotherapy and autologous stem cell transplant ( auto - sct ) , only 50% of patients survive in the long term . outcomes are even worse in patients with chemotherapy - resistant disease and for those ineligible for transplant because of age or comorbidities , with an expected survival of less than 1 year . allogeneic sct offers lower relapse rates compared to auto - sct , but the myeloablative pretransplant regimens are associated with high treatment - related mortality , which precludes its use in many patients.4 therefore , important challenges remain for the management of patients that fail complete tumor eradication postchemotherapies and/or are ineligible for transplant . specifically , for older or frailer patients , new less toxic strategies need to be developed and explored to overcome treatment failure.5 these therapies range from monoclonal antibodies ( mabs ) , ab drug conjugates , radioimmunotherapy , and small - molecule inhibitors targeting cell survival and growth pathways . rituximab ( the chimeric anti - cd20 ab ) is the pivotal example of mab therapy for nhl . thanks to its dramatic impact on the overall survival and response rate as front - line therapy , this drug is now part of the standard of care for patients with b - cell lymphomas.6 in the rituximab era , limited data are available on the efficacy of salvage therapy for relapsed / refractory nhl , and the role of rituximab in salvage regimens , when already included in primary therapy , remains unclear.7,8 in some cases , mabs are conjugated with cytotoxic agents to enhance the therapeutic efficacy of the original ab and ensure limited side effects.9 an example of ab drug conjugate therapy is represented by brentuximab vedotin , ( sgn-35 ) , an mab targeting cd30 used successfully in the last few years for nhls , such as anaplastic large - cell lymphoma or peripheral t - cell lymphomas , that express the cd30 molecule . this treatment has produced a 41% complete remission ( cr ) rate in relapsed patients , although the median duration of response has been often limited.10 alternatively , mabs can be chemically conjugated to radioactive isotopes for tumor targeting and delivery , or fragments of two mabs can be coupled to provide increased tumor - targeting specificity through binding of two tumor - specific antigens and enhanced cytotoxic efficacy by engagement of multiple effector mechanisms . the best example of this bispecific ab technology is blinatumomab , which couples cd19 ( a b - cell marker ) and cd3 ( a t - cell engager ) for recruitment of t - cell activity against b - cell malignancies . the first phase i study of 62 nhl patients demonstrated an overall response rate of 82% across nhl subtypes , maintained up to 3 years for 60% of responders . unfortunately , severe side effects ( encephalopathy , tremor , and aphasia ) require discontinuation of therapy in about 20% of patients.11 in parallel with the aforementioned approaches , strategies relying on restoring immune cell activities have been developed for the cure of nhl . in - depth knowledge on the specific characteristics of current t - cell - based approaches available in the clinic and on the advantages and weaknesses of each specific strategy can aid in the management of nhl patients postchemotherapy . t - cell - based approaches currently available to treat nhl are ( figure 1 and table 1 ) : ebv - specific t - cellstumor - associated antigen ( taa)-specific t - cellstransgenic t - cell receptor ( tcr)-engineered t - cellschimeric antigen receptor ( car)-engineered t - cells . tumor - associated antigen ( taa)-specific t - cells transgenic t - cell receptor ( tcr)-engineered t - cells chimeric antigen receptor ( car)-engineered t - cells . infections are known to play an important role in the development of some lymphomas by impairing multiple immune functions or inducing chronic inflammatory responses . this is a human 1-herpesvirus with oncogenic potential responsible for causing a self - limiting disease known as infectious mononucleosis when acquired during adolescence or an asymptomatic infection if acquired early in life . infection of b cells is accompanied by the expression of the highly immunogenic ebv - associated antigens ( latent ebnas and lytic antigens ) . cytotoxic t - cells ( ctls ) targeting these antigens limit the uncontrolled expansion of the infected b cells . however , the virus can be carried lifelong in the memory b - cell compartment in a latent state by expressing only few poorly immunogenic antigens ( ebna-1 and lmp1 and lmp2 ) . if t - cells are deficient in numbers or functionality , such as post hematopoietic stem cell or solid - organ transplantation , respectively , ebv reactivates and promotes the unrestricted proliferation of infected cells , leading to the development of a group of diseases collectively referred as ptlds.12 as these tumors express several of the same highly immunogenic antigens commonly present in ebv - infected b cells , the restoration of ctls targeting these antigens was anticipated as a potential cure . the approach was first explored at the memorial sloan kettering cancer center following the transfer of unmanipulated t lymphocytes from ebv - seropositive donors . although effective , this strategy was hampered by the high risk of graft - versus - host disease.13 a few years later , rooney et al refined the approach by specifically expanding t - cells targeting only ebv - associated antigens ( ebv - specific ctls ) , and showed that the infusion of ebv - specific ctls in patients with ptld was not only effective but also free from side effects.14 this pioneering work provided the rationale for extending the use of ebv - specific ctls to many more ebv - associated malignancies . as the etiology of approximately 30%40% of all nhl is recognized to be associated with ebv,15 it was important to apply the concept of ebv - ctl adoptive immunotherapy for nhl , though to be applicable for nhl , the process for the generation of the ebv - specific ctls had to be slightly modified . first , unlike ptld , which expresses the highly immunogenic ebv antigens , nhl almost exclusively expresses the less immunogenic lmp1 and lmp2 antigens . t - cell precursors targeting these weak antigens are usually very limited in the peripheral blood of individuals and patients , requiring the development of more complex protocols for ctl production . the relevance of lmp2 as a target antigen has been underlined by the high rate of clinical responses in patients infused with autologous ebv - specific ctls enriched in lmp2-specific precursors . of the eight nhl patients treated , none experienced immediate or long - term toxicities , four patients remained in cr , two patients treated with disease achieved durable cr ( for 9 and 10 months , respectively ) , and another one had a very good partial response , according to the response evaluation criteria in solid tumors.16 to further improve the efficacy of the approach , ebv - specific ctls were enriched for both lmp1 and lmp2 t - cell precursors , and in a comparative study , 33 patients were infused with ctls targeting both lmp1 and lmp2 antigens . at a median follow up of 3.1 years , none of the 29 patients treated in cr experienced disease relapse . the 2-year event - free survival ( efs ) was 82% , and deaths occurred from nonrelapse causes . of the 21 patients treated with clinical evidence of disease , eleven reached cr early after ctl infusion , while one achieved cr only after additional ctl infusions . the overall 2-year efs for patients treated with resistant / recurrent disease was 50% , and a trend for a better response was seen when ctls were targeting both lmp1 and lmp2 antigens.15 lack of toxicity and the reasonably effective profile of lmp - specific ctls render this approach quite appealing for application in patients who due to their age or presence of comorbidities had not completed or received full treatment and had failed or relapsed in their disease . for instance , the procedure is currently very time - consuming , since more than 2 months are necessary for the preparation of ebv lymphoblastoid cells and then production of virus - specific ctls . in addition , since the majority of nhl patients are treated with rituximab , which ablates circulating b cells for several months , the generation of ebv lymphoblastoid cells is frequently difficult to achieve . nevertheless , the use of artificial antigen - presenting cells ( apcs ) and peptide libraries as a source of specific antigens ( ebna-1 , lmp1/2 , and barf-1 ) may significantly simplify the manufacturing procedures.17 one nih - registered clinical trial based on this improved manufacturing approach is ongoing in our institution ( nct01555892 ) . as nhls are associated with ebv in only approximately 40% of cases , new antigens are needed for extending the benefits of ctl - based therapy to most nhl patients . the majority of well - characterized nonviral taas are , however , self - antigens and thus weak stimulators of t - cell immunity . in addition , self - reactive t - cells are typically deleted by thymic selection or frequently rendered unresponsive by tolerance mechanisms . within the many reported taas , cancer testis antigens ( ctas ) have attracted significant interest in the community , as they are consistently expressed by multiple tumors but are absent in healthy organs , with the exception of germ - line tissues.18 although sometimes demanding due to the low frequency of taa - ctl precursors in the peripheral blood , numerous strategies have been proposed to expand sufficient numbers of taa - ctls ex vivo . advances in the ebv or viral - associated antigen setting have significantly contributed to the taa - ctl field . protocols used for the generation of the former have been applied , with modifications , for the generation of the latter . apcs infected with adenoviruses coding the protein of interest or loaded with specific human leukocyte antigen ( hla)-restricted taa peptide or pepmixes have been used to generate cta- or taa - specific ctls ex vivo.19 most of the early clinical trials have been performed in patients with melanomas or leukemias and used cd8 t - cells targeting a single antigen ( most frequently mart-1,20 melanoma - associated antigen peptide tyrosinase , or gp10021 for melanoma and wt-122 for leukemia ) expressed in the context of specific major histocompatibility complex ( mhc ) class i molecules , generally hla - a2 and hla - b44 , and results are rather promising . as nhls frequently express a number of these ctas , like ssx2 , mage - a4 , prame , and ny - eso-1 , and the taa survivin , there is increasing interest for extending this therapy to patients with ebv - negative nhl . to expand this application to all patients , including those with less common hlas , pepmixes loaded to autologous apcs have proven very useful.23 as described earlier for ebv - specific ctls , the selection and expansion of ctl precursors for taas may benefit from improved manufacturing procedures based on the use of artificial apcs and pepmixes as the source of antigens . recently , our group developed a protocol that uses a combination of several pepmixes to generate ctl specific for multiple taas . ctl lines generated from lymphoma patients showed specific responses against a broad array of taas , specifically targeting ssx2 in four of eight lines , mage - a4 in six of eight lines , survivin in two of eight lines , prame in three of three lines , and ny - eso-1 in three of three lines . the safety , feasibility , and efficacy of this approach are currently being tested in an ongoing clinical trial enrolling patients with either hodgkin s lymphoma or nhl.23 as the physiological killing of t - cells occurs through the engagement of the tcr , the surface heterodimer between - and -chains that mediates recognition of short epitopes presented in the context of the mhc , the transfer of - and -tcr chains into t - cells is currently being explored as a means to reliably produce highly functionally avid t - cells in a very short period of time . gene - modified t - cells with tcrs cloned from single t - cells screened for high avidity have been used in clinical trials , and shown to be safe and feasible for the treatment of melanoma.24 although encouraging responses also have been reported in sarcomas , myeloma , and renal cancers through the targeting of ny - eso-1 , mage - a3 , p53 , and carcinoembryonic antigens , the major obstacle to a broad application of this strategy relates to the restricted number of studied hla molecules ( limited to the most common haplotypes ) , and therefore to the few known epitopes . the expression of mage - a and ny - eso-1 in 40%50% of nhl makes the infusion of transgenic tcrs appealing for patients with relapsed nhl . indeed , the surface expression of the desired tcrs can be hampered by the incorrect pairing of the transgenic chains with the endogenous chains , thus producing not only nonfunctional tcrs but also tcrs with potentially unwanted specificities , including induction of harmful recognition of self - antigens . second , the isolation of t - cell clones with high functional avidity from which cloning the -tcr chains invariably fails when the antigen expressed by the tumor cells is also present on healthy cells , as thymic selection eliminates these autoreactive clones . in these scenarios , functional avidities have been enhanced empirically by maturing the affinity of the engineered tcrs through phage - display technologies . while quite successful in generating improved antitumor activity , these approaches have recently raised concerns , as unwanted toxicities have occurred . in a recent case , new tcr specificities were generated in t - cells engineered to express a high - affinity enhanced tcr against an hla - a*01 restricted mage - a3 epitope.25,26 the first two patients treated with these engineered t - cells developed cardiac toxicity and died within days of the t - cell infusion . although heart tissues do not express mage - a3 , severe myocardial damage and robust proliferation of the transferred t - cells occurred due to the cross - recognition ( unexpectedly generated during the ex vivo affinity - enhancement process ) by the transgenic tcr of titin , a peptide derived from a striated muscle - specific protein.25,26 in other cases , affinity maturation can result in an increased tcr - threshold level of recognition , which can be dangerous when antigens , such as survivin , are expressed with different threshold between tumors and healthy cells . therefore , success with this strategy may be strictly dependent on targeting antigens expressed at different stages of tissue development , of cell activation , or overexpressed by tumor cells but no longer by healthy tissues , or derived from proteins that result from gene mutations or aberrations in tumor cells , making them uniquely expressed by tumor cells but not healthy tissues . due to the extensive gene manipulation required for the technology , this option for nhl patients finally , genotoxic risks , although not reported for t - cells , and complexities associated with gene manipulation render this technology still highly experimental . the overall major limitation of all the aforementioned tcr - mediated killing approaches remains the requirement for hla restriction and for functional antigen - processing machinery in tumor cells . however , to escape immune recognition , tumors invariably interfere with antigen processing or cause downregulation of mhc molecules . this phenomenon has been reported to occur in nhl , and is frequently the cause of relapse . a technical breakthrough was made in 1993 , when the antigen - specificity of mabs targeting a surface molecule essential or preserved in tumor cells was grafted on t - cells to confer lytic activity and prolonged persistence.27 these so - called car molecules provide t - cells with the capacity to recognize and kill malignant t - cells in an mhc - independent fashion and to recognize epitopes derived from carbohydrates and glycolipids , thus broadening the array of potential targets . the major advantage of cars over the corresponding mabs is represented by the direct activation of -chain signaling with rapid and highly efficient killing and recruitment of many other components of the immune systems . self - amplification and development of responses mediated by car t - cells are finally expected to be sustained in magnitude and prolonged in time , with the possibility to generate memory and thus long - term protection.25 as relapsed nhls often exhibit low hla expression , the setting of relapsed nhl disease has highly fueled the application of this approach . cd19 , cd20 , and cd30 are validated targets for ab - based therapy of nhl . numerous efforts have been aimed at the translation of car - based therapy toward these molecules for application in nhl . extensive preclinical work has been done to ensure that car - molecule engagement provides appropriate signaling to t - cells . this work has resulted in the incorporation of costimulatory molecules within the car for appropriate activation of these cells or in grafting the car on antigen - specific t - cells or central memory cells to guarantee long - term persistence . thus far , results on 26 patients with nhl receiving car - cd19 or car - cd20 t - cell therapy in the presence of active disease have been reported in the literature . because of gene manipulation and the potential for genotoxic risk , this approach is available only to patients that have exhausted other options . therefore , to date , only patients with multiple relapsed diseases have been enrolled to receive this therapy . most of the cars tested in the clinical setting for nhl incorporate the cd28 endodomain , an early costimulatory signal molecule that is usually supplied by professional apcs . although effective in improving in vivo expansion of car t - cells , persistence and antitumor efficacy in vivo have proven suboptimal , with one cr following auto - sct , and only a few transient partial responses ( 27% ) . as the incorporation of the 4 - 1bb endodomain ( also known as cd137 ) , a member of the tnf - receptor family involved in the late costimulatory signal of t - cells , has produced quite impressive responses in b - cell leukemias , its inclusion instead of or in addition to cd28 may be beneficial for car studies targeting nhl . importantly , the only crs observed so far in nhl have been in a pilot trial with a car that incorporated both the cd28 and 41bb endodomains or in patients heavily pretreated with b - cell - specific chemotherapy regimens prior to car - t - cells.28 further modifications are currently being investigated to achieve optimal expansion and clinical activity of car t - cells . therefore , the results of the many ongoing studies ( table 2 ) , although using cars generated under different culture conditions and different constructs , will be important to understand the efficacy and the impact of this approach for the treatment of nhl . as reported for tcr - based approaches and for patients treated with bispecific abs , some severe toxicities have been reported for car - based therapy , in terms of on - target / off - tumor effects and the rapid rise in serum proinflammatory cytokines , respectively . the expression of the cd20 and cd19 target antigens by healthy b cells is responsible for the occurrence of b - cell aplasia.29 while the administration of immunoglobulin can control this side effect in young patients , in older individuals , the prolonged b - cell aplasia when combined with the t - cell dysfunction following heavy chemotherapy regimens may be a fatal combination by predisposing them to life - threatening opportunistic infections . strategies to spare the normal b - cell compartment while delivering comparable antitumor activity are currently being explored.30 to deal with on - target / off - tumor toxicities of less tumor - selective cars , such as those targeting molecules like carcinoembryonic antigens or erbb2 , some investigators have proposed transiently expressing car through electroporation of t - cells with in vitro - transcribed ribonucleic acid,31 which warrants a self - limiting expression of the car . two patients have been treated with this approach , and although the infusion of these t - cells was safe and induced epitope spreading , the efficacy was very limited , mostly due to reduced persistence of the gene - modified t - cells . alternatively , groups are exploring the incorporation of a suicide gene into the construct encoding for the car to provide a rapid and effective method of controlling adverse events.32 the so - called cytokine - release storm ( crs ) is thus far the most common toxicity associated with the administration of car t - cells . although not all patients receiving car - t - cells experience crs , when it occurs , the severity of the clinical presentation is important , and fatalities have occurred.33 efforts are currently directed to reduce the occurrence of crs , but increasing evidence suggests that inhibiting t - cell activity too early in order to control crs may also impair their antitumor activity.34 while encouraging , car t - cell immunotherapy appears to be not as successful for nhl as it has been for acute lymphoblastic leukemia ( all ) . this experience suggests an important peculiarity for relapsed nhl . while the target antigen is identical , nhl and all have different tumor environments , and this may play a significant role in response to therapy . indeed , numerous evidence suggests that nhl tumors , especially when relapsed , display several immune - escape evasion tools , like upregulation of pdl1,35 infiltration by regulatory t - cells,36 and release of inhibitory molecules37,38 ( granzyme b inhibitor or protease inhibitor 9 ) that are absent in all . furthermore , t - cell activation following antigen engagement is often accompanied by the upregulation of pd1.39 the interaction between pd1 and pdl1 on many tumor cells can deliver a strong inhibitory signal that neutralizes the antitumor activity of residential lymphocytes , as well as of the adoptively transferred t - cells . as the administration of anti - pd1 abs has proven effective in highly immunogenic diseases in releasing t - cells from the inhibition induced through the pdl1pd1 axis , the combination with checkpoint inhibitors appears the most promising route to pursue to improve the efficacy of the adoptively transferred t - cells . lessons learned from these trials will be applicable for all previously described ( native or transgenic ) tcr - based immunotherapeutic approaches . infections are known to play an important role in the development of some lymphomas by impairing multiple immune functions or inducing chronic inflammatory responses . this is a human 1-herpesvirus with oncogenic potential responsible for causing a self - limiting disease known as infectious mononucleosis when acquired during adolescence or an asymptomatic infection if acquired early in life . infection of b cells is accompanied by the expression of the highly immunogenic ebv - associated antigens ( latent ebnas and lytic antigens ) . cytotoxic t - cells ( ctls ) targeting these antigens limit the uncontrolled expansion of the infected b cells . however , the virus can be carried lifelong in the memory b - cell compartment in a latent state by expressing only few poorly immunogenic antigens ( ebna-1 and lmp1 and lmp2 ) . if t - cells are deficient in numbers or functionality , such as post hematopoietic stem cell or solid - organ transplantation , respectively , ebv reactivates and promotes the unrestricted proliferation of infected cells , leading to the development of a group of diseases collectively referred as ptlds.12 as these tumors express several of the same highly immunogenic antigens commonly present in ebv - infected b cells , the restoration of ctls targeting these antigens was anticipated as a potential cure . the approach was first explored at the memorial sloan kettering cancer center following the transfer of unmanipulated t lymphocytes from ebv - seropositive donors . although effective , this strategy was hampered by the high risk of graft - versus - host disease.13 a few years later , rooney et al refined the approach by specifically expanding t - cells targeting only ebv - associated antigens ( ebv - specific ctls ) , and showed that the infusion of ebv - specific ctls in patients with ptld was not only effective but also free from side effects.14 this pioneering work provided the rationale for extending the use of ebv - specific ctls to many more ebv - associated malignancies . as the etiology of approximately 30%40% of all nhl is recognized to be associated with ebv,15 it was important to apply the concept of ebv - ctl adoptive immunotherapy for nhl , though to be applicable for nhl , the process for the generation of the ebv - specific ctls had to be slightly modified . first , unlike ptld , which expresses the highly immunogenic ebv antigens , nhl almost exclusively expresses the less immunogenic lmp1 and lmp2 antigens . t - cell precursors targeting these weak antigens are usually very limited in the peripheral blood of individuals and patients , requiring the development of more complex protocols for ctl production . the relevance of lmp2 as a target antigen has been underlined by the high rate of clinical responses in patients infused with autologous ebv - specific ctls enriched in lmp2-specific precursors . of the eight nhl patients treated , none experienced immediate or long - term toxicities , four patients remained in cr , two patients treated with disease achieved durable cr ( for 9 and 10 months , respectively ) , and another one had a very good partial response , according to the response evaluation criteria in solid tumors.16 to further improve the efficacy of the approach , ebv - specific ctls were enriched for both lmp1 and lmp2 t - cell precursors , and in a comparative study , 33 patients were infused with ctls targeting both lmp1 and lmp2 antigens . at a median follow up of 3.1 years , none of the 29 patients treated in cr experienced disease relapse . the 2-year event - free survival ( efs ) was 82% , and deaths occurred from nonrelapse causes . of the 21 patients treated with clinical evidence of disease , eleven reached cr early after ctl infusion , while one achieved cr only after additional ctl infusions . the overall 2-year efs for patients treated with resistant / recurrent disease was 50% , and a trend for a better response was seen when ctls were targeting both lmp1 and lmp2 antigens.15 lack of toxicity and the reasonably effective profile of lmp - specific ctls render this approach quite appealing for application in patients who due to their age or presence of comorbidities had not completed or received full treatment and had failed or relapsed in their disease . for instance , the procedure is currently very time - consuming , since more than 2 months are necessary for the preparation of ebv lymphoblastoid cells and then production of virus - specific ctls . in addition , since the majority of nhl patients are treated with rituximab , which ablates circulating b cells for several months , the generation of ebv lymphoblastoid cells is frequently difficult to achieve . nevertheless , the use of artificial antigen - presenting cells ( apcs ) and peptide libraries as a source of specific antigens ( ebna-1 , lmp1/2 , and barf-1 ) may significantly simplify the manufacturing procedures.17 one nih - registered clinical trial based on this improved manufacturing approach is ongoing in our institution ( nct01555892 ) . as nhls are associated with ebv in only approximately 40% of cases , new antigens are needed for extending the benefits of ctl - based therapy to most nhl patients . the majority of well - characterized nonviral taas are , however , self - antigens and thus weak stimulators of t - cell immunity . in addition , self - reactive t - cells are typically deleted by thymic selection or frequently rendered unresponsive by tolerance mechanisms . within the many reported taas , cancer testis antigens ( ctas ) have attracted significant interest in the community , as they are consistently expressed by multiple tumors but are absent in healthy organs , with the exception of germ - line tissues.18 although sometimes demanding due to the low frequency of taa - ctl precursors in the peripheral blood , numerous strategies have been proposed to expand sufficient numbers of taa - ctls ex vivo . advances in the ebv or viral - associated antigen setting have significantly contributed to the taa - ctl field . protocols used for the generation of the former have been applied , with modifications , for the generation of the latter . apcs infected with adenoviruses coding the protein of interest or loaded with specific human leukocyte antigen ( hla)-restricted taa peptide or pepmixes have been used to generate cta- or taa - specific ctls ex vivo.19 most of the early clinical trials have been performed in patients with melanomas or leukemias and used cd8 t - cells targeting a single antigen ( most frequently mart-1,20 melanoma - associated antigen peptide tyrosinase , or gp10021 for melanoma and wt-122 for leukemia ) expressed in the context of specific major histocompatibility complex ( mhc ) class i molecules , generally hla - a2 and hla - b44 , and results are rather promising . as nhls frequently express a number of these ctas , like ssx2 , mage - a4 , prame , and ny - eso-1 , and the taa survivin , there is increasing interest for extending this therapy to patients with ebv - negative nhl . to expand this application to all patients , including those with less common hlas , pepmixes loaded to autologous apcs have proven very useful.23 as described earlier for ebv - specific ctls , the selection and expansion of ctl precursors for taas may benefit from improved manufacturing procedures based on the use of artificial apcs and pepmixes as the source of antigens . recently , our group developed a protocol that uses a combination of several pepmixes to generate ctl specific for multiple taas . ctl lines generated from lymphoma patients showed specific responses against a broad array of taas , specifically targeting ssx2 in four of eight lines , mage - a4 in six of eight lines , survivin in two of eight lines , prame in three of three lines , and ny - eso-1 in three of three lines . the safety , feasibility , and efficacy of this approach are currently being tested in an ongoing clinical trial enrolling patients with either hodgkin s lymphoma or nhl.23 as the physiological killing of t - cells occurs through the engagement of the tcr , the surface heterodimer between - and -chains that mediates recognition of short epitopes presented in the context of the mhc , the transfer of - and -tcr chains into t - cells is currently being explored as a means to reliably produce highly functionally avid t - cells in a very short period of time . gene - modified t - cells with tcrs cloned from single t - cells screened for high avidity have been used in clinical trials , and shown to be safe and feasible for the treatment of melanoma.24 although encouraging responses also have been reported in sarcomas , myeloma , and renal cancers through the targeting of ny - eso-1 , mage - a3 , p53 , and carcinoembryonic antigens , the major obstacle to a broad application of this strategy relates to the restricted number of studied hla molecules ( limited to the most common haplotypes ) , and therefore to the few known epitopes . the expression of mage - a and ny - eso-1 in 40%50% of nhl makes the infusion of transgenic tcrs appealing for patients with relapsed nhl . indeed , the surface expression of the desired tcrs can be hampered by the incorrect pairing of the transgenic chains with the endogenous chains , thus producing not only nonfunctional tcrs but also tcrs with potentially unwanted specificities , including induction of harmful recognition of self - antigens . second , the isolation of t - cell clones with high functional avidity from which cloning the -tcr chains invariably fails when the antigen expressed by the tumor cells is also present on healthy cells , as thymic selection eliminates these autoreactive clones . in these scenarios , functional avidities have been enhanced empirically by maturing the affinity of the engineered tcrs through phage - display technologies . while quite successful in generating improved antitumor activity , these approaches have recently raised concerns , as unwanted toxicities have occurred . in a recent case , new tcr specificities were generated in t - cells engineered to express a high - affinity enhanced tcr against an hla - a*01 restricted mage - a3 epitope.25,26 the first two patients treated with these engineered t - cells developed cardiac toxicity and died within days of the t - cell infusion . although heart tissues do not express mage - a3 , severe myocardial damage and robust proliferation of the transferred t - cells occurred due to the cross - recognition ( unexpectedly generated during the ex vivo affinity - enhancement process ) by the transgenic tcr of titin , a peptide derived from a striated muscle - specific protein.25,26 in other cases , affinity maturation can result in an increased tcr - threshold level of recognition , which can be dangerous when antigens , such as survivin , are expressed with different threshold between tumors and healthy cells . therefore , success with this strategy may be strictly dependent on targeting antigens expressed at different stages of tissue development , of cell activation , or overexpressed by tumor cells but no longer by healthy tissues , or derived from proteins that result from gene mutations or aberrations in tumor cells , making them uniquely expressed by tumor cells but not healthy tissues . due to the extensive gene manipulation required for the technology , this option for nhl patients can be offered only in highly specialized facilities with extensive regulatory capacities . finally , genotoxic risks , although not reported for t - cells , and complexities associated with gene manipulation render this technology still highly experimental . the overall major limitation of all the aforementioned tcr - mediated killing approaches remains the requirement for hla restriction and for functional antigen - processing machinery in tumor cells . however , to escape immune recognition , tumors invariably interfere with antigen processing or cause downregulation of mhc molecules . this phenomenon has been reported to occur in nhl , and is frequently the cause of relapse . a technical breakthrough was made in 1993 , when the antigen - specificity of mabs targeting a surface molecule essential or preserved in tumor cells was grafted on t - cells to confer lytic activity and prolonged persistence.27 these so - called car molecules provide t - cells with the capacity to recognize and kill malignant t - cells in an mhc - independent fashion and to recognize epitopes derived from carbohydrates and glycolipids , thus broadening the array of potential targets . the major advantage of cars over the corresponding mabs is represented by the direct activation of -chain signaling with rapid and highly efficient killing and recruitment of many other components of the immune systems . self - amplification and development of responses mediated by car t - cells are finally expected to be sustained in magnitude and prolonged in time , with the possibility to generate memory and thus long - term protection.25 as relapsed nhls often exhibit low hla expression , the setting of relapsed nhl disease has highly fueled the application of this approach . cd19 , cd20 , and cd30 are validated targets for ab - based therapy of nhl . numerous efforts have been aimed at the translation of car - based therapy toward these molecules for application in nhl . extensive preclinical work has been done to ensure that car - molecule engagement provides appropriate signaling to t - cells . this work has resulted in the incorporation of costimulatory molecules within the car for appropriate activation of these cells or in grafting the car on antigen - specific t - cells or central memory cells to guarantee long - term persistence . thus far , results on 26 patients with nhl receiving car - cd19 or car - cd20 t - cell therapy in the presence of active disease have been reported in the literature . because of gene manipulation and the potential for genotoxic risk , this approach is available only to patients that have exhausted other options . therefore , to date , only patients with multiple relapsed diseases have been enrolled to receive this therapy . most of the cars tested in the clinical setting for nhl incorporate the cd28 endodomain , an early costimulatory signal molecule that is usually supplied by professional apcs . although effective in improving in vivo expansion of car t - cells , persistence and antitumor efficacy in vivo have proven suboptimal , with one cr following auto - sct , and only a few transient partial responses ( 27% ) . as the incorporation of the 4 - 1bb endodomain ( also known as cd137 ) , a member of the tnf - receptor family involved in the late costimulatory signal of t - cells , has produced quite impressive responses in b - cell leukemias , its inclusion instead of or in addition to cd28 may be beneficial for car studies targeting nhl . importantly , the only crs observed so far in nhl have been in a pilot trial with a car that incorporated both the cd28 and 41bb endodomains or in patients heavily pretreated with b - cell - specific chemotherapy regimens prior to car - t - cells.28 further modifications are currently being investigated to achieve optimal expansion and clinical activity of car t - cells . therefore , the results of the many ongoing studies ( table 2 ) , although using cars generated under different culture conditions and different constructs , will be important to understand the efficacy and the impact of this approach for the treatment of nhl . as reported for tcr - based approaches and for patients treated with bispecific abs , some severe toxicities have been reported for car - based therapy , in terms of on - target / off - tumor effects and the rapid rise in serum proinflammatory cytokines , respectively . the expression of the cd20 and cd19 target antigens by healthy b cells is responsible for the occurrence of b - cell aplasia.29 while the administration of immunoglobulin can control this side effect in young patients , in older individuals , the prolonged b - cell aplasia when combined with the t - cell dysfunction following heavy chemotherapy regimens may be a fatal combination by predisposing them to life - threatening opportunistic infections . strategies to spare the normal b - cell compartment while delivering comparable antitumor activity are currently being explored.30 to deal with on - target / off - tumor toxicities of less tumor - selective cars , such as those targeting molecules like carcinoembryonic antigens or erbb2 , some investigators have proposed transiently expressing car through electroporation of t - cells with in vitro - transcribed ribonucleic acid,31 which warrants a self - limiting expression of the car . two patients have been treated with this approach , and although the infusion of these t - cells was safe and induced epitope spreading , the efficacy was very limited , mostly due to reduced persistence of the gene - modified t - cells . alternatively , groups are exploring the incorporation of a suicide gene into the construct encoding for the car to provide a rapid and effective method of controlling adverse events.32 the so - called cytokine - release storm ( crs ) is thus far the most common toxicity associated with the administration of car t - cells . although not all patients receiving car - t - cells experience crs , when it occurs , the severity of the clinical presentation is important , and fatalities have occurred.33 efforts are currently directed to reduce the occurrence of crs , but increasing evidence suggests that inhibiting t - cell activity too early in order to control crs may also impair their antitumor activity.34 while encouraging , car t - cell immunotherapy appears to be not as successful for nhl as it has been for acute lymphoblastic leukemia ( all ) . while the target antigen is identical , nhl and all have different tumor environments , and this may play a significant role in response to therapy . indeed , numerous evidence suggests that nhl tumors , especially when relapsed , display several immune - escape evasion tools , like upregulation of pdl1,35 infiltration by regulatory t - cells,36 and release of inhibitory molecules37,38 ( granzyme b inhibitor or protease inhibitor 9 ) that are absent in all . furthermore , t - cell activation following antigen engagement is often accompanied by the upregulation of pd1.39 the interaction between pd1 and pdl1 on many tumor cells can deliver a strong inhibitory signal that neutralizes the antitumor activity of residential lymphocytes , as well as of the adoptively transferred t - cells . as the administration of anti - pd1 abs has proven effective in highly immunogenic diseases in releasing t - cells from the inhibition induced through the pdl1pd1 axis , the combination with checkpoint inhibitors appears the most promising route to pursue to improve the efficacy of the adoptively transferred t - cells . lessons learned from these trials will be applicable for all previously described ( native or transgenic ) tcr - based immunotherapeutic approaches . high hopes are held for the adoptive transfer of antigen - specific t - cells to complement the standard therapies for patients affected by nhl . although preliminary , available results from current and ongoing clinical trials support the administration of lmp1/2-specific ctls to consolidate crs in patients treated with no evidence of disease and to induce durable responses in those treated with measurable disease . the major benefit of this approach is its safety profile and great tolerability , particularly in more fragile or heavily pretreated patients . similar results are anticipated when using taa - ctl adoptive immunotherapy for nhl lacking ebv antigens . although data are promising , t - cells infused in nhl have shown limited persistence and expansion , explaining , at least in part , the more limited antitumor activity observed in nhl compared to patients affected by other diseases but treated with comparable t - cells / ctls in clinical trials . a more in - depth understanding of the biology of t - cell immune responses in nhl patients will certainly shed light into this diversity in clinical responses . several strategies have been developed to increase the effectiveness of adoptively transferred t - cells . on one side , investigators are exploring ex vivo strategies to increase the stemness of infused t - cells to improve persistence and proliferation . this can be achieved by reducing the length of the ex vivo culture and/or by manipulating the culture conditions using , eg , specific cytokine cocktails,40 or by selecting specific t - cell subpopulations41 or antigen - specific t - cells.42 in parallel , new costimulatory molecules or combinations of costimulatory pathways continue to be explored to facilitate prolonged car - t - cell persistence . on the other hand , in vivo tools are being used to deliver potent proliferative signals . specifically , in vivo proliferation can be driven by preconditioning regimens that induce profound lymphodepletion and as a consequence the release of significant amounts of homeostatic cytokines . fine tuning of these approaches is ongoing , as the massive proliferation associated with increased stemness and concentration of homeostatic cytokines has resulted in some cases in fatalities . although confounding for the interpretation of the relevance played by each component in the final clinical outcome , the systematic combination of adoptively transferred t - cells with standard therapies or with the new immunomodulatory drugs available in the clinical arena ( like checkpoint inhibitors , anti - ctla4 , or anti - pd1 abs ) may provide t - cells with the right environment to fulfill their potential and result in better control of the disease .

non - hodgkin s lymphoma ( nhl ) represents a heterogeneous group of malignancies with high diversity in terms of biology , clinical responses , and prognosis . standard therapy regimens produce a 5-year relative survival rate of only 69% , with the critical need to increase the treatment - success rate of this patient population presenting at diagnosis with a median age of 66 years and many comorbidities . the evidence that an impaired immune system favors the development of nhl has opened the stage for new therapeutics , and specifically for the adoptive transfer of ex vivo - expanded antigen - specific t - cells . in this review , we discuss how t - cells specific for viral - associated antigens , nonviral - associated antigens expressed by the tumor , t - cells redirected through the expression of chimeric antigen receptors , and transgenic t - cell receptors against tumor cells have been developed and used in clinical trials for the treatment of patients with nhls .

Background NHL and immunotherapy Adoptive T-cell therapy for NHL EpsteinBarr virus-specific T-cells for the treatment of NHL Tumor-associated antigen-specific T-cells for the treatment of NHL Transgenic TCR-engineered T-cells for the treatment of NHL Chimeric antigen receptor-engineered T-cells for the treatment of NHL Conclusion

non - hodgkin s lymphomas ( nhls ) comprise a heterogeneous group of malignancies of the lymphoid tissues . this has been at least in part attributed to the strict relationship between the occurrence of nhl and a competent immune system , with diseases or treatments that weaken the immune system often favoring the development of nhl . barr virus ( ebv ) is strongly associated with the occurrence of burkitt s lymphoma , nasal nk - t lymphomas , and posttransplant lymphoproliferative diseases ( ptlds ) , helicobacter pylori is a risk factor for gastric mucosal associated lymphomas , hepatitis c virus is associated with splenic marginal - zone lymphomas , borrelia burgdorferi with cutaneous mucosa - associated lymphoid - tissue lymphomas , and chlamydophila psittaci with ocular adrenal lymphomas.1 all these conditions are associated , to different extents , with a defect of the b - cell , t - cell , or nk - cell compartments that play a central role in patrolling the body and preventing the proliferation of transformed cell clones . for lymphomas associated with conditions that directly affect the immune system ( hiv , primary immunodeficiency , chemotherapy ) , a failure in immunosurveillance leads to the development of nhl , while in the case of infection - associated lymphomas , dysfunctional immunosurveillance needs to be associated with chronic antigen exposure and the presence of oncogenic viruses . in this case , genetics and defective deoxyribonucleic acid - damage responses play a relevant role in the pathogenesis of the diseases.2 the median age at diagnosis for nhl is 66 years , with more than 9% of patients over the age of 85 years . thanks to its dramatic impact on the overall survival and response rate as front - line therapy , this drug is now part of the standard of care for patients with b - cell lymphomas.6 in the rituximab era , limited data are available on the efficacy of salvage therapy for relapsed / refractory nhl , and the role of rituximab in salvage regimens , when already included in primary therapy , remains unclear.7,8 in some cases , mabs are conjugated with cytotoxic agents to enhance the therapeutic efficacy of the original ab and ensure limited side effects.9 an example of ab drug conjugate therapy is represented by brentuximab vedotin , ( sgn-35 ) , an mab targeting cd30 used successfully in the last few years for nhls , such as anaplastic large - cell lymphoma or peripheral t - cell lymphomas , that express the cd30 molecule . unfortunately , severe side effects ( encephalopathy , tremor , and aphasia ) require discontinuation of therapy in about 20% of patients.11 in parallel with the aforementioned approaches , strategies relying on restoring immune cell activities have been developed for the cure of nhl . t - cell - based approaches currently available to treat nhl are ( figure 1 and table 1 ) : ebv - specific t - cellstumor - associated antigen ( taa)-specific t - cellstransgenic t - cell receptor ( tcr)-engineered t - cellschimeric antigen receptor ( car)-engineered t - cells . tumor - associated antigen ( taa)-specific t - cells transgenic t - cell receptor ( tcr)-engineered t - cells chimeric antigen receptor ( car)-engineered t - cells . infection of b cells is accompanied by the expression of the highly immunogenic ebv - associated antigens ( latent ebnas and lytic antigens ) . if t - cells are deficient in numbers or functionality , such as post hematopoietic stem cell or solid - organ transplantation , respectively , ebv reactivates and promotes the unrestricted proliferation of infected cells , leading to the development of a group of diseases collectively referred as ptlds.12 as these tumors express several of the same highly immunogenic antigens commonly present in ebv - infected b cells , the restoration of ctls targeting these antigens was anticipated as a potential cure . although effective , this strategy was hampered by the high risk of graft - versus - host disease.13 a few years later , rooney et al refined the approach by specifically expanding t - cells targeting only ebv - associated antigens ( ebv - specific ctls ) , and showed that the infusion of ebv - specific ctls in patients with ptld was not only effective but also free from side effects.14 this pioneering work provided the rationale for extending the use of ebv - specific ctls to many more ebv - associated malignancies . of the eight nhl patients treated , none experienced immediate or long - term toxicities , four patients remained in cr , two patients treated with disease achieved durable cr ( for 9 and 10 months , respectively ) , and another one had a very good partial response , according to the response evaluation criteria in solid tumors.16 to further improve the efficacy of the approach , ebv - specific ctls were enriched for both lmp1 and lmp2 t - cell precursors , and in a comparative study , 33 patients were infused with ctls targeting both lmp1 and lmp2 antigens . within the many reported taas , cancer testis antigens ( ctas ) have attracted significant interest in the community , as they are consistently expressed by multiple tumors but are absent in healthy organs , with the exception of germ - line tissues.18 although sometimes demanding due to the low frequency of taa - ctl precursors in the peripheral blood , numerous strategies have been proposed to expand sufficient numbers of taa - ctls ex vivo . apcs infected with adenoviruses coding the protein of interest or loaded with specific human leukocyte antigen ( hla)-restricted taa peptide or pepmixes have been used to generate cta- or taa - specific ctls ex vivo.19 most of the early clinical trials have been performed in patients with melanomas or leukemias and used cd8 t - cells targeting a single antigen ( most frequently mart-1,20 melanoma - associated antigen peptide tyrosinase , or gp10021 for melanoma and wt-122 for leukemia ) expressed in the context of specific major histocompatibility complex ( mhc ) class i molecules , generally hla - a2 and hla - b44 , and results are rather promising . the safety , feasibility , and efficacy of this approach are currently being tested in an ongoing clinical trial enrolling patients with either hodgkin s lymphoma or nhl.23 as the physiological killing of t - cells occurs through the engagement of the tcr , the surface heterodimer between - and -chains that mediates recognition of short epitopes presented in the context of the mhc , the transfer of - and -tcr chains into t - cells is currently being explored as a means to reliably produce highly functionally avid t - cells in a very short period of time . gene - modified t - cells with tcrs cloned from single t - cells screened for high avidity have been used in clinical trials , and shown to be safe and feasible for the treatment of melanoma.24 although encouraging responses also have been reported in sarcomas , myeloma , and renal cancers through the targeting of ny - eso-1 , mage - a3 , p53 , and carcinoembryonic antigens , the major obstacle to a broad application of this strategy relates to the restricted number of studied hla molecules ( limited to the most common haplotypes ) , and therefore to the few known epitopes . second , the isolation of t - cell clones with high functional avidity from which cloning the -tcr chains invariably fails when the antigen expressed by the tumor cells is also present on healthy cells , as thymic selection eliminates these autoreactive clones . although heart tissues do not express mage - a3 , severe myocardial damage and robust proliferation of the transferred t - cells occurred due to the cross - recognition ( unexpectedly generated during the ex vivo affinity - enhancement process ) by the transgenic tcr of titin , a peptide derived from a striated muscle - specific protein.25,26 in other cases , affinity maturation can result in an increased tcr - threshold level of recognition , which can be dangerous when antigens , such as survivin , are expressed with different threshold between tumors and healthy cells . due to the extensive gene manipulation required for the technology , this option for nhl patients finally , genotoxic risks , although not reported for t - cells , and complexities associated with gene manipulation render this technology still highly experimental . a technical breakthrough was made in 1993 , when the antigen - specificity of mabs targeting a surface molecule essential or preserved in tumor cells was grafted on t - cells to confer lytic activity and prolonged persistence.27 these so - called car molecules provide t - cells with the capacity to recognize and kill malignant t - cells in an mhc - independent fashion and to recognize epitopes derived from carbohydrates and glycolipids , thus broadening the array of potential targets . self - amplification and development of responses mediated by car t - cells are finally expected to be sustained in magnitude and prolonged in time , with the possibility to generate memory and thus long - term protection.25 as relapsed nhls often exhibit low hla expression , the setting of relapsed nhl disease has highly fueled the application of this approach . this work has resulted in the incorporation of costimulatory molecules within the car for appropriate activation of these cells or in grafting the car on antigen - specific t - cells or central memory cells to guarantee long - term persistence . importantly , the only crs observed so far in nhl have been in a pilot trial with a car that incorporated both the cd28 and 41bb endodomains or in patients heavily pretreated with b - cell - specific chemotherapy regimens prior to car - t - cells.28 further modifications are currently being investigated to achieve optimal expansion and clinical activity of car t - cells . therefore , the results of the many ongoing studies ( table 2 ) , although using cars generated under different culture conditions and different constructs , will be important to understand the efficacy and the impact of this approach for the treatment of nhl . the expression of the cd20 and cd19 target antigens by healthy b cells is responsible for the occurrence of b - cell aplasia.29 while the administration of immunoglobulin can control this side effect in young patients , in older individuals , the prolonged b - cell aplasia when combined with the t - cell dysfunction following heavy chemotherapy regimens may be a fatal combination by predisposing them to life - threatening opportunistic infections . furthermore , t - cell activation following antigen engagement is often accompanied by the upregulation of pd1.39 the interaction between pd1 and pdl1 on many tumor cells can deliver a strong inhibitory signal that neutralizes the antitumor activity of residential lymphocytes , as well as of the adoptively transferred t - cells . infection of b cells is accompanied by the expression of the highly immunogenic ebv - associated antigens ( latent ebnas and lytic antigens ) . if t - cells are deficient in numbers or functionality , such as post hematopoietic stem cell or solid - organ transplantation , respectively , ebv reactivates and promotes the unrestricted proliferation of infected cells , leading to the development of a group of diseases collectively referred as ptlds.12 as these tumors express several of the same highly immunogenic antigens commonly present in ebv - infected b cells , the restoration of ctls targeting these antigens was anticipated as a potential cure . although effective , this strategy was hampered by the high risk of graft - versus - host disease.13 a few years later , rooney et al refined the approach by specifically expanding t - cells targeting only ebv - associated antigens ( ebv - specific ctls ) , and showed that the infusion of ebv - specific ctls in patients with ptld was not only effective but also free from side effects.14 this pioneering work provided the rationale for extending the use of ebv - specific ctls to many more ebv - associated malignancies . t - cell precursors targeting these weak antigens are usually very limited in the peripheral blood of individuals and patients , requiring the development of more complex protocols for ctl production . within the many reported taas , cancer testis antigens ( ctas ) have attracted significant interest in the community , as they are consistently expressed by multiple tumors but are absent in healthy organs , with the exception of germ - line tissues.18 although sometimes demanding due to the low frequency of taa - ctl precursors in the peripheral blood , numerous strategies have been proposed to expand sufficient numbers of taa - ctls ex vivo . apcs infected with adenoviruses coding the protein of interest or loaded with specific human leukocyte antigen ( hla)-restricted taa peptide or pepmixes have been used to generate cta- or taa - specific ctls ex vivo.19 most of the early clinical trials have been performed in patients with melanomas or leukemias and used cd8 t - cells targeting a single antigen ( most frequently mart-1,20 melanoma - associated antigen peptide tyrosinase , or gp10021 for melanoma and wt-122 for leukemia ) expressed in the context of specific major histocompatibility complex ( mhc ) class i molecules , generally hla - a2 and hla - b44 , and results are rather promising . the safety , feasibility , and efficacy of this approach are currently being tested in an ongoing clinical trial enrolling patients with either hodgkin s lymphoma or nhl.23 as the physiological killing of t - cells occurs through the engagement of the tcr , the surface heterodimer between - and -chains that mediates recognition of short epitopes presented in the context of the mhc , the transfer of - and -tcr chains into t - cells is currently being explored as a means to reliably produce highly functionally avid t - cells in a very short period of time . gene - modified t - cells with tcrs cloned from single t - cells screened for high avidity have been used in clinical trials , and shown to be safe and feasible for the treatment of melanoma.24 although encouraging responses also have been reported in sarcomas , myeloma , and renal cancers through the targeting of ny - eso-1 , mage - a3 , p53 , and carcinoembryonic antigens , the major obstacle to a broad application of this strategy relates to the restricted number of studied hla molecules ( limited to the most common haplotypes ) , and therefore to the few known epitopes . second , the isolation of t - cell clones with high functional avidity from which cloning the -tcr chains invariably fails when the antigen expressed by the tumor cells is also present on healthy cells , as thymic selection eliminates these autoreactive clones . although heart tissues do not express mage - a3 , severe myocardial damage and robust proliferation of the transferred t - cells occurred due to the cross - recognition ( unexpectedly generated during the ex vivo affinity - enhancement process ) by the transgenic tcr of titin , a peptide derived from a striated muscle - specific protein.25,26 in other cases , affinity maturation can result in an increased tcr - threshold level of recognition , which can be dangerous when antigens , such as survivin , are expressed with different threshold between tumors and healthy cells . a technical breakthrough was made in 1993 , when the antigen - specificity of mabs targeting a surface molecule essential or preserved in tumor cells was grafted on t - cells to confer lytic activity and prolonged persistence.27 these so - called car molecules provide t - cells with the capacity to recognize and kill malignant t - cells in an mhc - independent fashion and to recognize epitopes derived from carbohydrates and glycolipids , thus broadening the array of potential targets . self - amplification and development of responses mediated by car t - cells are finally expected to be sustained in magnitude and prolonged in time , with the possibility to generate memory and thus long - term protection.25 as relapsed nhls often exhibit low hla expression , the setting of relapsed nhl disease has highly fueled the application of this approach . this work has resulted in the incorporation of costimulatory molecules within the car for appropriate activation of these cells or in grafting the car on antigen - specific t - cells or central memory cells to guarantee long - term persistence . thus far , results on 26 patients with nhl receiving car - cd19 or car - cd20 t - cell therapy in the presence of active disease have been reported in the literature . importantly , the only crs observed so far in nhl have been in a pilot trial with a car that incorporated both the cd28 and 41bb endodomains or in patients heavily pretreated with b - cell - specific chemotherapy regimens prior to car - t - cells.28 further modifications are currently being investigated to achieve optimal expansion and clinical activity of car t - cells . therefore , the results of the many ongoing studies ( table 2 ) , although using cars generated under different culture conditions and different constructs , will be important to understand the efficacy and the impact of this approach for the treatment of nhl . the expression of the cd20 and cd19 target antigens by healthy b cells is responsible for the occurrence of b - cell aplasia.29 while the administration of immunoglobulin can control this side effect in young patients , in older individuals , the prolonged b - cell aplasia when combined with the t - cell dysfunction following heavy chemotherapy regimens may be a fatal combination by predisposing them to life - threatening opportunistic infections . furthermore , t - cell activation following antigen engagement is often accompanied by the upregulation of pd1.39 the interaction between pd1 and pdl1 on many tumor cells can deliver a strong inhibitory signal that neutralizes the antitumor activity of residential lymphocytes , as well as of the adoptively transferred t - cells . high hopes are held for the adoptive transfer of antigen - specific t - cells to complement the standard therapies for patients affected by nhl . although data are promising , t - cells infused in nhl have shown limited persistence and expansion , explaining , at least in part , the more limited antitumor activity observed in nhl compared to patients affected by other diseases but treated with comparable t - cells / ctls in clinical trials . a more in - depth understanding of the biology of t - cell immune responses in nhl patients will certainly shed light into this diversity in clinical responses . several strategies have been developed to increase the effectiveness of adoptively transferred t - cells . on one side , investigators are exploring ex vivo strategies to increase the stemness of infused t - cells to improve persistence and proliferation . this can be achieved by reducing the length of the ex vivo culture and/or by manipulating the culture conditions using , eg , specific cytokine cocktails,40 or by selecting specific t - cell subpopulations41 or antigen - specific t - cells.42 in parallel , new costimulatory molecules or combinations of costimulatory pathways continue to be explored to facilitate prolonged car - t - cell persistence . although confounding for the interpretation of the relevance played by each component in the final clinical outcome , the systematic combination of adoptively transferred t - cells with standard therapies or with the new immunomodulatory drugs available in the clinical arena ( like checkpoint inhibitors , anti - ctla4 , or anti - pd1 abs ) may provide t - cells with the right environment to fulfill their potential and result in better control of the disease .

unless otherwise noted , all reagents were purchased from sigma aldrich ( st . louis , mo , usa ) and used without further purification . boc - gly , merrifield resin and boc - lys - ome were purchased from bachem ( torrance , ca , usa ) . boc - ala - oh , boc - lys - oh , boc - phe - oh and boc - val - oh were purchased from novabiochem ( merck kgaa , darmstadt , germany ) . 3-(4-phenylacetic acid)-1,2,4,5-tetrazine ( tz - cooh ) and ( e)-cyclooct-4-enyl 2,5-dioxopyrrolidin-1-yl carbonate ( tco - nhs ) were synthesized as described before . , hplc analyses were performed using a shimadzu hplc equipped with 2 lc-10at pumps , a spd - m10avp photodiode array detector , a flow count pin diode radiodetector from bioscan and a waters atlantis t3 column ( 6250 mm , 5 m ) . a gradient of 95:50:100 h2o / ch3cn supplemented with 0.1 % tfa over 18 min at 1 ml min was used . for preparative hplcs , a phenomenex jupiter ( 250100 mm , 5 m ) , and flowrates of 5 ml min were used . radioactivity measurements were performed with a capintec crc1243 dose calibrator ( capintec , ramsay , nj , usa ) . for tlc measurements with radioactive materials , a bioscan ar2000 ( bioscan , inc . , washington , dc , usa ) was used . microwave irradiations were carried out using a biotage initiator microwave synthesizer ( biotage , llc , charlotte , nc , usa ) . lrms were recorded with a waters acquity uplc with electrospray ionization sq detector ( esi ) . hrms were recorded with a waters lct premier system ( esi ) . h nmr spectra were recorded on a bruker aviii ( 500 mhz ) spectrometer . chemical shifts for protons are reported in parts per million ( ppm ) and are referenced against the residual proton resonance of deuterated solvents ( cdcl3 : h , 7.26 ppm ; [ d4]meoh : h , 3.31 ppm ; [ d4]dmso : h , 2.50 ppm ) . nmr data are reported as follows : chemical shift , multiplicity ( s = singlet , d = doublet , t = triplet , m = multiplet ) , coupling constants ( hz ) and integration . 3-(4-phenylacetic acid)-1,2,4,5-tetrazine succinimidyl ester ( tz - nhs ) : n - hydroxysuccinimide ( 163 mg , 1.42 mmol ) and et3n ( 495 l , 3.55 mmol ) were added to a mixture tz - cooh ( 150 mg , 0.71 mmol ) and n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride ( edci , 544 mg , 2.84 mmol ) in ch2cl2 ( 15 ml ) , and the reaction mixture was stirred for 6 h at rt . the mixture was extracted with acetic acid ( 1 m , 210 ml ) and h2o ( 210 ml ) , dried ( mgso4 ) , and volatiles were removed in vacuo . the crude product was purified by column chromatography ( ch2cl2/meoh , 97.5:2.5 ) , yielding tz - nhs as a pink solid ( 67.3 mg , 0.22 mmol , 31 % ): h nmr ( 500 mhz , [ d4]meoh ) : =10.60 ( s , 1 h ) , 8.51 ( d , j=8.3 , 2 h ) , 7.55 ( d , j=8.3 , 2 h ) , 7.20 ( d , j=7.7 , 1 h ) , 4.32 ( s , 2 h ) , 2.36 ( s , 4 h ) ; lc ms ( esi ) : m / z ( % ) : 313.1 ( 100 ) [ mh ] , 312.1 ( 100 ) [ 2 mh ] . n-(tert - butoxycarbonyl)-n-(tetrazine)-l - lysine ( boc--oh , 1 ) : tz - nhs ( 55.0 mg , 0.18 mmol ) was dissolved in a mixture of boc - l - lys - oh ( 53.2 mg , 0.36 mmol ) and et3n ( 63 l , 0.45 mmol ) in meoh ( 6 ml ) and stirred for 2 h. the reaction mixture was dried in vacuo , and the resulting crude product purified by column chromatography ( 525 % meoh in ch2cl2 ) , yielding 1 as a pink film ( 40.3 mg , 0.09 mmol , 50 % ): h nmr ( 500 mhz , [ d4]meoh ) : =10.31 ( s , 1 h ) , 8.55 ( d , j=8.4 hz , 2 h ) , 7.56 ( d , j=8.4 hz , 2 h ) , 4.05 ( dd , j=8.7 , 4.7 hz , 1 h ) , 3.22 ( t , j=6.9 hz , 2 h ) , 1.861.61 ( m , 2 h ) , 1.581.38 ( m , 13 h ) ; lc ms ( esi+ ) : m / z ( % ) : 467.3 ( 30 ) [ m+na ] , 911.6 ( 10 ) [ 2 m+na ] ; lc ms(esi ) : m / z ( % ) : 443.3 ( 10 ) [ mh ] , 887.6 ( 20 ) [ 2 mh ] ; hrms ( esi ) m / z [ m+na]calcd for c21h28n6o5na : 467.2005 , found : 467.2019 . methyl n-(tert - butoxycarbonyl)-n-(tetrazine)-l - lysinate ( boc--ome , 2 ) : n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride ( 51.8 mg , 0.27 mol ) was added to a mixture of 3-(4-phenylacetic acid)-1,2,4,5-tetrazine succinimidyl ester ( 20 mg , 0.09 mmol ) , boc - l - lys - oh ( 28.6 mg , 0.11 mmol ) , et3n ( 63 l , 0.45 mmol ) in ch2cl2 ( 5 ml ) , and the reaction mixture was stirred over night at rt , before it was dried in vacuo . the resulting crude product was purified by column chromatography ( 5 % meoh in dcm ) , yielding 2 as a pink film ( 20.8 mg , 0.05 mmol , 56 % ): h nmr ( 500 mhz , [ d6]dmso ) : =10.57 ( s , 1 h ) , 8.44 ( d , j=8.2 , 2 h ) , 8.13 ( t , j=5.5 hz , 1 h ) , 7.55 ( d , j=8.2 hz , 2 h ) , 7.20 ( d , j=7.7 hz , 1 h ) , 3.933.88 ( m , 1 h ) , 3.61 ( s , 3 h ) , 3.55 ( s , 2 h ) , 3.05 ( q , j=6.5 hz , 2 h ) , 1.651.52 ( m , 2 h ) , 1.431.27 ( m , 13 h ) ; lc ms ( esi+ ) m / z ( % ) : 359.3 ( 75 ) [ mboc+h ] , 459.3 ( 40 ) [ m+h ] , 917.7 ( 30 ) [ 2 m+h ] ; hrms ( esi ) : m / z [ m+h ] calcd for c22h30n6o5na : 481.2171 , found : 481.2175 . n-(tetrazine)-l - lysine ( h--oh , 3 ) : boc--oh 1 ( 10 mg , 22.5 mol ) was dissolved in a mixture of tfa / ch2cl2 ( 1:1 ) and vigorously stirred for 1.5 h. volatiles were removed in vacuo , and the resulting crude material purified using hplc , yielding 3 as a pink solid ( 5.6 mg , 16.3 mol , 72 % ): h nmr ( 500 mhz , [ d4]meoh ) : =10.32 ( s , 1 h ) , 8.55 ( d , j=8.3 , 2 h ) , 7.57 ( d , j=8.3 , 2 h ) , 3.92 ( t , j=6.3 , 2 h ) , 3.65 ( s , 2 h ) , 3.24 ( dt , j=6.3 , 3.2 hz , 2 h ) , 2.001.84 ( m , 2 h ) , 1.621.42 ( m , 2 h ) ; lc ms ( esi+ ) : m / z ( % ) : 345.2 ( 100 ) [ m+h ] , 367.2 ( 15 ) [ m+na ] ; hrms ( esi ) : m / z [ m+h ] calcd for c16h21n6o3 : 345.1668 , found : 345.1675 . pentapeptide gafv ( 4 ) : the synthesis was carried out on a 0.010.02 mmol scale in a fritted syringe ( 3 ml volume ) , using a merrifield resin , which was preloaded with boc - gly - oh ( 20 mg , 0.51.0 mmol g ) . the resin was swollen by washing with dmf ( 22 ml ) and ch2cl2 ( 22 ml ) . n-boc groups were removed by addition of a solution of tfa / ch2cl2 ( 1:1 ; 15 min , 2 ml and 120 min , 2 ml ) , followed by washing with ch2cl2 ( 22 ml ) and dmf ( 22 ml ) . amino acids were coupled ( 1 h , rt ) without prior neutralization of the resin using a freshly prepared mixture of bop ( 0.04 mmol , 18 mg ) , hnigs base ( n , n-diisopropylethylamine , diea ; 0.12 mmol , 16 mg ) and amino acid ( 0.08 mmol ) in dmf ( 2.5 ml ) . after coupling , the resin was washed with dmf ( 32 ml ) and ch2cl2 ( 32 ml ) . after completion of the deprotection / coupling sequence , the peptide was transferred to a microwave vessel , suspended in a solution of tfa / h2o ( 97.5:2.5 , 500 l ) , and microwaved ( 25 min , 50 hz ) . the resin was removed by filtration and et2o ( 10 ml ) added to the supernatant . the resulting crude precipitate was filtered and purified using hplc to yield 4 as a pink solid ( 0.9 mg , 1.3 mol , 7 % yield ) : lc ms ( esi+ ) : m / z ( % ) : 719.5 ( 20 ) [ m+h ] ; lc ms ( esi ) : m / z ( % ) : 717.5 ( 10 ) [ mh ] ; hrms ( esi ) : m / z calcd for [ m+h ] c35h47n10o7 : 719.3633 , found : 719.3629 . trans - cyclooctene desferrioxamine conjugate ( tco dfo ) : tco - nhs ( 12 mg , 45 mol ) was added to a solution deferrioxamine mesylate salt ( 20 mg , 30 mol ) and et3n ( 21 l , 0.15 mmol ) in ch3cn / h2o ( 1:1 , 1 ml ) and stirred at rt for 90 min . subsequently , volatiles were removed in vacuo , and the crude product was purified using hplc , yielding tco dfo as a white solid ( 6.6 mg , 9 mol , 30 % ): h nmr ( 500 mhz , [ d6]dmso ) : =9.689.54 ( m , 2 h ) , 7.787.74 ( m , 1 h ) , 6.936.88 ( m , 1 h ) , 5.605.40 ( m , 2 h ) , 4.214.17 ( m , 1 h ) , 3.483.41 ( m , 6 h ) , 3.032.87 ( m , 6 h ) , 2.612.53 ( m , 4 h ) , 2.292.21 ( m , 7 h ) , 1.96 ( s , 3 h ) , 1.911.85 ( m , 4 h ) , 1.561.15 ( m , 22 h ) ; lc ms ( esi+ ) : m / z ( % ) : 357.4 ( 10 ) [ m+2 h ] , 713.6 ( 100 ) [ m+h ] , 735.5 ( 70 ) [ m+na ] , 759.4 ( 10 ) [ m+hcoo ] ; hrms ( esi ) : m / z calcd for [ m+h ] c34h61n6o10 : 713.4474 , found : 713.4449 . zr - trans - cyclooctene ( zr - tco , 5 ) : [ zr]zr - oxalate ( 59.274 mbq , 1.6002.000 ci ) in oxalic acid ( 1.0 m , 150 l ) was adjusted to ph 7.28.0 with na2co3 ( 1.0 m , 150 l ) . after the evolution of co2(g ) stops , the zr was added to a solution of tco dfo ( 1 mm , 100 l ) in pbs / dmso ( 9:1 ) . the reaction solution was stirred at 37 c for 30 min , before the reaction progress was assayed using instant thin - layer chromatography ( itlc ) with an eluent of 1 m citric acid . in the itlc experiments , zr - tco remains at the baseline , while free zr ions and other [ zr]-citrate complexes elute with high rf values . the crude reaction mixture was purified using hplc , and volatiles were removed in vacuo , yielding 5 in > 98 % radiochemical purity , 39 % uncorrected isolated radiochemical yield ( rcy ) , and a specific activity of > 5.59 mci mol ( n=3 ) . zr - gafv ( 6 ) : gafv ( 4 , 20 nmol , 20 l , 1 mm in dmso ) was added to zr - tco ( 56 ci , 10 l , 5.56 mci ml in 1:1 ch3cn / h2o with 0.05 % tfa ) , and the mixture was agitated at rt for 20 min , before purification using hplc , yielding 6 in > 95 % purity , 65 % uncorrected isolated rcy , and a specific activity of > 3.00 mci mol ( n=3 ) . boc - deprotection conditions : tfa ( 760 l ) was added to a solution of 3 ( 5 mm , 20 l , dmso ) and coumarin ( 5 mm , 20 l , dmso ) and the resulting mixture stirred at room temperature . a control sample consisted of dmso ( 760 l ) , which was added to a solution of 3 ( 5 mm , 20 l , dmso ) and coumarin ( 5 mm , 20 l , dmso ) . the amount of 3 relative to coumarin was measured at t=0 , 0.5 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 and 9 h using hplc analysis , and the results were compared to the control sample . coupling conditions : solutions of ( benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate ( bop , 10 mm , 25 l , dmf ) and diea ( 100 mm , 25 l , dmf ) were added to a solution of 2 ( 5 mm , 25 l , dmf ) and coumarin ( 5 mm , 25 l , dmf ) . a control sample consisted of dmf ( 50 l ) , which was added to a solution of 2 ( 5 mm , 25 l , dmf ) and coumarin ( 5 mm , 25 l , dmf ) . the amount of 3 relative to coumarin was measured at t=0 , 0.5 , 1 h , 2 h , 3 h , 4 h , 5 h , 6 h , 7 h , 8 h and 9 h using hplc analysis , and the results were compared to the control sample . resin - cleavage conditions : tfa ( 950 l ) and deionized h2o ( 25 l ) were added to a solution of 3 ( 5 mm , 20 l , dmso ) and coumarin ( 5 mm , 5 l , dmso ) , and the resulting mixture was exposed to sequential microwave irradiation intervals ( 0112.5 min , 50 w ) . a control sample consisted of dmso ( 975 l ) , which was added to a solution of 3 ( 5 mm , 20 l , dmso ) and coumarin ( 5 mm , 5 l , dmso ) . at the end of each microwave irradiation interval , a sample was drawn , the amount of 3 relative to coumarin was measured using hplc analysis , and the results were compared to the control sample . 3-(4-phenylacetic acid)-1,2,4,5-tetrazine succinimidyl ester ( tz - nhs ) : n - hydroxysuccinimide ( 163 mg , 1.42 mmol ) and et3n ( 495 l , 3.55 mmol ) were added to a mixture tz - cooh ( 150 mg , 0.71 mmol ) and n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride ( edci , 544 mg , 2.84 mmol ) in ch2cl2 ( 15 ml ) , and the reaction mixture was stirred for 6 h at rt . the mixture was extracted with acetic acid ( 1 m , 210 ml ) and h2o ( 210 ml ) , dried ( mgso4 ) , and volatiles were removed in vacuo . the crude product was purified by column chromatography ( ch2cl2/meoh , 97.5:2.5 ) , yielding tz - nhs as a pink solid ( 67.3 mg , 0.22 mmol , 31 % ): h nmr ( 500 mhz , [ d4]meoh ) : =10.60 ( s , 1 h ) , 8.51 ( d , j=8.3 , 2 h ) , 7.55 ( d , j=8.3 , 2 h ) , 7.20 ( d , j=7.7 , 1 h ) , 4.32 ( s , 2 h ) , 2.36 ( s , 4 h ) ; lc ms ( esi ) : m / z ( % ) : 313.1 ( 100 ) [ mh ] , 312.1 ( 100 ) [ 2 mh ] . n-(tert - butoxycarbonyl)-n-(tetrazine)-l - lysine ( boc--oh , 1 ) : tz - nhs ( 55.0 mg , 0.18 mmol ) was dissolved in a mixture of boc - l - lys - oh ( 53.2 mg , 0.36 mmol ) and et3n ( 63 l , 0.45 mmol ) in meoh ( 6 ml ) and stirred for 2 h. the reaction mixture was dried in vacuo , and the resulting crude product purified by column chromatography ( 525 % meoh in ch2cl2 ) , yielding 1 as a pink film ( 40.3 mg , 0.09 mmol , 50 % ): h nmr ( 500 mhz , [ d4]meoh ) : =10.31 ( s , 1 h ) , 8.55 ( d , j=8.4 hz , 2 h ) , 7.56 ( d , j=8.4 hz , 2 h ) , 4.05 ( dd , j=8.7 , 4.7 hz , 1 h ) , 3.22 ( t , j=6.9 hz , 2 h ) , 1.861.61 ( m , 2 h ) , 1.581.38 ( m , 13 h ) ; lc ms ( esi+ ) : m / z ( % ) : 467.3 ( 30 ) [ m+na ] , 911.6 ( 10 ) [ 2 m+na ] ; lc ms(esi ) : m / z ( % ) : 443.3 ( 10 ) [ mh ] , 887.6 ( 20 ) [ 2 mh ] ; hrms ( esi ) m / z [ m+na]calcd for c21h28n6o5na : 467.2005 , found : 467.2019 . methyl n-(tert - butoxycarbonyl)-n-(tetrazine)-l - lysinate ( boc--ome , 2 ) : n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride ( 51.8 mg , 0.27 mol ) was added to a mixture of 3-(4-phenylacetic acid)-1,2,4,5-tetrazine succinimidyl ester ( 20 mg , 0.09 mmol ) , boc - l - lys - oh ( 28.6 mg , 0.11 mmol ) , et3n ( 63 l , 0.45 mmol ) in ch2cl2 ( 5 ml ) , and the reaction mixture was stirred over night at rt , before it was dried in vacuo . the resulting crude product was purified by column chromatography ( 5 % meoh in dcm ) , yielding 2 as a pink film ( 20.8 mg , 0.05 mmol , 56 % ): h nmr ( 500 mhz , [ d6]dmso ) : =10.57 ( s , 1 h ) , 8.44 ( d , j=8.2 , 2 h ) , 8.13 ( t , j=5.5 hz , 1 h ) , 7.55 ( d , j=8.2 hz , 2 h ) , 7.20 ( d , j=7.7 hz , 1 h ) , 3.933.88 ( m , 1 h ) , 3.61 ( s , 3 h ) , 3.55 ( s , 2 h ) , 3.05 ( q , j=6.5 hz , 2 h ) , 1.651.52 ( m , 2 h ) , 1.431.27 ( m , 13 h ) ; lc ms ( esi+ ) m / z ( % ) : 359.3 ( 75 ) [ mboc+h ] , 459.3 ( 40 ) [ m+h ] , 917.7 ( 30 ) [ 2 m+h ] ; hrms ( esi ) : m / z [ m+h ] calcd for c22h30n6o5na : 481.2171 , found : 481.2175 . n-(tetrazine)-l - lysine ( h--oh , 3 ) : boc--oh 1 ( 10 mg , 22.5 mol ) was dissolved in a mixture of tfa / ch2cl2 ( 1:1 ) and vigorously stirred for 1.5 h. volatiles were removed in vacuo , and the resulting crude material purified using hplc , yielding 3 as a pink solid ( 5.6 mg , 16.3 mol , 72 % ): h nmr ( 500 mhz , [ d4]meoh ) : =10.32 ( s , 1 h ) , 8.55 ( d , j=8.3 , 2 h ) , 7.57 ( d , j=8.3 , 2 h ) , 3.92 ( t , j=6.3 , 2 h ) , 3.65 ( s , 2 h ) , 3.24 ( dt , j=6.3 , 3.2 hz , 2 h ) , 2.001.84 ( m , 2 h ) , 1.621.42 ( m , 2 h ) ; lc ms ( esi+ ) : m / z ( % ) : 345.2 ( 100 ) [ m+h ] , 367.2 ( 15 ) [ m+na ] ; hrms ( esi ) : m / z [ m+h ] calcd for c16h21n6o3 : 345.1668 , found : 345.1675 . pentapeptide gafv ( 4 ) : the synthesis was carried out on a 0.010.02 mmol scale in a fritted syringe ( 3 ml volume ) , using a merrifield resin , which was preloaded with boc - gly - oh ( 20 mg , 0.51.0 mmol g ) . the resin was swollen by washing with dmf ( 22 ml ) and ch2cl2 ( 22 ml ) . n-boc groups were removed by addition of a solution of tfa / ch2cl2 ( 1:1 ; 15 min , 2 ml and 120 min , 2 ml ) , followed by washing with ch2cl2 ( 22 ml ) and dmf ( 22 ml ) . amino acids were coupled ( 1 h , rt ) without prior neutralization of the resin using a freshly prepared mixture of bop ( 0.04 mmol , 18 mg ) , hnigs base ( n , n-diisopropylethylamine , diea ; 0.12 mmol , 16 mg ) and amino acid ( 0.08 mmol ) in dmf ( 2.5 ml ) . after coupling , the resin was washed with dmf ( 32 ml ) and ch2cl2 ( 32 ml ) . after completion of the deprotection / coupling sequence , the peptide was transferred to a microwave vessel , suspended in a solution of tfa / h2o ( 97.5:2.5 , 500 l ) , and microwaved ( 25 min , 50 hz ) . the resin was removed by filtration and et2o ( 10 ml ) added to the supernatant . the resulting crude precipitate was filtered and purified using hplc to yield 4 as a pink solid ( 0.9 mg , 1.3 mol , 7 % yield ) : lc ms ( esi+ ) : m / z ( % ) : 719.5 ( 20 ) [ m+h ] ; lc ms ( esi ) : m / z ( % ) : 717.5 ( 10 ) [ mh ] ; hrms ( esi ) : m / z calcd for [ m+h ] c35h47n10o7 : 719.3633 , found : 719.3629 . trans - cyclooctene desferrioxamine conjugate ( tco dfo ) : tco - nhs ( 12 mg , 45 mol ) was added to a solution deferrioxamine mesylate salt ( 20 mg , 30 mol ) and et3n ( 21 l , 0.15 mmol ) in ch3cn / h2o ( 1:1 , 1 ml ) and stirred at rt for 90 min . subsequently , volatiles were removed in vacuo , and the crude product was purified using hplc , yielding tco dfo as a white solid ( 6.6 mg , 9 mol , 30 % ): h nmr ( 500 mhz , [ d6]dmso ) : =9.689.54 ( m , 2 h ) , 7.787.74 ( m , 1 h ) , 6.936.88 ( m , 1 h ) , 5.605.40 ( m , 2 h ) , 4.214.17 ( m , 1 h ) , 3.483.41 ( m , 6 h ) , 3.032.87 ( m , 6 h ) , 2.612.53 ( m , 4 h ) , 2.292.21 ( m , 7 h ) , 1.96 ( s , 3 h ) , 1.911.85 ( m , 4 h ) , 1.561.15 ( m , 22 h ) ; lc ms ( esi+ ) : m / z ( % ) : 357.4 ( 10 ) [ m+2 h ] , 713.6 ( 100 ) [ m+h ] , 735.5 ( 70 ) [ m+na ] , 759.4 ( 10 ) [ m+hcoo ] ; hrms ( esi ) : m / z calcd for [ m+h ] c34h61n6o10 : 713.4474 , found : 713.4449 . zr - trans - cyclooctene ( zr - tco , 5 ) : [ zr]zr - oxalate ( 59.274 mbq , 1.6002.000 ci ) in oxalic acid ( 1.0 m , 150 l ) was adjusted to ph 7.28.0 with na2co3 ( 1.0 m , 150 l ) . after the evolution of co2(g ) stops , the zr was added to a solution of tco dfo ( 1 mm , 100 l ) in pbs / dmso ( 9:1 ) . the reaction solution was stirred at 37 c for 30 min , before the reaction progress was assayed using instant thin - layer chromatography ( itlc ) with an eluent of 1 m citric acid . in the itlc experiments , zr - tco remains at the baseline , while free zr ions and other [ zr]-citrate complexes elute with high rf values . the crude reaction mixture was purified using hplc , and volatiles were removed in vacuo , yielding 5 in > 98 % radiochemical purity , 39 % uncorrected isolated radiochemical yield ( rcy ) , and a specific activity of > 5.59 mci mol ( n=3 ) . zr - gafv ( 6 ) : gafv ( 4 , 20 nmol , 20 l , 1 mm in dmso ) was added to zr - tco ( 56 ci , 10 l , 5.56 mci ml in 1:1 ch3cn / h2o with 0.05 % tfa ) , and the mixture was agitated at rt for 20 min , before purification using hplc , yielding 6 in > 95 % purity , 65 % uncorrected isolated rcy , and a specific activity of > 3.00 mci mol ( n=3 ) . boc - deprotection conditions : tfa ( 760 l ) was added to a solution of 3 ( 5 mm , 20 l , dmso ) and coumarin ( 5 mm , 20 l , dmso ) and the resulting mixture stirred at room temperature . a control sample consisted of dmso ( 760 l ) , which was added to a solution of 3 ( 5 mm , 20 l , dmso ) and coumarin ( 5 mm , 20 l , dmso ) . the amount of 3 relative to coumarin was measured at t=0 , 0.5 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 and 9 h using hplc analysis , and the results were compared to the control sample . coupling conditions : solutions of ( benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate ( bop , 10 mm , 25 l , dmf ) and diea ( 100 mm , 25 l , dmf ) were added to a solution of 2 ( 5 mm , 25 l , dmf ) and coumarin ( 5 mm , 25 l , dmf ) . a control sample consisted of dmf ( 50 l ) , which was added to a solution of 2 ( 5 mm , 25 l , dmf ) and coumarin ( 5 mm , 25 l , dmf ) . the amount of 3 relative to coumarin was measured at t=0 , 0.5 , 1 h , 2 h , 3 h , 4 h , 5 h , 6 h , 7 h , 8 h and 9 h using hplc analysis , and the results were compared to the control sample . resin - cleavage conditions : tfa ( 950 l ) and deionized h2o ( 25 l ) were added to a solution of 3 ( 5 mm , 20 l , dmso ) and coumarin ( 5 mm , 5 l , dmso ) , and the resulting mixture was exposed to sequential microwave irradiation intervals ( 0112.5 min , 50 w ) . a control sample consisted of dmso ( 975 l ) , which was added to a solution of 3 ( 5 mm , 20 l , dmso ) and coumarin ( 5 mm , 5 l , dmso ) . at the end of each microwave irradiation interval , a sample was drawn , the amount of 3 relative to coumarin was measured using hplc analysis , and the results were compared to the control sample .

the need for post - synthetic modifications and reactive prosthetic groups has long been a limiting factor in the synthesis and study of peptidic and peptidomimetic imaging agents . in this regard , the application of biologically and chemically orthogonal reactions to the design and development of novel radiotracers has the potential to have far - reaching implications in both the laboratory and the clinic . herein , we report the synthesis and development of a series of modular and versatile building blocks for inverse electron - demand diels alder copper - free click chemistry : tetrazine - functionalized artificial amino acids . following the development of a novel peptide coupling protocol for peptide synthesis in the presence of tetrazines , we successfully demonstrated its effectiveness and applicability . this versatile methodology has the potential to have a transformational impact , opening the door for the rapid , facile , and modular synthesis of bioorthogonally reactive peptide probes .

Experimental Section Synthesis Tetrazine stability determination

3-(4-phenylacetic acid)-1,2,4,5-tetrazine succinimidyl ester ( tz - nhs ) : n - hydroxysuccinimide ( 163 mg , 1.42 mmol ) and et3n ( 495 l , 3.55 mmol ) were added to a mixture tz - cooh ( 150 mg , 0.71 mmol ) and n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride ( edci , 544 mg , 2.84 mmol ) in ch2cl2 ( 15 ml ) , and the reaction mixture was stirred for 6 h at rt . the mixture was extracted with acetic acid ( 1 m , 210 ml ) and h2o ( 210 ml ) , dried ( mgso4 ) , and volatiles were removed in vacuo . n-(tert - butoxycarbonyl)-n-(tetrazine)-l - lysine ( boc--oh , 1 ) : tz - nhs ( 55.0 mg , 0.18 mmol ) was dissolved in a mixture of boc - l - lys - oh ( 53.2 mg , 0.36 mmol ) and et3n ( 63 l , 0.45 mmol ) in meoh ( 6 ml ) and stirred for 2 h. the reaction mixture was dried in vacuo , and the resulting crude product purified by column chromatography ( 525 % meoh in ch2cl2 ) , yielding 1 as a pink film ( 40.3 mg , 0.09 mmol , 50 % ): h nmr ( 500 mhz , [ d4]meoh ) : =10.31 ( s , 1 h ) , 8.55 ( d , j=8.4 hz , 2 h ) , 7.56 ( d , j=8.4 hz , 2 h ) , 4.05 ( dd , j=8.7 , 4.7 hz , 1 h ) , 3.22 ( t , j=6.9 hz , 2 h ) , 1.861.61 ( m , 2 h ) , 1.581.38 ( m , 13 h ) ; lc ms ( esi+ ) : m / z ( % ) : 467.3 ( 30 ) [ m+na ] , 911.6 ( 10 ) [ 2 m+na ] ; lc ms(esi ) : m / z ( % ) : 443.3 ( 10 ) [ mh ] , 887.6 ( 20 ) [ 2 mh ] ; hrms ( esi ) m / z [ m+na]calcd for c21h28n6o5na : 467.2005 , found : 467.2019 . methyl n-(tert - butoxycarbonyl)-n-(tetrazine)-l - lysinate ( boc--ome , 2 ) : n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride ( 51.8 mg , 0.27 mol ) was added to a mixture of 3-(4-phenylacetic acid)-1,2,4,5-tetrazine succinimidyl ester ( 20 mg , 0.09 mmol ) , boc - l - lys - oh ( 28.6 mg , 0.11 mmol ) , et3n ( 63 l , 0.45 mmol ) in ch2cl2 ( 5 ml ) , and the reaction mixture was stirred over night at rt , before it was dried in vacuo . n-(tetrazine)-l - lysine ( h--oh , 3 ) : boc--oh 1 ( 10 mg , 22.5 mol ) was dissolved in a mixture of tfa / ch2cl2 ( 1:1 ) and vigorously stirred for 1.5 h. volatiles were removed in vacuo , and the resulting crude material purified using hplc , yielding 3 as a pink solid ( 5.6 mg , 16.3 mol , 72 % ): h nmr ( 500 mhz , [ d4]meoh ) : =10.32 ( s , 1 h ) , 8.55 ( d , j=8.3 , 2 h ) , 7.57 ( d , j=8.3 , 2 h ) , 3.92 ( t , j=6.3 , 2 h ) , 3.65 ( s , 2 h ) , 3.24 ( dt , j=6.3 , 3.2 hz , 2 h ) , 2.001.84 ( m , 2 h ) , 1.621.42 ( m , 2 h ) ; lc ms ( esi+ ) : m / z ( % ) : 345.2 ( 100 ) [ m+h ] , 367.2 ( 15 ) [ m+na ] ; hrms ( esi ) : m / z [ m+h ] calcd for c16h21n6o3 : 345.1668 , found : 345.1675 . after coupling , the resin was washed with dmf ( 32 ml ) and ch2cl2 ( 32 ml ) . after completion of the deprotection / coupling sequence , the peptide was transferred to a microwave vessel , suspended in a solution of tfa / h2o ( 97.5:2.5 , 500 l ) , and microwaved ( 25 min , 50 hz ) . subsequently , volatiles were removed in vacuo , and the crude product was purified using hplc , yielding tco dfo as a white solid ( 6.6 mg , 9 mol , 30 % ): h nmr ( 500 mhz , [ d6]dmso ) : =9.689.54 ( m , 2 h ) , 7.787.74 ( m , 1 h ) , 6.936.88 ( m , 1 h ) , 5.605.40 ( m , 2 h ) , 4.214.17 ( m , 1 h ) , 3.483.41 ( m , 6 h ) , 3.032.87 ( m , 6 h ) , 2.612.53 ( m , 4 h ) , 2.292.21 ( m , 7 h ) , 1.96 ( s , 3 h ) , 1.911.85 ( m , 4 h ) , 1.561.15 ( m , 22 h ) ; lc ms ( esi+ ) : m / z ( % ) : 357.4 ( 10 ) [ m+2 h ] , 713.6 ( 100 ) [ m+h ] , 735.5 ( 70 ) [ m+na ] , 759.4 ( 10 ) [ m+hcoo ] ; hrms ( esi ) : m / z calcd for [ m+h ] c34h61n6o10 : 713.4474 , found : 713.4449 . the crude reaction mixture was purified using hplc , and volatiles were removed in vacuo , yielding 5 in > 98 % radiochemical purity , 39 % uncorrected isolated radiochemical yield ( rcy ) , and a specific activity of > 5.59 mci mol ( n=3 ) . zr - gafv ( 6 ) : gafv ( 4 , 20 nmol , 20 l , 1 mm in dmso ) was added to zr - tco ( 56 ci , 10 l , 5.56 mci ml in 1:1 ch3cn / h2o with 0.05 % tfa ) , and the mixture was agitated at rt for 20 min , before purification using hplc , yielding 6 in > 95 % purity , 65 % uncorrected isolated rcy , and a specific activity of > 3.00 mci mol ( n=3 ) . boc - deprotection conditions : tfa ( 760 l ) was added to a solution of 3 ( 5 mm , 20 l , dmso ) and coumarin ( 5 mm , 20 l , dmso ) and the resulting mixture stirred at room temperature . the amount of 3 relative to coumarin was measured at t=0 , 0.5 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 and 9 h using hplc analysis , and the results were compared to the control sample . the amount of 3 relative to coumarin was measured at t=0 , 0.5 , 1 h , 2 h , 3 h , 4 h , 5 h , 6 h , 7 h , 8 h and 9 h using hplc analysis , and the results were compared to the control sample . resin - cleavage conditions : tfa ( 950 l ) and deionized h2o ( 25 l ) were added to a solution of 3 ( 5 mm , 20 l , dmso ) and coumarin ( 5 mm , 5 l , dmso ) , and the resulting mixture was exposed to sequential microwave irradiation intervals ( 0112.5 min , 50 w ) . at the end of each microwave irradiation interval , a sample was drawn , the amount of 3 relative to coumarin was measured using hplc analysis , and the results were compared to the control sample . 3-(4-phenylacetic acid)-1,2,4,5-tetrazine succinimidyl ester ( tz - nhs ) : n - hydroxysuccinimide ( 163 mg , 1.42 mmol ) and et3n ( 495 l , 3.55 mmol ) were added to a mixture tz - cooh ( 150 mg , 0.71 mmol ) and n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride ( edci , 544 mg , 2.84 mmol ) in ch2cl2 ( 15 ml ) , and the reaction mixture was stirred for 6 h at rt . n-(tert - butoxycarbonyl)-n-(tetrazine)-l - lysine ( boc--oh , 1 ) : tz - nhs ( 55.0 mg , 0.18 mmol ) was dissolved in a mixture of boc - l - lys - oh ( 53.2 mg , 0.36 mmol ) and et3n ( 63 l , 0.45 mmol ) in meoh ( 6 ml ) and stirred for 2 h. the reaction mixture was dried in vacuo , and the resulting crude product purified by column chromatography ( 525 % meoh in ch2cl2 ) , yielding 1 as a pink film ( 40.3 mg , 0.09 mmol , 50 % ): h nmr ( 500 mhz , [ d4]meoh ) : =10.31 ( s , 1 h ) , 8.55 ( d , j=8.4 hz , 2 h ) , 7.56 ( d , j=8.4 hz , 2 h ) , 4.05 ( dd , j=8.7 , 4.7 hz , 1 h ) , 3.22 ( t , j=6.9 hz , 2 h ) , 1.861.61 ( m , 2 h ) , 1.581.38 ( m , 13 h ) ; lc ms ( esi+ ) : m / z ( % ) : 467.3 ( 30 ) [ m+na ] , 911.6 ( 10 ) [ 2 m+na ] ; lc ms(esi ) : m / z ( % ) : 443.3 ( 10 ) [ mh ] , 887.6 ( 20 ) [ 2 mh ] ; hrms ( esi ) m / z [ m+na]calcd for c21h28n6o5na : 467.2005 , found : 467.2019 . methyl n-(tert - butoxycarbonyl)-n-(tetrazine)-l - lysinate ( boc--ome , 2 ) : n-(3-dimethylaminopropyl)-n-ethylcarbodiimide hydrochloride ( 51.8 mg , 0.27 mol ) was added to a mixture of 3-(4-phenylacetic acid)-1,2,4,5-tetrazine succinimidyl ester ( 20 mg , 0.09 mmol ) , boc - l - lys - oh ( 28.6 mg , 0.11 mmol ) , et3n ( 63 l , 0.45 mmol ) in ch2cl2 ( 5 ml ) , and the reaction mixture was stirred over night at rt , before it was dried in vacuo . n-(tetrazine)-l - lysine ( h--oh , 3 ) : boc--oh 1 ( 10 mg , 22.5 mol ) was dissolved in a mixture of tfa / ch2cl2 ( 1:1 ) and vigorously stirred for 1.5 h. volatiles were removed in vacuo , and the resulting crude material purified using hplc , yielding 3 as a pink solid ( 5.6 mg , 16.3 mol , 72 % ): h nmr ( 500 mhz , [ d4]meoh ) : =10.32 ( s , 1 h ) , 8.55 ( d , j=8.3 , 2 h ) , 7.57 ( d , j=8.3 , 2 h ) , 3.92 ( t , j=6.3 , 2 h ) , 3.65 ( s , 2 h ) , 3.24 ( dt , j=6.3 , 3.2 hz , 2 h ) , 2.001.84 ( m , 2 h ) , 1.621.42 ( m , 2 h ) ; lc ms ( esi+ ) : m / z ( % ) : 345.2 ( 100 ) [ m+h ] , 367.2 ( 15 ) [ m+na ] ; hrms ( esi ) : m / z [ m+h ] calcd for c16h21n6o3 : 345.1668 , found : 345.1675 . pentapeptide gafv ( 4 ) : the synthesis was carried out on a 0.010.02 mmol scale in a fritted syringe ( 3 ml volume ) , using a merrifield resin , which was preloaded with boc - gly - oh ( 20 mg , 0.51.0 mmol g ) . after completion of the deprotection / coupling sequence , the peptide was transferred to a microwave vessel , suspended in a solution of tfa / h2o ( 97.5:2.5 , 500 l ) , and microwaved ( 25 min , 50 hz ) . subsequently , volatiles were removed in vacuo , and the crude product was purified using hplc , yielding tco dfo as a white solid ( 6.6 mg , 9 mol , 30 % ): h nmr ( 500 mhz , [ d6]dmso ) : =9.689.54 ( m , 2 h ) , 7.787.74 ( m , 1 h ) , 6.936.88 ( m , 1 h ) , 5.605.40 ( m , 2 h ) , 4.214.17 ( m , 1 h ) , 3.483.41 ( m , 6 h ) , 3.032.87 ( m , 6 h ) , 2.612.53 ( m , 4 h ) , 2.292.21 ( m , 7 h ) , 1.96 ( s , 3 h ) , 1.911.85 ( m , 4 h ) , 1.561.15 ( m , 22 h ) ; lc ms ( esi+ ) : m / z ( % ) : 357.4 ( 10 ) [ m+2 h ] , 713.6 ( 100 ) [ m+h ] , 735.5 ( 70 ) [ m+na ] , 759.4 ( 10 ) [ m+hcoo ] ; hrms ( esi ) : m / z calcd for [ m+h ] c34h61n6o10 : 713.4474 , found : 713.4449 . the crude reaction mixture was purified using hplc , and volatiles were removed in vacuo , yielding 5 in > 98 % radiochemical purity , 39 % uncorrected isolated radiochemical yield ( rcy ) , and a specific activity of > 5.59 mci mol ( n=3 ) . zr - gafv ( 6 ) : gafv ( 4 , 20 nmol , 20 l , 1 mm in dmso ) was added to zr - tco ( 56 ci , 10 l , 5.56 mci ml in 1:1 ch3cn / h2o with 0.05 % tfa ) , and the mixture was agitated at rt for 20 min , before purification using hplc , yielding 6 in > 95 % purity , 65 % uncorrected isolated rcy , and a specific activity of > 3.00 mci mol ( n=3 ) . the amount of 3 relative to coumarin was measured at t=0 , 0.5 , 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 and 9 h using hplc analysis , and the results were compared to the control sample . the amount of 3 relative to coumarin was measured at t=0 , 0.5 , 1 h , 2 h , 3 h , 4 h , 5 h , 6 h , 7 h , 8 h and 9 h using hplc analysis , and the results were compared to the control sample . resin - cleavage conditions : tfa ( 950 l ) and deionized h2o ( 25 l ) were added to a solution of 3 ( 5 mm , 20 l , dmso ) and coumarin ( 5 mm , 5 l , dmso ) , and the resulting mixture was exposed to sequential microwave irradiation intervals ( 0112.5 min , 50 w ) . at the end of each microwave irradiation interval , a sample was drawn , the amount of 3 relative to coumarin was measured using hplc analysis , and the results were compared to the control sample .

the interviews were completed as the first stage of a realist review of the evidence of what supports effective working between health care providers and care homes in england . realist review is a systematic , theory - driven approach for making sense of diverse evidence about complex interventions applied in different settings . to achieve this , it brings together multiple sources of evidence to develop possible explanations for the way in which particular interventions are thought to work and the way in which change occurs because of an intervention . programme theories that explain how key elements within health service provision to care homes works . to complement a scoping of the relevant policy and evidence on how health care services support care homes , the stakeholder interviews explored the necessary preconditions for improving health care for older people resident in care homes . the purpose of the interviews was to identify these theory areas and linked questions so as to frame how the evidence on health care interventions for care homes would then be interrogated . these either had responsibility for the commissioning , organization , or monitoring of nhs provision to care homes or direct experience as care recipients . to capture a range of experience that reflected regional , historical , and organizational differences , we identified a purposive sample of nhs and local authority commissioners , senior managers from care home organizations , and the care regulator for england ( the care quality commission [ cqc ] ) ( table 1 ) . they were recruited and defined as stakeholders on the basis that they were able to characterize the view of a group or organization and would enable us to capture a range of relevant views . the sample was chosen to be able to speak authoritatively about the organization of health care to care homes and to theorize about what achieved the best health - related outcomes for residents , while acknowledging competing explanations . the sample was identified through the cqc ; national care home provider representative organizations , residents , and relatives ' representative groups ; and national health and local authority commissioners for care homes in the east of england and the midlands . a small sample of care home residents was also interviewed . participants were specifically asked to provide a stakeholder view ; in other words , to use their experience and expertise as , for example , a care home manager to inform what a good service should look like rather than provide a solely personal account . to facilitate this , residents ' prompts focused on what they believed good health care to care homes should comprise . interviews asked about examples of success and failure , how continuity of care was achieved , what good working between nhs services and care homes looked like , and the mechanisms or particular service models necessary to achieve the desired outcomes . the resident interviews conducted for this study were supplemented with a secondary analysis of 34 resident interviews from an earlier study that had focused on access to nhs health care . these interviews had specifically asked about health and the health care services received and what was seen as effective . this additional sample was included because of the challenges of identifying residents who had the capacity to participate and their difficulties in extrapolating from their own experience of health care to consider what good health care provision for care homes should look like . this being the case , rather than conduct more interviews , we decided to use the previously conducted interviews from the earlier study that also had asked about residents ' health care experiences . first , there was a process of familiarization and decontextualization and segmenting of data into separate and defined categories close to participants ' categories . second , comparison was made within and between categories and the identification of preoccupations , difference , and themes . the third stage of interpretation was the identification of relationships and emergent hypotheses about how the favored approaches worked , and what was necessary to support their implementation . the protocol was reviewed and supported by the university of hertfordshire ethics committee reference ( ref hsk / ss / nhs/00040 ) . all the stakeholders stated that residents are entitled to the same health care that older people who live at home receive . the following are the 3 overlapping themes of what was perceived as central to the provision of quality health care:interventions that supported relational working between nhs practitioners and care home staffprovision of age - appropriate carecontractual and governance systems to guarantee care homes ' access to nhs services . interventions that supported relational working between nhs practitioners and care home staff provision of age - appropriate care contractual and governance systems to guarantee care homes ' access to nhs services . across the 3 themes there were shared elements ; for example , access to a gp when needed and the importance of a person - centered care approach , particularly for people with dementia . however , the 3 themes could be differentiated by the emphasis on what had to be done to provide effective health care and what supported or enabled that process . thus , a person - centered care approach or access to a gp was important for residents ' health , but they were described as an enabler rather than the driver for quality health care delivery to care homes . the different emphases on what was contextual and what was seen as essential informed how effective models of service delivery were conceptualized . nhs service delivery models that supported relational working were seen as addressing the difficulties of working across health and social care systems , agreeing what was publicly funded health care and what came under the jurisdiction of the care home , maintaining continuity of information and reconciling the different priorities of care homes and health care providers . to achieve relational working required investment of resources , dedicated staff time , and the creation of formal and informal opportunities for health care and care home staff to work together . in the examples given , this could happen organically over time or as an explicit intervention with resources allocated to facilitate the process . relational working could thus be realized by the identification of key people to work within the care home , the creation of care home specialist roles to provide ongoing support to care home staff , or the maintenance of working relationships within existing models of working with primary care services . examples focused on models of care in which health care staff visited regularly and predictably , provided teaching and support to care home staff , and were accessible for advice . one local authority commissioner identified the importance of nominated care home staff ( champions ) being allocated to work with visiting nhs staff to structure how they collaborate together and enable resolution of residents ' health problems in situations of pressure and limited resources.i think there has to be buy in on all sides , there 's got to be an understanding that certainly in this day and age care home settings are very tight on staff and budgets and sometimes it 's getting the right people from these particular homesto have that spare time to come along and get involved ... it'shaving champions , it 's making sure thateach home has their particular championon particular ( health ) topics and the they 've got ownership of that particular subject ( local authority commissioner ) i think there has to be buy in on all sides , there 's got to be an understanding that certainly in this day and age care home settings are very tight on staff and budgets and sometimes it 's getting the right people from these particular homesto have that spare time to come along and get involved ... it'shaving champions , it 's making sure thateach home has their particular championon particular ( health ) topics and the they 've got ownership of that particular subject ( local authority commissioner ) similarly a gp commissioner drawing on personal experience saw that it was possible to maintain continuity and the desired outcomes of care , such as avoiding unplanned hospital admissions , if visiting nhs staff knew that there were what she called designated care coordinators to work with . this role was seen as fostering integrated working.i think the key to successes , individuals are absolutely key , in care homes having a key professional , someone who takes responsibility for each patient ( sic ) , you know you need someone who is trained enough to coordinate that care , which is about integrated care and about , as i say , having patients ' co - coordinators , a key worker , whatever you want to call them . ( gp commissioner ) i think the key to successes , individuals are absolutely key , in care homes having a key professional , someone who takes responsibility for each patient ( sic ) , you know you need someone who is trained enough to coordinate that care , which is about integrated care and about , as i say , having patients ' co - coordinators , a key worker , whatever you want to call them . ( gp commissioner ) a less formalized method of supporting relational working was described by one care home manager ( sh9 ) as working like a team . team working involved shared training events with district nursing staff , care home staff being invited to the nhs provider meetings , and mutual confidence built on previous experience of having jointly resolved problems . she gave the example of being able to work with a liaison nurse to access residents ' notes when they were admitted to hospital . similarly , another care home manager described how residents ' needs were addressed and hospital admission rates were kept low because she knew that gps would come when asked , listen to their assessment of residents ' needs , and support care home staff to provide care . they had , however , learned how to work together over a prolonged period of time . the perceived success was predicated on the fact that there was continuity in their working relationship . although the gp was identified as the lynchpin , it was the quality of the association that was emphasized as key.but they ( gps ) are very good ; they come out and respond , we can ask the gp to phone us , we have a good relationshipinterviewer what makes it such a good service?i think theclinical knowledge of the nursesand the fact thatwe are in a very rural community and we have had families of gps as patientsand they chose us because of our sound clinical knowledge . we can phone and say abc , we have ruled out a uti , we have listened to his chest and the blood sugar is normal , can you come and have a look ? ( care home manager ) but they ( gps ) are very good ; they come out and respond , we can ask the gp to phone us , we have a good relationship interviewer what makes it such a good service ? i think theclinical knowledge of the nursesand the fact thatwe are in a very rural community and we have had families of gps as patientsand they chose us because of our sound clinical knowledge . we can phone and say abc , we have ruled out a uti , we have listened to his chest and the blood sugar is normal , can you come and have a look ? other stakeholders saw good working relationships as important but secondary to ensuring that residents received age - appropriate health care . this was expressed in terms of providing health care services equipped to address the health care needs of residents who were frail . the ability of a person or a service to integrate health and social care , or promote relational working , was not seen as sufficient to achieve the desired outcomes of improved resident health and reduced use of secondary services . for these stakeholders , it was the clinical expertise of the visiting health care professionals and equity of access that was important . there was a need to redress what were known to be serious gaps and failures in how services were currently organized for care homes . a model of health care provision was needed that was sufficiently specialist to assess and address a frail older person 's health care needs . it was also an issue of access and equity.people are very worried about paying extra for physio , for chiropody , for the kind of services that are intrinsic to people 's conditions . people are very worried about poststroke patients not having the kind of rehab they 'd get if they were at home or staying longer in hospital you know you really need people like geriatricians who are specialist in the care of older people the people who specialize in old age psychiatry also need to have a key role ( stakeholder from resident representative organization ) people are very worried about paying extra for physio , for chiropody , for the kind of services that are intrinsic to people 's conditions . people are very worried about poststroke patients not having the kind of rehab they 'd get if they were at home or staying longer in hospital you know you really need people like geriatricians who are specialist in the care of older people the people who specialize in old age psychiatry also need to have a key role ( stakeholder from resident representative organization ) the 3 residents interviewed struggled to extrapolate from their personal experience of nhs service delivery to how it should be provided generally to care homes . nevertheless , their interviews and the secondary analysis of how residents had talked in general about their experiences of health care reiterated the theme about expertise , how seeing the practitioner who understood their health care needs meant that problems were resolved . for example , this resident wanted to see a physiotherapist to help her regain some independence : i ca n't stand , but i have said to the manager that if i had proper physio i think they could get me to stand , but nothing has happened about that . before , i used to get from this chair to my wheelchair and move myself along to the loo and back again . then when i got this shingles it seemed to take the stuffing out of me . ( resident ) i ca n't stand , but i have said to the manager that if i had proper physio i think they could get me to stand , but nothing has happened about that . before , i used to get from this chair to my wheelchair and move myself along to the loo and back again . then when i got this shingles it seemed to take the stuffing out of me . ( resident ) the secondary data analysis of residents ' interviews presented a picture of residents in the center of a flow of visiting health care professionals who may or may not be able to address their particular health care needs . this was linked both to limited access to the clinicians whom they wanted and limited control over how or when they could be referred to services . this resident saw that her needs were secondary to the needs of other possibly more ill residents . she described a process that could be quite protracted , whereby progressively more senior staff decided if a health care professional should be called to look at her leg wound.well that i do n't know . i just feel i 'm on a sort of , waiting , i 'm not as ill as a lot of people so i think i 'm just left to tick over .... this morning , i was seeing the senior nurse who comes with the others ( care staff ) , and tell her and she 's had a look and she 's going to be in touch , get in touch with somebody else who is higher up still , who is going to look at it this afternoon . i just feel i 'm on a sort of , waiting , i 'm not as ill as a lot of people so i think i 'm just left to tick over .... this morning , i was seeing the senior nurse who comes with the others ( care staff ) , and tell her and she 's had a look and she 's going to be in touch , get in touch with somebody else who is higher up still , who is going to look at it this afternoon . it was an assumption of the residents interviewed that information would be shared but they did not know for sure , or the process by which it was done , and as this quote shows , they did not always feel able to participate in the decision making . one representative of care home providers suggested that the reason the provision of age - appropriate care did not occur was because of agism and stigma . residents ' needs were not recognized or prioritized because older people and care homes were not valued by society . she compared provision to children 's residential care homes and suggested that there was inequity across the age groups and that commissioners and providers had considerable discretion in the number and type of services offered . she advocated medical consultant led services both for their expertise and status within the medical hierarchy and gave an example of where it had been effective : it was a multidisciplinary team . headed by a consultant geriatrician and of course he had the status to be able to pull other people into his work , whereas i think if you do n't have that status in medicine you would find that you would struggle . one of the things he did was corral the gps and force them to do things that they were n't doing before . if you have got a consultant geriatrician saying you should have visited , it 's a bit of grit in the oyster . ( stakeholder from care home representative organization ) it was a multidisciplinary team . headed by a consultant geriatrician and of course he had the status to be able to pull other people into his work , whereas i think if you do n't have that status in medicine you would find that you would struggle . one of the things he did was corral the gps and force them to do things that they were n't doing before . if you have got a consultant geriatrician saying you should have visited , it 's a bit of grit in the oyster . stakeholders who had responsibilities for the monitoring and regulation of services to and within care homes emphasized interventions that reinforced good health care practice either through the use of incentives or performance management . if the proper governance were in place , this would guarantee residents ' access to evidence - based , age - appropriate health care , as well as continuity of care . an awareness of concerns about elder abuse , the need for vigilance , and examples of suboptimal care and avoidable deaths were given as the reasons why such systems needed to be in place . outcomes , such as the prevalence of pressure sores and reduction of prescribing antipsychotics for people with dementia were emphasized as important within such frameworks . this commissioner with responsibility for commissioning health and social care services for care homes gave the example of how using existing audit processes to improve end - of - life care for care home residents could improve health - related outcomes : end - of - life care is a health care issue , but what we do say in the ( contract with the care home ) specification is that we would expect that care homes are well versed around end - of - life care , that their staff are appropriately trained , and that 's part and parcel of the audit . so our annual audit looks at how geared up a care home is in meeting the needs of patients who are at the end of life . end - of - life care is a health care issue , but what we do say in the ( contract with the care home ) specification is that we would expect that care homes are well versed around end - of - life care , that their staff are appropriately trained , and that 's part and parcel of the audit . so our annual audit looks at how geared up a care home is in meeting the needs of patients who are at the end of life . these systems were seen as setting a minimum standard for how services should be provided to care homes . this stakeholder worked for the cqc ( the regulator ) and acknowledged the importance of a good gp care home relationships but saw that this was consistently achieved only when there were explicit agreements in place and a planned approach : it 's where they 've got a proper agreed arrangement with that gp surgery around , you know , they visit at certain times of the week and they can be contacted if there are any problems ... ensuring then that people have a properly planned package of care that is really focused on their needs it 's where they 've got a proper agreed arrangement with that gp surgery around , you know , they visit at certain times of the week and they can be contacted if there are any problems ... ensuring then that people have a properly planned package of care that is really focused on their needs one gp commissioner saw financial incentives for gps as essential in recognition of the extra work that was entailed and drew on personal experience : this care home has 41 beds and those are not the equivalent of 41 patients . i have been doing a surgery once a week , colleagues have been in most days , they have multiple conditions and changing needs , i have greater responsibility ; there is no way this is the equivalent to the work of 41 patients at home . ( commissioner ) this care home has 41 beds and those are not the equivalent of 41 patients . i have been doing a surgery once a week , colleagues have been in most days , they have multiple conditions and changing needs , i have greater responsibility ; there is no way this is the equivalent to the work of 41 patients at home . first , to recompense extra work that care homes generate , and second , to provide gps with the incentive to learn how the system works and improve current practice . he gave the example of where a specialist nurse working with the gp had reduced prescribing costs , and supported more residents to die at home . this could be rolled out further to help gps work more efficiently with other services : you are lookingto pay for the process of change , once the average gp haslearnedthe system andlearnedwho does what , then they should be able to come into the system and do what they do well and not much else , and work more effectively in an integrated system . ( commissioner ) you are lookingto pay for the process of change , once the average gp haslearnedthe system andlearnedwho does what , then they should be able to come into the system and do what they do well and not much else , and work more effectively in an integrated system . the basic unit of analysis in a realist review is not the intervention but the ideas and assumptions that underpin it , also referred to as program theories . the interviews identified 3 provisional theories ( mechanisms ) of health service delivery to care homes and likely barriers and enablers ( contexts ) to improving residents ' health care in specific areas ( outcomes ) . thus , what one stakeholder hypothesized as essential to achieving improved resident outcomes , for example , a contractual framework for clinician involvement in care homes , another identified as the enabler for activities that promoted shared learning and relational working . table 2 summarizes how these were configured by the participants and the contending positions of the different stakeholders . these hypothesized context mechanism outcome configurations are the basis for the next step of interrogating the evidence and provide a framework for how the effectiveness of different models of service delivery to care homes is understood . the english model of health care provision to care homes with visiting physicians is common across the world . these data show that there was a broad consensus about what were the elements or characteristics of quality health care provision to care homes by the nhs . these were as follows : consistent and predictable access to expertise in geriatric medicine , investment in resources , and good working relationships and structured approaches to care that could be monitored . we found , however , that different individuals accorded different weights to these elements and had different views about what embeds good practice , what was essential , and what was desirable . for example , there was less agreement about whether specific professionals needed to be closely monitored and performance - managed or if investment in the training of care home staff should always be a joint enterprise with visiting care home professionals . it also was unclear if the use of financial incentives and sanctions , or the creation of sustained trusting working relationships , were the drivers ( or mechanisms ) for effective working or whether these represented enablers ( or contextual factors ) supporting residents ' access to health care . for some participants , a single element , for example access to expertise in geriatric medicine , was sufficient in and of itself to improve quality of care ; however , this was not the predominant view . where this was seen as one of several core elements , it was accorded differing importance , or weighting , by different respondents . we were not able to explore why assumptions were held to be more or less true by different respondents as part of this study . what combination of elements and the dose of each are required to achieve optimal outcomes for care home resident(s ) has implications for how services are designed , prioritized , and resourced and who is identified within the system as responsible for service delivery . it was what stakeholders believed should work and reflected a range of theories about how to implement evidence - based practice and manage risk in primary health care , about frailty , approaches to comprehensive geriatric assessment , and what supports integrated working between health and social care . the findings have captured the range of perspectives and interested parties : commissioners , providers , and recipients of care . although only 3 older people living in care homes participated in the study , the study was fortunate to have access to 34 previously conducted interviews that enabled the study to obtain a resident - focused perspective on what good care looked like . the findings from this study reinforce bowman and meyer 's observation that the organization of care for older people resident in care homes represents an emerging medical space . the 3 models , and how they are exemplified as working , given what is known about the heterogeneity of care home markets and their residents and the range of context - sensitive variables that shape how services are provided , these findings mean that it is unlikely that a particular service specification for health service delivery can promote effective working for all care homes . rather , there will be key features or explanatory mechanisms , already manifest within multiple models and potentially applicable across multiple models , that will influence the delivery of optimal care . the next step is to test and refine these theories by a review of the relevant evidence to identify the necessary conditions under which the provision of health care services to care homes optimizes residents ' health .

objectivesto explore what commissioners of care , regulators , providers , and care home residents in england identify as the key mechanisms or components of different service delivery models that support the provision of national health service ( nhs ) provision to independent care homes.methodsqualitative , semistructured interviews with a purposive sample of people with direct experience of commissioning , providing , and regulating health care provision in care homes and care home residents . data from interviews were augmented by a secondary analysis of previous interviews with care home residents on their personal experience of and priorities for access to health care . analysis was framed by the assumptions of realist evaluation and drew on the constant comparative method to identify key themes about what is required to achieve quality health care provision to care homes and resident health.resultsparticipants identified 3 overlapping approaches to the provision of nhs that they believed supported access to health care for older people in care homes : ( 1 ) investment in relational working that fostered continuity and shared learning between visiting nhs staff and care home staff , ( 2 ) the provision of age - appropriate clinical services , and ( 3 ) governance arrangements that used contractual and financial incentives to specify a minimum service that care homes should receive.conclusionthe 3 approaches , and how they were typified as working , provide a rich picture of the stakeholder perspectives and the underlying assumptions about how service delivery models should work with care homes . the findings inform how evidence on effective working in care homes will be interrogated to identify how different approaches , or specifically key elements of those approaches , achieve different health - related outcomes in different situations for residents and associated health and social care organizations .

Methods Results Relational Working Provision of Age-Appropriate Care Governance and Incentives Context Mechanism Outcomes: What Works When In What Setting With What Outcomes? Discussion Conclusion

the interviews were completed as the first stage of a realist review of the evidence of what supports effective working between health care providers and care homes in england . to achieve this , it brings together multiple sources of evidence to develop possible explanations for the way in which particular interventions are thought to work and the way in which change occurs because of an intervention . programme theories that explain how key elements within health service provision to care homes works . to complement a scoping of the relevant policy and evidence on how health care services support care homes , the stakeholder interviews explored the necessary preconditions for improving health care for older people resident in care homes . the purpose of the interviews was to identify these theory areas and linked questions so as to frame how the evidence on health care interventions for care homes would then be interrogated . these either had responsibility for the commissioning , organization , or monitoring of nhs provision to care homes or direct experience as care recipients . to capture a range of experience that reflected regional , historical , and organizational differences , we identified a purposive sample of nhs and local authority commissioners , senior managers from care home organizations , and the care regulator for england ( the care quality commission [ cqc ] ) ( table 1 ) . they were recruited and defined as stakeholders on the basis that they were able to characterize the view of a group or organization and would enable us to capture a range of relevant views . the sample was chosen to be able to speak authoritatively about the organization of health care to care homes and to theorize about what achieved the best health - related outcomes for residents , while acknowledging competing explanations . the sample was identified through the cqc ; national care home provider representative organizations , residents , and relatives ' representative groups ; and national health and local authority commissioners for care homes in the east of england and the midlands . a small sample of care home residents was also interviewed . participants were specifically asked to provide a stakeholder view ; in other words , to use their experience and expertise as , for example , a care home manager to inform what a good service should look like rather than provide a solely personal account . to facilitate this , residents ' prompts focused on what they believed good health care to care homes should comprise . interviews asked about examples of success and failure , how continuity of care was achieved , what good working between nhs services and care homes looked like , and the mechanisms or particular service models necessary to achieve the desired outcomes . the resident interviews conducted for this study were supplemented with a secondary analysis of 34 resident interviews from an earlier study that had focused on access to nhs health care . these interviews had specifically asked about health and the health care services received and what was seen as effective . this additional sample was included because of the challenges of identifying residents who had the capacity to participate and their difficulties in extrapolating from their own experience of health care to consider what good health care provision for care homes should look like . second , comparison was made within and between categories and the identification of preoccupations , difference , and themes . the third stage of interpretation was the identification of relationships and emergent hypotheses about how the favored approaches worked , and what was necessary to support their implementation . all the stakeholders stated that residents are entitled to the same health care that older people who live at home receive . the following are the 3 overlapping themes of what was perceived as central to the provision of quality health care:interventions that supported relational working between nhs practitioners and care home staffprovision of age - appropriate carecontractual and governance systems to guarantee care homes ' access to nhs services . interventions that supported relational working between nhs practitioners and care home staff provision of age - appropriate care contractual and governance systems to guarantee care homes ' access to nhs services . across the 3 themes there were shared elements ; for example , access to a gp when needed and the importance of a person - centered care approach , particularly for people with dementia . however , the 3 themes could be differentiated by the emphasis on what had to be done to provide effective health care and what supported or enabled that process . thus , a person - centered care approach or access to a gp was important for residents ' health , but they were described as an enabler rather than the driver for quality health care delivery to care homes . nhs service delivery models that supported relational working were seen as addressing the difficulties of working across health and social care systems , agreeing what was publicly funded health care and what came under the jurisdiction of the care home , maintaining continuity of information and reconciling the different priorities of care homes and health care providers . to achieve relational working required investment of resources , dedicated staff time , and the creation of formal and informal opportunities for health care and care home staff to work together . relational working could thus be realized by the identification of key people to work within the care home , the creation of care home specialist roles to provide ongoing support to care home staff , or the maintenance of working relationships within existing models of working with primary care services . examples focused on models of care in which health care staff visited regularly and predictably , provided teaching and support to care home staff , and were accessible for advice . one local authority commissioner identified the importance of nominated care home staff ( champions ) being allocated to work with visiting nhs staff to structure how they collaborate together and enable resolution of residents ' health problems in situations of pressure and limited resources.i think there has to be buy in on all sides , there 's got to be an understanding that certainly in this day and age care home settings are very tight on staff and budgets and sometimes it 's getting the right people from these particular homesto have that spare time to come along and get involved ... it'shaving champions , it 's making sure thateach home has their particular championon particular ( health ) topics and the they 've got ownership of that particular subject ( local authority commissioner ) i think there has to be buy in on all sides , there 's got to be an understanding that certainly in this day and age care home settings are very tight on staff and budgets and sometimes it 's getting the right people from these particular homesto have that spare time to come along and get involved ... it'shaving champions , it 's making sure thateach home has their particular championon particular ( health ) topics and the they 've got ownership of that particular subject ( local authority commissioner ) similarly a gp commissioner drawing on personal experience saw that it was possible to maintain continuity and the desired outcomes of care , such as avoiding unplanned hospital admissions , if visiting nhs staff knew that there were what she called designated care coordinators to work with . this role was seen as fostering integrated working.i think the key to successes , individuals are absolutely key , in care homes having a key professional , someone who takes responsibility for each patient ( sic ) , you know you need someone who is trained enough to coordinate that care , which is about integrated care and about , as i say , having patients ' co - coordinators , a key worker , whatever you want to call them . ( gp commissioner ) i think the key to successes , individuals are absolutely key , in care homes having a key professional , someone who takes responsibility for each patient ( sic ) , you know you need someone who is trained enough to coordinate that care , which is about integrated care and about , as i say , having patients ' co - coordinators , a key worker , whatever you want to call them . ( gp commissioner ) a less formalized method of supporting relational working was described by one care home manager ( sh9 ) as working like a team . team working involved shared training events with district nursing staff , care home staff being invited to the nhs provider meetings , and mutual confidence built on previous experience of having jointly resolved problems . she gave the example of being able to work with a liaison nurse to access residents ' notes when they were admitted to hospital . similarly , another care home manager described how residents ' needs were addressed and hospital admission rates were kept low because she knew that gps would come when asked , listen to their assessment of residents ' needs , and support care home staff to provide care . although the gp was identified as the lynchpin , it was the quality of the association that was emphasized as key.but they ( gps ) are very good ; they come out and respond , we can ask the gp to phone us , we have a good relationshipinterviewer what makes it such a good service?i think theclinical knowledge of the nursesand the fact thatwe are in a very rural community and we have had families of gps as patientsand they chose us because of our sound clinical knowledge . ( care home manager ) but they ( gps ) are very good ; they come out and respond , we can ask the gp to phone us , we have a good relationship interviewer what makes it such a good service ? we can phone and say abc , we have ruled out a uti , we have listened to his chest and the blood sugar is normal , can you come and have a look ? other stakeholders saw good working relationships as important but secondary to ensuring that residents received age - appropriate health care . the ability of a person or a service to integrate health and social care , or promote relational working , was not seen as sufficient to achieve the desired outcomes of improved resident health and reduced use of secondary services . for these stakeholders , it was the clinical expertise of the visiting health care professionals and equity of access that was important . a model of health care provision was needed that was sufficiently specialist to assess and address a frail older person 's health care needs . people are very worried about poststroke patients not having the kind of rehab they 'd get if they were at home or staying longer in hospital you know you really need people like geriatricians who are specialist in the care of older people the people who specialize in old age psychiatry also need to have a key role ( stakeholder from resident representative organization ) people are very worried about paying extra for physio , for chiropody , for the kind of services that are intrinsic to people 's conditions . people are very worried about poststroke patients not having the kind of rehab they 'd get if they were at home or staying longer in hospital you know you really need people like geriatricians who are specialist in the care of older people the people who specialize in old age psychiatry also need to have a key role ( stakeholder from resident representative organization ) the 3 residents interviewed struggled to extrapolate from their personal experience of nhs service delivery to how it should be provided generally to care homes . nevertheless , their interviews and the secondary analysis of how residents had talked in general about their experiences of health care reiterated the theme about expertise , how seeing the practitioner who understood their health care needs meant that problems were resolved . ( resident ) i ca n't stand , but i have said to the manager that if i had proper physio i think they could get me to stand , but nothing has happened about that . ( resident ) the secondary data analysis of residents ' interviews presented a picture of residents in the center of a flow of visiting health care professionals who may or may not be able to address their particular health care needs . this was linked both to limited access to the clinicians whom they wanted and limited control over how or when they could be referred to services . i just feel i 'm on a sort of , waiting , i 'm not as ill as a lot of people so i think i 'm just left to tick over .... this morning , i was seeing the senior nurse who comes with the others ( care staff ) , and tell her and she 's had a look and she 's going to be in touch , get in touch with somebody else who is higher up still , who is going to look at it this afternoon . i just feel i 'm on a sort of , waiting , i 'm not as ill as a lot of people so i think i 'm just left to tick over .... this morning , i was seeing the senior nurse who comes with the others ( care staff ) , and tell her and she 's had a look and she 's going to be in touch , get in touch with somebody else who is higher up still , who is going to look at it this afternoon . it was an assumption of the residents interviewed that information would be shared but they did not know for sure , or the process by which it was done , and as this quote shows , they did not always feel able to participate in the decision making . one representative of care home providers suggested that the reason the provision of age - appropriate care did not occur was because of agism and stigma . residents ' needs were not recognized or prioritized because older people and care homes were not valued by society . she compared provision to children 's residential care homes and suggested that there was inequity across the age groups and that commissioners and providers had considerable discretion in the number and type of services offered . headed by a consultant geriatrician and of course he had the status to be able to pull other people into his work , whereas i think if you do n't have that status in medicine you would find that you would struggle . one of the things he did was corral the gps and force them to do things that they were n't doing before . one of the things he did was corral the gps and force them to do things that they were n't doing before . stakeholders who had responsibilities for the monitoring and regulation of services to and within care homes emphasized interventions that reinforced good health care practice either through the use of incentives or performance management . if the proper governance were in place , this would guarantee residents ' access to evidence - based , age - appropriate health care , as well as continuity of care . an awareness of concerns about elder abuse , the need for vigilance , and examples of suboptimal care and avoidable deaths were given as the reasons why such systems needed to be in place . outcomes , such as the prevalence of pressure sores and reduction of prescribing antipsychotics for people with dementia were emphasized as important within such frameworks . this commissioner with responsibility for commissioning health and social care services for care homes gave the example of how using existing audit processes to improve end - of - life care for care home residents could improve health - related outcomes : end - of - life care is a health care issue , but what we do say in the ( contract with the care home ) specification is that we would expect that care homes are well versed around end - of - life care , that their staff are appropriately trained , and that 's part and parcel of the audit . end - of - life care is a health care issue , but what we do say in the ( contract with the care home ) specification is that we would expect that care homes are well versed around end - of - life care , that their staff are appropriately trained , and that 's part and parcel of the audit . so our annual audit looks at how geared up a care home is in meeting the needs of patients who are at the end of life . these systems were seen as setting a minimum standard for how services should be provided to care homes . this stakeholder worked for the cqc ( the regulator ) and acknowledged the importance of a good gp care home relationships but saw that this was consistently achieved only when there were explicit agreements in place and a planned approach : it 's where they 've got a proper agreed arrangement with that gp surgery around , you know , they visit at certain times of the week and they can be contacted if there are any problems ... ensuring then that people have a properly planned package of care that is really focused on their needs it 's where they 've got a proper agreed arrangement with that gp surgery around , you know , they visit at certain times of the week and they can be contacted if there are any problems ... ensuring then that people have a properly planned package of care that is really focused on their needs one gp commissioner saw financial incentives for gps as essential in recognition of the extra work that was entailed and drew on personal experience : this care home has 41 beds and those are not the equivalent of 41 patients . first , to recompense extra work that care homes generate , and second , to provide gps with the incentive to learn how the system works and improve current practice . this could be rolled out further to help gps work more efficiently with other services : you are lookingto pay for the process of change , once the average gp haslearnedthe system andlearnedwho does what , then they should be able to come into the system and do what they do well and not much else , and work more effectively in an integrated system . the interviews identified 3 provisional theories ( mechanisms ) of health service delivery to care homes and likely barriers and enablers ( contexts ) to improving residents ' health care in specific areas ( outcomes ) . thus , what one stakeholder hypothesized as essential to achieving improved resident outcomes , for example , a contractual framework for clinician involvement in care homes , another identified as the enabler for activities that promoted shared learning and relational working . table 2 summarizes how these were configured by the participants and the contending positions of the different stakeholders . these hypothesized context mechanism outcome configurations are the basis for the next step of interrogating the evidence and provide a framework for how the effectiveness of different models of service delivery to care homes is understood . the english model of health care provision to care homes with visiting physicians is common across the world . these data show that there was a broad consensus about what were the elements or characteristics of quality health care provision to care homes by the nhs . these were as follows : consistent and predictable access to expertise in geriatric medicine , investment in resources , and good working relationships and structured approaches to care that could be monitored . we found , however , that different individuals accorded different weights to these elements and had different views about what embeds good practice , what was essential , and what was desirable . for example , there was less agreement about whether specific professionals needed to be closely monitored and performance - managed or if investment in the training of care home staff should always be a joint enterprise with visiting care home professionals . it also was unclear if the use of financial incentives and sanctions , or the creation of sustained trusting working relationships , were the drivers ( or mechanisms ) for effective working or whether these represented enablers ( or contextual factors ) supporting residents ' access to health care . for some participants , a single element , for example access to expertise in geriatric medicine , was sufficient in and of itself to improve quality of care ; however , this was not the predominant view . what combination of elements and the dose of each are required to achieve optimal outcomes for care home resident(s ) has implications for how services are designed , prioritized , and resourced and who is identified within the system as responsible for service delivery . it was what stakeholders believed should work and reflected a range of theories about how to implement evidence - based practice and manage risk in primary health care , about frailty , approaches to comprehensive geriatric assessment , and what supports integrated working between health and social care . the findings have captured the range of perspectives and interested parties : commissioners , providers , and recipients of care . although only 3 older people living in care homes participated in the study , the study was fortunate to have access to 34 previously conducted interviews that enabled the study to obtain a resident - focused perspective on what good care looked like . the findings from this study reinforce bowman and meyer 's observation that the organization of care for older people resident in care homes represents an emerging medical space . the 3 models , and how they are exemplified as working , given what is known about the heterogeneity of care home markets and their residents and the range of context - sensitive variables that shape how services are provided , these findings mean that it is unlikely that a particular service specification for health service delivery can promote effective working for all care homes . the next step is to test and refine these theories by a review of the relevant evidence to identify the necessary conditions under which the provision of health care services to care homes optimizes residents ' health .

ze wzgldu na skpo doniesie naukowych dotyczcych diagnostyki nerek za pomoc elastografii impulsu mocy promieniowania akustycznego oraz niejednoznaczno prezentowanych wynikw pomiarw prdkoci propagacji fali cinajcej autorzy postanowili wykorzysta fizyczne modele tkankowe nerek w celu sprawdzenia wiarygodnoci samej metody w kontrolowanych warunkach , zblionych do panujcych podczas diagnostyki tego narzdu . dwa o normalnej i dwa o cienkiej warstwie miszowej , kady na gbokoci dostosowanej do pacjenta o normalnej budowie i otyego . zostay one przeprowadzone przy pomocy wbudowanej funkcji virtual touch tissue quantification ultrasonografu siemens acuson s2000 , wyposaonego w gowic 6c1 hd ( siemens mountainview , usa ) . rednie wartoci prdkoci fali cinajcej zarejestrowane we wszystkich punktach zainteresowania zawieray si w zakresie od 2,445 do 3,941 m / s , z odchyleniem standardowym przekraczajcym 0,1 m / s tylko w jednym z 29 punktw pomiarowych . uzyskane wyniki pokazuj , e metoda bdca przedmiotem zainteresowania jest wysoce wiarygodna pod warunkiem , e w objtoci pomiarowej znajduje si jednorodna struktura . jeli okno pomiarowe obejmuje choby czciowo region o innych waciwociach , to wynik pomiaru zostaje znieksztacony . co wicej , na wariancj wynikw pomiarw prdkoci fali cinajcej nie ma wpywu gboko , na jakiej pomiary s dokonywane . acoustic radiation force impulse ( arfi ) technique is a new ultrasound ( us ) method for assessing tissue stiffness . this technique uses a focused ultrasonic pulse to generate a shear wave spreading sideways from the focus point . tissue behavior , while the wave propagates , is registered based on a principle of classical strain imaging ( elastography ) , where tissue displacements are identified using correlation based methods . in practice the shear wave generating pulse this single , focused , high intensity pulse delivers mechanical energy to the examined tissue due to the relatively large absorption contributing to overall attenuation , as compared to scattering . this force impulse causes local displacement of tissue on the magnitude of micrometers . this displaced tissue velocity at which this wave travels across tissue is directly related to the tissue density and its shear modulus g. shear modulus is one of the quantities describing mechanical stiffness of materials it is the ratio between the shear stress and shear strain in deformed material , similarly as the young s modulus e is the ratio between the axial stress and strain . since the values of soft tissues mass densities are relatively close to the density of water and do not differ significantly from tissue to tissue , the values of shear wave velocities ( swv ) provide a valuable , quantitative information about tissue stiffness . 0.5 to 5 m / s . based on the arfi principle two main techniques have been developed qualitative imaging where various shear wave related quantities are used to create an image reflecting the mechanical properties of the tissue . this technique has been implemented by siemens medical solutions under the name virtual touch tissue imaging . the second technique is the quantitative measurement of the shear wave velocity where usually a time - of - flight methods are applied for assessing the velocity from the displacement data . this quantitative technique has been implemented by siemens medical solutions under the name virtual touch tissue quantification . in this case , b - mode imaging is supplemented by a measurement of the shear wave velocity for an operator - chosen region of interest ( roi ) covering approximately 1 cm . arfi techniques have been successfully applied to various tissues including liver , breast , prostate , kidneys and gastrointestinal tract providing new diagnostic capabilities for various diseases . the aim of the study was to evaluate the ability of arfi method to measure swv values of renal cortex in patients with different renal cortex thickness and different weight . in overweight patients the depth of measurement may exceed 8 cm , considered a maximum depth of arfi study , and create a challenge to perform proper examination . on the other hand the cortex thickness may be lower than 1 cm , which is the usual size of arfi measurement window . in patients with chronic kidney disease ( ckd ) changes in renal tissue both in us and fibrosis leads to reduction of renal cortex thickness , that normally presents values from 15 to 20 mm . the values of renal cortex thickness in ckd patients may vary from normal to values less than 10 mm . thin renal cortex is a challenging tissue for swv measurements according to examination guidelines and examination window size . examination guidelines state that in order to obtain correct results the measurement window must be placed entirely on examined structure , which is assumed to be uniform . in clinical practice this condition is not always achieved and measurements are imprecise due to : patient s respiratory movements ; limited stability of manually placing the measurement window ; natural variability of renal cortex thickness ; variable measurement depth ( distance between the probe and the measurement window ) depending on the weight of the patient . kidney phantom - based experiments were planned in order to assess the influence of mentioned limitations on the accuracy of renal cortex elasticity measurements . the window placement accuracy and the depth of measurements were of main interest . use of various materials for ultrasonography and elastography phantoms has been reported . for the purpose of this study gelatin phantoms were chosen , as this material has been widely accepted for ultrasonics including accoustic radiation force impulse technique , and has been examined in other works . gelatin phantoms in previous experiments were designed to obtain certain elastic ( young s ) modulus values . in this study shear shear wave velocity vs depends on the shear modulus g of the material and its density as given by the equation 1 . shear modulus can be related to elastic modulus using the poisson s ratio of the material as described by the equation 2 . it has been shown that elastic modulus of gelatin gel is linearly dependent on the concentration of gelatin . based on this property and measurements carried out during experiments described in a formula for expected young modulus of the material was derived equation 3 where cp is the concentration of gelatin , given as a relative value ( 0 to 1 ) and resulting e in kpa . for the gelatin mass concentrations calculations , based on literature , poisson s ratio was assumed to have a value of 0,49 . for poisson s ratios ranging from 0,48 to 0,495 , the expected young modulus differed by less than 1% , thus this approximation is sufficient for our purpose . mass density of the gelatin phantom material is usually assumed to be 1,0 g / cm but since experience shows that it is greater than density of water it was assumed to be 1,05 g / cm . for the purpose of this study phantom materials with two different shear wave velocities the renal cortex shear wave velocity was set at approximately 3 m / s , although average swv s of 1,75 m / s were reported too . the surrounding tissue swv was set at 4,5 m / s which corresponds to gelatin concentrations of 10,6% . attenuation or absorption of ultrasonic wave in the gelatin material , which is crucial in the arfi technique , was not measured in this study . overall attenuation can be estimated based on a classic study published by madsen , with the utilized composition it should be at a level close to 0,33 db / cm / mhz . this is a relatively low value and does not provide direct information on the absorption of the ultrasound in the phantom material . results presented below and by other authors prove that the absorption of the beam in gelatin based phantoms is sufficient for the arfi technique . components used for phantom manufacturing included : distilled water , gelatin ( polskie odczynniki chemiczne s.a . , poland ) , 5 m% graphite flakes median size 710 micron ( alfa aesar , germany ) , 0.2 m% glutaral aldehyde ( merck - schuchardt , germany ) and 0.2 m% edta ( carl roth , germany ) . the addition of n - propanol increases the sound velocity to the expected value of 1540 m / s , graphite flakes provide scattering and attenuation similar to what is observed in tissue , glutaral aldehyde accelerates the process of gelatin solidification and improves its resistance to melting , edta is used as a preservative . phantoms were manufactured by first dissolving the edta in preheated distilled water and alcohol mixture at approx . 60c , followed by dissolving the gelatin . after obtaining a clear solution of gelatin , finally , the aldehyde was added and then immediately the solution was poured into prepared molds . experience with gelatin phantoms shows that contact between areas with different gelatin concentrations results in water transportation due to different osmotic pressures . this causes significant swallowing of the material with higher concentration and shrinking of the material with lower gelatin concentration . to prevent this phenomenon in the phantoms used in this work , different regions were separated by a thin layer of latex membrane . for this purpose trans - vaginal latex ultrasonic probe covers ( plp , malaysia ) , cleared of the lubricating substance were used . four phantoms imitating different clinical situations were created , their description is given in tab . 1 . list of phantoms used in the study experiments were carried out in a setup configured as shown in fig . probe stand , c water container , d ultrasound probe ( siemens 6c1 hd ) , e phantom , f thermometer the ultrasound probe ( siemens 6c1 hd transducer , siemens mountainview , usa ) working at 4 mhz , was fixed in a stand above the phantom and thus its position remained constant for each measurement point . a siemens acuson s2000 ( siemens mountainview , usa ) was used for the shear wave velocity measurements the build - in virtual touch tissue quantification tool was applied for this purpose . the measurement gate for this scanner has fixed dimensions with the height of 10 mm . maximum depth for its placement is 80 mm . after placing the phantom in the container and fixing the probe in a position 20 measurements of swv for each point results obtained for every point were tested for normality of distribution using the jarque - bera test ( with the null hypothesis that the results come from a normal distribution with unknown mean and variance ) . ultrasound image of phantom a with shear wave velocity measurement in point 1 use of various materials for ultrasonography and elastography phantoms has been reported . for the purpose of this study gelatin phantoms were chosen , as this material has been widely accepted for ultrasonics including accoustic radiation force impulse technique , and has been examined in other works . gelatin phantoms in previous experiments were designed to obtain certain elastic ( young s ) modulus values . in this study shear shear wave velocity vs depends on the shear modulus g of the material and its density as given by the equation 1 . shear modulus can be related to elastic modulus using the poisson s ratio of the material as described by the equation 2 . it has been shown that elastic modulus of gelatin gel is linearly dependent on the concentration of gelatin . based on this property and measurements carried out during experiments described in a formula for expected young modulus of the material was derived equation 3 where cp is the concentration of gelatin , given as a relative value ( 0 to 1 ) and resulting e in kpa . for the gelatin mass concentrations calculations , based on literature , poisson s ratio was assumed to have a value of 0,49 . for poisson s ratios ranging from 0,48 to 0,495 , the expected young modulus differed by less than 1% , thus this approximation is sufficient for our purpose . mass density of the gelatin phantom material is usually assumed to be 1,0 g / cm but since experience shows that it is greater than density of water it was assumed to be 1,05 g / cm . for the purpose of this study phantom materials with two different shear wave velocities the renal cortex shear wave velocity was set at approximately 3 m / s , although average swv s of 1,75 m / s were reported too . the surrounding tissue swv was set at 4,5 m / s which corresponds to gelatin concentrations of 10,6% . attenuation or absorption of ultrasonic wave in the gelatin material , which is crucial in the arfi technique , was not measured in this study . overall attenuation can be estimated based on a classic study published by madsen , with the utilized composition it should be at a level close to 0,33 db / cm / mhz . this is a relatively low value and does not provide direct information on the absorption of the ultrasound in the phantom material . results presented below and by other authors prove that the absorption of the beam in gelatin based phantoms is sufficient for the arfi technique . components used for phantom manufacturing included : distilled water , gelatin ( polskie odczynniki chemiczne s.a . , poland ) , 5 m% graphite flakes median size 710 micron ( alfa aesar , germany ) , 0.2 m% glutaral aldehyde ( merck - schuchardt , germany ) and 0.2 m% edta ( carl roth , germany ) . the addition of n - propanol increases the sound velocity to the expected value of 1540 m / s , graphite flakes provide scattering and attenuation similar to what is observed in tissue , glutaral aldehyde accelerates the process of gelatin solidification and improves its resistance to melting , edta is used as a preservative . phantoms were manufactured by first dissolving the edta in preheated distilled water and alcohol mixture at approx . 60c , followed by dissolving the gelatin . after obtaining a clear solution of gelatin , finally , the aldehyde was added and then immediately the solution was poured into prepared molds . experience with gelatin phantoms shows that contact between areas with different gelatin concentrations results in water transportation due to different osmotic pressures . this causes significant swallowing of the material with higher concentration and shrinking of the material with lower gelatin concentration . to prevent this phenomenon in the phantoms used in this work , different regions were separated by a thin layer of latex membrane . for this purpose trans - vaginal latex ultrasonic probe covers ( plp , malaysia ) , cleared of the lubricating substance were used . four phantoms imitating different clinical situations were created , their description is given in tab . probe stand , c water container , d ultrasound probe ( siemens 6c1 hd ) , e phantom , f thermometer the ultrasound probe ( siemens 6c1 hd transducer , siemens mountainview , usa ) working at 4 mhz , was fixed in a stand above the phantom and thus its position remained constant for each measurement point . a siemens acuson s2000 ( siemens mountainview , usa ) was used for the shear wave velocity measurements the build - in virtual touch tissue quantification tool was applied for this purpose . the measurement gate for this scanner has fixed dimensions with the height of 10 mm . maximum depth for its placement is 80 mm . after placing the phantom in the container and fixing the probe in a position 20 measurements of swv for each point results obtained for every point were tested for normality of distribution using the jarque - bera test ( with the null hypothesis that the results come from a normal distribution with unknown mean and variance ) . ultrasound image of phantom a with shear wave velocity measurement in point 1 3 . table 3 lists depths at which the measurement window was placed for each measurement point . measurement points for each kidney phantom measurement window placement depths for each point and phantom in centimeters phantoms configuration with marked measurement points for each point shown in the fig . 3 , 20 measurements of shear wave propagation velocity were taken . each set of results was tested for normality of distribution using a jarque - bera test . in three cases this test returned p - values under 0,001 due to a single erroneous measurement mean shear wave propagation velocities for all measurement points measurements results for the same points , within different phantoms , were tested with the student s t - tests to check if they can be assumed to be equal ( the null hypothesis that their mean values are equal ) . in all cases this hypothesis was rejected with maximum obtained p - value 0,0076 for point 2 in phantoms b and c , while for other points p - value was less than 10 . this test shows that , although very close shear wave velocity values were recorded for points 1 , 2 , 3 and 5 , due to low standard deviation values , they are statistically different , what was illustrated in fig . 4 . shear wave velocities for each measurement point , phantoms a thru d results for each point in every phantom 5 . median ( red ) shear wave velocities for each point in all phantoms . full range to assess the influence of window placement a plot of standard deviations of velocities for each point number for all phantoms was created ( fig . 6 ) . in order to assess the influence of depth on measurement accuracy , standard deviations of velocities were plotted against the measurement depths fig . 7 . a linear model was fitted to the data of variation values ( solid line in fig . 7 ) with the values of r - squared 0.099 and adjusted r - squared 0.0598 . standard deviation of shear wave velocity measurements for different point numbers , all phantoms standard deviation of shear wave velocity measurements as a function of depth 3 . table 3 lists depths at which the measurement window was placed for each measurement point . measurement points for each kidney phantom measurement window placement depths for each point and phantom in centimeters phantoms configuration with marked measurement points each set of results was tested for normality of distribution using a jarque - bera test . in three cases this test returned p - values under 0,001 due to a single erroneous measurement clearly visible during visual inspection of results . after removing outliers from results , mean shear wave propagation velocities for all measurement points measurements results for the same points , within different phantoms , were tested with the student s t - tests to check if they can be assumed to be equal ( the null hypothesis that their mean values are equal ) . in all cases this hypothesis was rejected with maximum obtained p - value 0,0076 for point 2 in phantoms b and c , while for other points p - value was less than 10 . this test shows that , although very close shear wave velocity values were recorded for points 1 , 2 , 3 and 5 , due to low standard deviation values , they are statistically different , what was illustrated in fig . 4 . shear wave velocities for each measurement point , phantoms a thru d results for each point in every phantom 5 . median ( red ) shear wave velocities for each point in all phantoms . boxes show the 25 to 75 percentile , whiskers full range to assess the influence of window placement a plot of standard deviations of velocities for each point number for all phantoms 6 ) . in order to assess the influence of depth on measurement accuracy , standard deviations of velocities were plotted against the measurement depths fig . 7 . a linear model was fitted to the data of variation values ( solid line in fig . 7 ) with the values of r - squared 0.099 and adjusted r - squared 0.0598 . standard deviation of shear wave velocity measurements for different point numbers , all phantoms standard deviation of shear wave velocity measurements as a function of depth measurement points were chosen by hand , so in case of each point and each phantom they were different . all measurements for a single point were , on the other hand , well stabilized by the probe mounting frame and thus were taken exactly in the same area of the phantom material with precisely the same speckle pattern , which resulted in a very low variation of measured velocity values ( fig . the variation of results strongly increases when the results for each point are taken together from all phantoms ( fig . 5 ) , which corresponds more closely the situation of freehand examinations on a patient . both cases show that catching two different materials in the measurement window ( points 25 , fig . 4 and 5 ) affects measured velocity value . this assessment is only qualitative since precise measurement of the percentage of window covering the it is very important to note , that the measurement window has also a third dimension since the ultrasonic beam in the direction of elevation has a thickness of couple to over 10 mm . relation between the uniformity of material scanned and the variation of velocity measurements can be assessed by comparing the variation for points 1 , 6 and 7 obtained from a homogenous area versus variation for points 2 , 3 , 4 and 5 obtained for windows catching two different areas ( fig . 6 ) . the visual inspection of the plot shows no effect of the homogeneity of the material covered by the measurement window on the variation of results of measurements while the average variation of results is relatively low for point 1 in all phantoms , it proves to be high for point 6 and 7 . for inhomogeneous points they can be high ( points 2 and 4 ) or low ( point 5 ) . increase in average variation ( solid line in fig . 7 ) with increase in measurement depth suggests a correlation between those quantities , but low values of r - squared and adjusted r - squared makes this relation uncertain . this may result from the fact that at higher depths the arfi becomes weakand are thus expected to be less operator dependent . methods have been developed that utilize impulsive ( i.e. < 1 ms ( amplitude of the pulse is low ) and tracking of material movements due to the shear wave becomes difficult . an experiment providing more points for this assessment , using a uniform phantom could probably provide more precise results . maintaining the whole window area within the examined tissue is the most important precondition that has to be met during examinations . this also assumes the homogeneity of the examined tissue which is scarcely the case . in practice , the obtained results are averaged over the measurement volume ( which includes also the out - of - plane dimension ) . the examination of standard deviations shows that this averaging is consistent and does not introduce a higher variability of results . an increase in depth at which measurements are taken does not clearly affect the results , but might increase the variation of results . in this study , this relation was weak and does not allow to conclude that the depth of measurement has a significant impact on measurements reliability . overall conclusions from this study are that the arfi method is precise and reliable as long as the object is suitable measurements can be taken in a uniform / homogenous volume that is representative for the tissue of interest . with this limitations arfi work was supported from the statutory funds of the faculty of mechatronics , warsaw university of technology .

aim of the studysince there have been only few works reporting the diagnosis of kidneys using acoustic radiation force impulse technique and those works do not provide consistent results of shear wave velocity measurements in renal tissue , we have decided to use kidney phantoms with known properties to examine the reliability of the method itself in a controlled setup similar to kidneys examination.materials and methodsfour gelatin - based phantoms imitating different clinical situations were manufactured two with thick and two with thin renal cortex , each type at a depth similar to a normal - weight or overweight patient . for each phantom , a series of interest points was chosen and for each point 20 shear wave velocity measurements were taken using the build - in virtual touch tissue quantification tool in a siemens acuson s2000 ultrasound scanner equipped with a 6c1 hd transducer ( siemens mountainview , usa).resultsmean shear wave velocity values obtained for all the examined points ranged from 2.445 to 3.941 m / s , with standard deviation exceeding 0.1 in only one case out of 29 points , but differing significantly between all points.conclusionsthe obtained results indicate that the method is highly reliable as long as the measurement volume contains a uniform tissue region . if the measurement window covers a region with different properties even partially , the obtained results are affected . the variance of measured values on the other hand is not affected by the said non - uniformity of material under examination . furthermore , the variance of measured values does not show a clear dependency on the depth at which the shear wave velocities are measured .

Cel pracy Materiay i metody Wyniki Wnioski Introduction Materials and methods Phantoms preparation Examination setup and procedure Results Acquired data Shear wave velocities Discussion Conclusions Conflict of interest

zostay one przeprowadzone przy pomocy wbudowanej funkcji virtual touch tissue quantification ultrasonografu siemens acuson s2000 , wyposaonego w gowic 6c1 hd ( siemens mountainview , usa ) . rednie wartoci prdkoci fali cinajcej zarejestrowane we wszystkich punktach zainteresowania zawieray si w zakresie od 2,445 do 3,941 m / s , z odchyleniem standardowym przekraczajcym 0,1 m / s tylko w jednym z 29 punktw pomiarowych . acoustic radiation force impulse ( arfi ) technique is a new ultrasound ( us ) method for assessing tissue stiffness . this technique uses a focused ultrasonic pulse to generate a shear wave spreading sideways from the focus point . in practice the shear wave generating pulse this single , focused , high intensity pulse delivers mechanical energy to the examined tissue due to the relatively large absorption contributing to overall attenuation , as compared to scattering . this force impulse causes local displacement of tissue on the magnitude of micrometers . this displaced tissue velocity at which this wave travels across tissue is directly related to the tissue density and its shear modulus g. shear modulus is one of the quantities describing mechanical stiffness of materials it is the ratio between the shear stress and shear strain in deformed material , similarly as the young s modulus e is the ratio between the axial stress and strain . since the values of soft tissues mass densities are relatively close to the density of water and do not differ significantly from tissue to tissue , the values of shear wave velocities ( swv ) provide a valuable , quantitative information about tissue stiffness . 0.5 to 5 m / s . based on the arfi principle two main techniques have been developed qualitative imaging where various shear wave related quantities are used to create an image reflecting the mechanical properties of the tissue . this technique has been implemented by siemens medical solutions under the name virtual touch tissue imaging . the second technique is the quantitative measurement of the shear wave velocity where usually a time - of - flight methods are applied for assessing the velocity from the displacement data . this quantitative technique has been implemented by siemens medical solutions under the name virtual touch tissue quantification . in this case , b - mode imaging is supplemented by a measurement of the shear wave velocity for an operator - chosen region of interest ( roi ) covering approximately 1 cm . arfi techniques have been successfully applied to various tissues including liver , breast , prostate , kidneys and gastrointestinal tract providing new diagnostic capabilities for various diseases . the aim of the study was to evaluate the ability of arfi method to measure swv values of renal cortex in patients with different renal cortex thickness and different weight . in overweight patients the depth of measurement may exceed 8 cm , considered a maximum depth of arfi study , and create a challenge to perform proper examination . on the other hand the cortex thickness may be lower than 1 cm , which is the usual size of arfi measurement window . in patients with chronic kidney disease ( ckd ) changes in renal tissue both in us and fibrosis leads to reduction of renal cortex thickness , that normally presents values from 15 to 20 mm . the values of renal cortex thickness in ckd patients may vary from normal to values less than 10 mm . thin renal cortex is a challenging tissue for swv measurements according to examination guidelines and examination window size . examination guidelines state that in order to obtain correct results the measurement window must be placed entirely on examined structure , which is assumed to be uniform . in clinical practice this condition is not always achieved and measurements are imprecise due to : patient s respiratory movements ; limited stability of manually placing the measurement window ; natural variability of renal cortex thickness ; variable measurement depth ( distance between the probe and the measurement window ) depending on the weight of the patient . kidney phantom - based experiments were planned in order to assess the influence of mentioned limitations on the accuracy of renal cortex elasticity measurements . the window placement accuracy and the depth of measurements were of main interest . for the purpose of this study gelatin phantoms were chosen , as this material has been widely accepted for ultrasonics including accoustic radiation force impulse technique , and has been examined in other works . in this study shear shear wave velocity vs depends on the shear modulus g of the material and its density as given by the equation 1 . shear modulus can be related to elastic modulus using the poisson s ratio of the material as described by the equation 2 . it has been shown that elastic modulus of gelatin gel is linearly dependent on the concentration of gelatin . based on this property and measurements carried out during experiments described in a formula for expected young modulus of the material was derived equation 3 where cp is the concentration of gelatin , given as a relative value ( 0 to 1 ) and resulting e in kpa . for the gelatin mass concentrations calculations , based on literature , poisson s ratio was assumed to have a value of 0,49 . for poisson s ratios ranging from 0,48 to 0,495 , the expected young modulus differed by less than 1% , thus this approximation is sufficient for our purpose . mass density of the gelatin phantom material is usually assumed to be 1,0 g / cm but since experience shows that it is greater than density of water it was assumed to be 1,05 g / cm . for the purpose of this study phantom materials with two different shear wave velocities the renal cortex shear wave velocity was set at approximately 3 m / s , although average swv s of 1,75 m / s were reported too . the surrounding tissue swv was set at 4,5 m / s which corresponds to gelatin concentrations of 10,6% . overall attenuation can be estimated based on a classic study published by madsen , with the utilized composition it should be at a level close to 0,33 db / cm / mhz . this is a relatively low value and does not provide direct information on the absorption of the ultrasound in the phantom material . results presented below and by other authors prove that the absorption of the beam in gelatin based phantoms is sufficient for the arfi technique . components used for phantom manufacturing included : distilled water , gelatin ( polskie odczynniki chemiczne s.a . the addition of n - propanol increases the sound velocity to the expected value of 1540 m / s , graphite flakes provide scattering and attenuation similar to what is observed in tissue , glutaral aldehyde accelerates the process of gelatin solidification and improves its resistance to melting , edta is used as a preservative . phantoms were manufactured by first dissolving the edta in preheated distilled water and alcohol mixture at approx . after obtaining a clear solution of gelatin , finally , the aldehyde was added and then immediately the solution was poured into prepared molds . experience with gelatin phantoms shows that contact between areas with different gelatin concentrations results in water transportation due to different osmotic pressures . this causes significant swallowing of the material with higher concentration and shrinking of the material with lower gelatin concentration . for this purpose trans - vaginal latex ultrasonic probe covers ( plp , malaysia ) , cleared of the lubricating substance were used . four phantoms imitating different clinical situations were created , their description is given in tab . 1 . list of phantoms used in the study experiments were carried out in a setup configured as shown in fig . probe stand , c water container , d ultrasound probe ( siemens 6c1 hd ) , e phantom , f thermometer the ultrasound probe ( siemens 6c1 hd transducer , siemens mountainview , usa ) working at 4 mhz , was fixed in a stand above the phantom and thus its position remained constant for each measurement point . a siemens acuson s2000 ( siemens mountainview , usa ) was used for the shear wave velocity measurements the build - in virtual touch tissue quantification tool was applied for this purpose . the measurement gate for this scanner has fixed dimensions with the height of 10 mm . after placing the phantom in the container and fixing the probe in a position 20 measurements of swv for each point results obtained for every point were tested for normality of distribution using the jarque - bera test ( with the null hypothesis that the results come from a normal distribution with unknown mean and variance ) . ultrasound image of phantom a with shear wave velocity measurement in point 1 use of various materials for ultrasonography and elastography phantoms has been reported . for the purpose of this study gelatin phantoms were chosen , as this material has been widely accepted for ultrasonics including accoustic radiation force impulse technique , and has been examined in other works . in this study shear shear wave velocity vs depends on the shear modulus g of the material and its density as given by the equation 1 . shear modulus can be related to elastic modulus using the poisson s ratio of the material as described by the equation 2 . it has been shown that elastic modulus of gelatin gel is linearly dependent on the concentration of gelatin . based on this property and measurements carried out during experiments described in a formula for expected young modulus of the material was derived equation 3 where cp is the concentration of gelatin , given as a relative value ( 0 to 1 ) and resulting e in kpa . for the gelatin mass concentrations calculations , based on literature , poisson s ratio was assumed to have a value of 0,49 . for poisson s ratios ranging from 0,48 to 0,495 , the expected young modulus differed by less than 1% , thus this approximation is sufficient for our purpose . mass density of the gelatin phantom material is usually assumed to be 1,0 g / cm but since experience shows that it is greater than density of water it was assumed to be 1,05 g / cm . for the purpose of this study phantom materials with two different shear wave velocities the renal cortex shear wave velocity was set at approximately 3 m / s , although average swv s of 1,75 m / s were reported too . the surrounding tissue swv was set at 4,5 m / s which corresponds to gelatin concentrations of 10,6% . attenuation or absorption of ultrasonic wave in the gelatin material , which is crucial in the arfi technique , was not measured in this study . overall attenuation can be estimated based on a classic study published by madsen , with the utilized composition it should be at a level close to 0,33 db / cm / mhz . this is a relatively low value and does not provide direct information on the absorption of the ultrasound in the phantom material . results presented below and by other authors prove that the absorption of the beam in gelatin based phantoms is sufficient for the arfi technique . , poland ) , 5 m% graphite flakes median size 710 micron ( alfa aesar , germany ) , 0.2 m% glutaral aldehyde ( merck - schuchardt , germany ) and 0.2 m% edta ( carl roth , germany ) . the addition of n - propanol increases the sound velocity to the expected value of 1540 m / s , graphite flakes provide scattering and attenuation similar to what is observed in tissue , glutaral aldehyde accelerates the process of gelatin solidification and improves its resistance to melting , edta is used as a preservative . phantoms were manufactured by first dissolving the edta in preheated distilled water and alcohol mixture at approx . 60c , followed by dissolving the gelatin . after obtaining a clear solution of gelatin , finally , the aldehyde was added and then immediately the solution was poured into prepared molds . experience with gelatin phantoms shows that contact between areas with different gelatin concentrations results in water transportation due to different osmotic pressures . this causes significant swallowing of the material with higher concentration and shrinking of the material with lower gelatin concentration . for this purpose trans - vaginal latex ultrasonic probe covers ( plp , malaysia ) , cleared of the lubricating substance were used . four phantoms imitating different clinical situations were created , their description is given in tab . probe stand , c water container , d ultrasound probe ( siemens 6c1 hd ) , e phantom , f thermometer the ultrasound probe ( siemens 6c1 hd transducer , siemens mountainview , usa ) working at 4 mhz , was fixed in a stand above the phantom and thus its position remained constant for each measurement point . a siemens acuson s2000 ( siemens mountainview , usa ) was used for the shear wave velocity measurements the build - in virtual touch tissue quantification tool was applied for this purpose . the measurement gate for this scanner has fixed dimensions with the height of 10 mm . after placing the phantom in the container and fixing the probe in a position 20 measurements of swv for each point results obtained for every point were tested for normality of distribution using the jarque - bera test ( with the null hypothesis that the results come from a normal distribution with unknown mean and variance ) . ultrasound image of phantom a with shear wave velocity measurement in point 1 3 . table 3 lists depths at which the measurement window was placed for each measurement point . measurement points for each kidney phantom measurement window placement depths for each point and phantom in centimeters phantoms configuration with marked measurement points for each point shown in the fig . 3 , 20 measurements of shear wave propagation velocity were taken . in three cases this test returned p - values under 0,001 due to a single erroneous measurement mean shear wave propagation velocities for all measurement points measurements results for the same points , within different phantoms , were tested with the student s t - tests to check if they can be assumed to be equal ( the null hypothesis that their mean values are equal ) . this test shows that , although very close shear wave velocity values were recorded for points 1 , 2 , 3 and 5 , due to low standard deviation values , they are statistically different , what was illustrated in fig . shear wave velocities for each measurement point , phantoms a thru d results for each point in every phantom 5 . median ( red ) shear wave velocities for each point in all phantoms . full range to assess the influence of window placement a plot of standard deviations of velocities for each point number for all phantoms was created ( fig . in order to assess the influence of depth on measurement accuracy , standard deviations of velocities were plotted against the measurement depths fig . standard deviation of shear wave velocity measurements for different point numbers , all phantoms standard deviation of shear wave velocity measurements as a function of depth 3 . table 3 lists depths at which the measurement window was placed for each measurement point . measurement points for each kidney phantom measurement window placement depths for each point and phantom in centimeters phantoms configuration with marked measurement points each set of results was tested for normality of distribution using a jarque - bera test . in three cases this test returned p - values under 0,001 due to a single erroneous measurement clearly visible during visual inspection of results . after removing outliers from results , mean shear wave propagation velocities for all measurement points measurements results for the same points , within different phantoms , were tested with the student s t - tests to check if they can be assumed to be equal ( the null hypothesis that their mean values are equal ) . this test shows that , although very close shear wave velocity values were recorded for points 1 , 2 , 3 and 5 , due to low standard deviation values , they are statistically different , what was illustrated in fig . shear wave velocities for each measurement point , phantoms a thru d results for each point in every phantom 5 . median ( red ) shear wave velocities for each point in all phantoms . boxes show the 25 to 75 percentile , whiskers full range to assess the influence of window placement a plot of standard deviations of velocities for each point number for all phantoms 6 ) . in order to assess the influence of depth on measurement accuracy , standard deviations of velocities were plotted against the measurement depths fig . 7 . standard deviation of shear wave velocity measurements for different point numbers , all phantoms standard deviation of shear wave velocity measurements as a function of depth measurement points were chosen by hand , so in case of each point and each phantom they were different . all measurements for a single point were , on the other hand , well stabilized by the probe mounting frame and thus were taken exactly in the same area of the phantom material with precisely the same speckle pattern , which resulted in a very low variation of measured velocity values ( fig . the variation of results strongly increases when the results for each point are taken together from all phantoms ( fig . both cases show that catching two different materials in the measurement window ( points 25 , fig . this assessment is only qualitative since precise measurement of the percentage of window covering the it is very important to note , that the measurement window has also a third dimension since the ultrasonic beam in the direction of elevation has a thickness of couple to over 10 mm . relation between the uniformity of material scanned and the variation of velocity measurements can be assessed by comparing the variation for points 1 , 6 and 7 obtained from a homogenous area versus variation for points 2 , 3 , 4 and 5 obtained for windows catching two different areas ( fig . 6 ) . the visual inspection of the plot shows no effect of the homogeneity of the material covered by the measurement window on the variation of results of measurements while the average variation of results is relatively low for point 1 in all phantoms , it proves to be high for point 6 and 7 . 7 ) with increase in measurement depth suggests a correlation between those quantities , but low values of r - squared and adjusted r - squared makes this relation uncertain . methods have been developed that utilize impulsive ( i.e. < 1 ms ( amplitude of the pulse is low ) and tracking of material movements due to the shear wave becomes difficult . an experiment providing more points for this assessment , using a uniform phantom could probably provide more precise results . maintaining the whole window area within the examined tissue is the most important precondition that has to be met during examinations . this also assumes the homogeneity of the examined tissue which is scarcely the case . in practice , the obtained results are averaged over the measurement volume ( which includes also the out - of - plane dimension ) . the examination of standard deviations shows that this averaging is consistent and does not introduce a higher variability of results . an increase in depth at which measurements are taken does not clearly affect the results , but might increase the variation of results . in this study , this relation was weak and does not allow to conclude that the depth of measurement has a significant impact on measurements reliability . overall conclusions from this study are that the arfi method is precise and reliable as long as the object is suitable measurements can be taken in a uniform / homogenous volume that is representative for the tissue of interest . with this limitations arfi work was supported from the statutory funds of the faculty of mechatronics , warsaw university of technology .

neuropathic pain is a common condition that affects up to 8% of the european population.13 established treatments for neuropathic pain are limited as they provide only partial pain relief in an estimated 40%60% of patients , and many are associated with a variety of unwanted systemic effects and intensive daily regimens.47 capsaicin , the main active capsaicinoid ingredient of chilies ( capsicum spp . ) , is an agonist of the transient receptor potential vanilloid 1 ( trpv1 ) receptor,8 which is highly expressed on nociceptors.5,9,10 ngx-4010 is a localized dermal delivery system containing high - concentration capsaicin ( 8% w / w ) that is designed to rapidly deliver therapeutic doses of capsaicin locally into the skin . this results in defunctionalization of trpv1-expressing sensory nerve endings and reduced epidermal nerve fiber density.1114 prolonged relief of neuropathic pain for up to 12 weeks following a single ngx-4010 application has been observed in patients with post - herpetic neuralgia ( phn ) or painful hiv - associated distal sensory polyneuropathy ( hiv - dsp ) in phase ii and iii studies.1520 due to the irritancy of capsaicin , topical application is associated with pain , erythema , and other application - site reactions.1520 because of this , a topical anesthetic is applied to reduce application - site discomfort prior to administration of ngx-4010 . in the above clinical studies , a 4% lidocaine topical anesthetic cream ( l.m.x.4/ela - max4 ; ferndale laboratories inc , ferndale , mi ) was applied for 60 minutes and removed prior to ngx-4010 application . the current open - label study sought to determine whether similar tolerability could be achieved with other commonly available 4% lidocaine formulations and assessed the safety , tolerability , and efficacy of ngx-4010 following pretreatment with either l.m.x.4 or the alternative products ( topicaine gel [ esba laboratories inc , jupiter , fl ] or betacaine enhanced gel 4 [ theraderm inc , tampa , fl ] ) . the study was approved by a central institutional review board ( biomed irb , san diego , ca ) or a local institutional review board at participating sites and conducted in accordance with the ethical principles of the declaration of helsinki , good clinical practice guidelines , and applicable regulatory requirements . written informed consent was obtained from all participating patients before initiating study - related procedures . patients were at least 18 years old with moderate to severe neuropathic pain ( average numeric pain rating scale [ nprs ] score of 38 inclusive ) for at least 3 months secondary to painful diabetic neuropathy ( pdn ) , phn , or hiv - dsp . the nprs is an 11-point scale ( 010 ) , with 0 indicating no pain and 10 indicating the worst possible pain.21 patients taking chronic pain medications ( anticonvulsants , nonselective serotonin reuptake inhibitor [ ssri ] antidepressants , or opioids ) had to be on a stable dose for at least 21 days before study patch application and remain on a stable dose throughout the 12-week study . exclusion criteria included the following : use of any topically applied pain medication on the painful area within 21 days before study patch application ; history of diabetic foot ulcerations and/or status post - amputation ; any implanted medical device for the treatment of neuropathic pain ; significant ongoing or recurrent pain of another etiology that interfered with judging neuropathic pain ; evidence of another contributing or immunologic cause for the sensory neuropathy ; and neuropathic pain areas located only on the face , above the scalp hairline , or near mucous membranes . patients were randomized to receive pretreatment for 60 minutes with one of three lidocaine 4%-based topical anesthetics : l.m.x.4 , topicaine gel , or betacaine enhanced gel 4 followed by a 60- or 90-minute treatment with ngx-4010 ( qutenza ; neurogesx inc , san mateo , ca ) . up to four ngx-4010 patches of 280 cm could be used ( maximum treatment area of 1120 cm ) . patients were randomly assigned to receive 60 or 90 minutes of treatment with ngx-4010 and allocated to one of three topical anesthetic arms according to a 1:1:1:1:1:1 randomization scheme generated by icon clinical research ( redwood city , ca ) . oxycodone oral solution ( 1 mg / ml ) could be administered at the onset of treatment - associated discomfort and as needed in the clinic on treatment days . after patch removal , local cooling ( such as cold compresses ) could also be used to relieve treatment - associated discomfort . in addition , patients could take hydrocodone bitartrate/ acetaminophen ( 5 mg/500 mg ) for up to 5 days after patch application for treatment - associated discomfort as needed . topical pain medications were not permitted . throughout the study , patients were allowed to take acetaminophen up to 2 g / day as needed for pain . safety was assessed by adverse events ( aes ) , which were coded using the medical dictionary for regulatory activities , version 7.0 . treatment - associated erythema , discomfort , and pain on the day of treatment were not captured as aes but reported as dermal assessment scores or nprs scores . dermal assessment scores ( 0- to 7-point severity score)22 were recorded on the day of treatment before and after application of topical anesthetic , and 5 and 120 minutes after patch removal and at each study visit . the distribution of maximum score between the pooled l.m.x.4 group and the other two pooled anesthetic groups was compared using the cochran mantel nprs scores were recorded immediately prior to anesthetic application ; 30 and 55 minutes after anesthetic application ; 25 , 55 , and 85 ( if applicable ) minutes after patch application ; and 5 , 25 , 55 , 85 , and 115 minutes after patch removal . the change in nprs scores from the pre - anesthetic time point , change in vital signs ( systolic blood pressure , diastolic blood pressure , heart rate , and respiratory rate ) from the pre - patch time point , the proportion of patients with a 33% increase in nprs score from baseline during the first 48 hours after ngx-4010 treatment , and the proportion of patients with < 90% of intended patch application duration were summarized . a cochran mantel haenszel test adjusted for treatment duration ( 60 or 90 minutes ) was used to test for a difference in the proportion of patients with a 33% increase in nprs score from baseline during the first 48 hours between the pooled l.m.x.4 group and the other two pooled anesthetic groups . descriptive statistics were calculated for laboratory parameters and vital signs at screening , termination , and change from screening to termination . a chi - squared test was performed to test for a difference in the proportion of patients using medication for treatment - related discomfort between the pooled l.m.x.4 group and the other two pooled anesthetic groups . the sample size was determined based on a chi - squared continuity corrected test to detect a difference of 20% in the proportion of subjects completing at least 90% of intended duration between the l.m.x.4-treated group and each of the other topical anesthetic groups at the 0.05 significance level with 80% power . efficacy was evaluated using nprs scores for average pain for the past 24 hours patient global impression of change ( pgic ; patients reported how they felt after treatment as compared with before treatment on a scale of 3 , indicating very much worse , to + 3 , indicating very much improved , with 0 being no change ) and the investigator - rated clinical global impression of change ( cgic)23 were evaluated at weeks 2 , 6 , and 12 . the primary efficacy endpoint was the percentage change in average pain for the past 24 hours nprs scores from baseline to weeks 2 through 12 . to avoid the potential confounding effect of allowed opioid medications during days 05 , week 1 scores were not included . other efficacy measures included : mean absolute changes in nprs scores and the proportion of patients with a 30% or 50% reduction in nprs score from baseline to weeks 2 through 12 ; and the percentage of patients considered improved ( slightly , much , or very much ) on the pgic or cgic at week 12 . all patients who received any study treatment and had at least 3 days of available nprs scores during the baseline period were included in the efficacy analyses . an analysis of covariance model with baseline pain score as the covariate was used to test for differences in change from baseline to weeks 2 through 12 between the pooled l.m.x.4 group and each of the other two pooled anesthetic groups . a logistic regression model , with the baseline nprs score as covariate , was performed to test for a difference in the proportion of patients reaching 30% and 50% decreases from baseline between the pooled l.m.x.4 group and each of the other two pooled anesthetic groups . the study was approved by a central institutional review board ( biomed irb , san diego , ca ) or a local institutional review board at participating sites and conducted in accordance with the ethical principles of the declaration of helsinki , good clinical practice guidelines , and applicable regulatory requirements . written informed consent was obtained from all participating patients before initiating study - related procedures . patients were at least 18 years old with moderate to severe neuropathic pain ( average numeric pain rating scale [ nprs ] score of 38 inclusive ) for at least 3 months secondary to painful diabetic neuropathy ( pdn ) , phn , or hiv - dsp . the nprs is an 11-point scale ( 010 ) , with 0 indicating no pain and 10 indicating the worst possible pain.21 patients taking chronic pain medications ( anticonvulsants , nonselective serotonin reuptake inhibitor [ ssri ] antidepressants , or opioids ) had to be on a stable dose for at least 21 days before study patch application and remain on a stable dose throughout the 12-week study . exclusion criteria included the following : use of any topically applied pain medication on the painful area within 21 days before study patch application ; history of diabetic foot ulcerations and/or status post - amputation ; any implanted medical device for the treatment of neuropathic pain ; significant ongoing or recurrent pain of another etiology that interfered with judging neuropathic pain ; evidence of another contributing or immunologic cause for the sensory neuropathy ; and neuropathic pain areas located only on the face , above the scalp hairline , or near mucous membranes . patients were randomized to receive pretreatment for 60 minutes with one of three lidocaine 4%-based topical anesthetics : l.m.x.4 , topicaine gel , or betacaine enhanced gel 4 followed by a 60- or 90-minute treatment with ngx-4010 ( qutenza ; neurogesx inc , san mateo , ca ) . up to four ngx-4010 patches of 280 cm could be used ( maximum treatment area of 1120 cm ) . patients were randomly assigned to receive 60 or 90 minutes of treatment with ngx-4010 and allocated to one of three topical anesthetic arms according to a 1:1:1:1:1:1 randomization scheme generated by icon clinical research ( redwood city , ca ) . oxycodone oral solution ( 1 mg / ml ) could be administered at the onset of treatment - associated discomfort and as needed in the clinic on treatment days . after patch removal , local cooling ( such as cold compresses ) could also be used to relieve treatment - associated discomfort . in addition , patients could take hydrocodone bitartrate/ acetaminophen ( 5 mg/500 mg ) for up to 5 days after patch application for treatment - associated discomfort as needed . topical pain medications were not permitted . throughout the study , patients were allowed to take acetaminophen up to 2 g / day as needed for pain . safety was assessed by adverse events ( aes ) , which were coded using the medical dictionary for regulatory activities , version 7.0 . treatment - associated erythema , discomfort , and pain on the day of treatment were not captured as aes but reported as dermal assessment scores or nprs scores . dermal assessment scores ( 0- to 7-point severity score)22 were recorded on the day of treatment before and after application of topical anesthetic , and 5 and 120 minutes after patch removal and at each study visit . the distribution of maximum score between the pooled l.m.x.4 group and the other two pooled anesthetic groups was compared using the cochran mantel nprs scores were recorded immediately prior to anesthetic application ; 30 and 55 minutes after anesthetic application ; 25 , 55 , and 85 ( if applicable ) minutes after patch application ; and 5 , 25 , 55 , 85 , and 115 minutes after patch removal . the change in nprs scores from the pre - anesthetic time point , change in vital signs ( systolic blood pressure , diastolic blood pressure , heart rate , and respiratory rate ) from the pre - patch time point , the proportion of patients with a 33% increase in nprs score from baseline during the first 48 hours after ngx-4010 treatment , and the proportion of patients with < 90% of intended patch application duration were summarized . haenszel test adjusted for treatment duration ( 60 or 90 minutes ) was used to test for a difference in the proportion of patients with a 33% increase in nprs score from baseline during the first 48 hours between the pooled l.m.x.4 group and the other two pooled anesthetic groups . descriptive statistics were calculated for laboratory parameters and vital signs at screening , termination , and change from screening to termination . a chi - squared test was performed to test for a difference in the proportion of patients using medication for treatment - related discomfort between the pooled l.m.x.4 group and the other two pooled anesthetic groups . the sample size was determined based on a chi - squared continuity corrected test to detect a difference of 20% in the proportion of subjects completing at least 90% of intended duration between the l.m.x.4-treated group and each of the other topical anesthetic groups at the 0.05 significance level with 80% power . efficacy was evaluated using nprs scores for average pain for the past 24 hours recorded daily at 9 pm in a paper diary throughout the study period . patient global impression of change ( pgic ; patients reported how they felt after treatment as compared with before treatment on a scale of 3 , indicating very much worse , to + 3 , indicating very much improved , with 0 being no change ) and the investigator - rated clinical global impression of change ( cgic)23 were evaluated at weeks 2 , 6 , and 12 . the primary efficacy endpoint was the percentage change in average pain for the past 24 hours nprs scores from baseline to weeks 2 through 12 . to avoid the potential confounding effect of allowed opioid medications during days 05 , week 1 scores were not included . other efficacy measures included : mean absolute changes in nprs scores and the proportion of patients with a 30% or 50% reduction in nprs score from baseline to weeks 2 through 12 ; and the percentage of patients considered improved ( slightly , much , or very much ) on the pgic or cgic at week 12 . all patients who received any study treatment and had at least 3 days of available nprs scores during the baseline period were included in the efficacy analyses . an analysis of covariance model with baseline pain score as the covariate was used to test for differences in change from baseline to weeks 2 through 12 between the pooled l.m.x.4 group and each of the other two pooled anesthetic groups . a logistic regression model , with the baseline nprs score as covariate , was performed to test for a difference in the proportion of patients reaching 30% and 50% decreases from baseline between the pooled l.m.x.4 group and each of the other two pooled anesthetic groups . a total of 117 patients were enrolled and received ngx- 4010 treatment : 39 were pretreated with l.m.x.4 , 38 with topicaine , and 40 with betacaine ( figure 1 ) . fourteen patients ( 12% ) terminated the study early ; the number of early- terminating patients was similar among the three groups . six patients terminated early because of unsatisfactory therapeutic response , two from each topical anesthetic group . the average age of patients enrolled in the studies ranged from 58 to 63 years ( table 1 ) . the average duration of pain ranged from 3.8 to 5.3 years , and baseline average pain scores ranged from 5.4 to 5.9 . slightly more than half of the patients were receiving concomitant neuropathic pain treatment consisting of anticonvulsants , non - ssri antidepressants , or opioids at baseline . ngx-4010 was well tolerated regardless of the pretreatment used . the proportion of patients completing at least 90% of the planned ngx-4010 patch application duration was 100% for betacaine , 97% for l.m.x.4 , and 97% for topicaine . one patient pretreated with l.m.x.4 had ngx- 4010 inadvertently removed after 55 minutes instead of the intended 90 minutes , and one patient pretreated with topicaine had ngx-4010 removed after 30 minutes instead of the intended 60 minutes , due to intolerability . the proportion of patients with at least one ae ranged from 50% to 59% ( table 2 ) . aes were primarily capsaicin- related application - site events , which were reported by 30%37% of patients . common application - site events included application - site burning and application - site pain . application - site events were transient , resolved within 1 or 2 days , and were mostly mild or moderate . the proportion of patients with severe application - site events was slightly greater in the topicaine group ( 7 out of 38 , 18% ) compared with the l.m.x.4 ( 4 out of 39 , 10% ) or betacaine ( 5 out of 40 , 13% ) groups and mostly consisted of application - site burning ( data not shown ) . no serious ae was considered related to treatment , and no patients died during the study . on the day of treatment , mean pain now nprs scores decreased following topical anesthetic application and increased following patch application but on average did not or only slightly exceeded pre - anesthetic treatment values ( table 3 ) . no significant differences in the proportion of patients reporting a 33% pain increase from baseline during the first 48 hours were observed between the l.m.x.4 and topicaine or betacaine groups . the proportion of patients using oral analgesics , including opioids , for treatment - related discomfort on days 0 through 5 was slightly greater in the topicaine group ( 66% ) compared with the l.m.x.4 ( 51% ) or betacaine ( 50% ) groups , but the differences were not statistically significant . the majority of patients had a maximum dermal assessment score of 2 or less , indicative of minor dermal irritation ( table 4 ) . maximum scores were typically recorded within 2 hours after patch removal ; no significant differences were observed between the l.m.x.4 and topicaine or betacaine groups . regardless of the pretreatment used , there were no clinically relevant changes in vital signs or any laboratory parameters evaluated across treatment groups . small , transient blood pressure changes were observed during and shortly after patch application and appeared to be associated with treatment - related changes in pain . patients reported a mean 27.2%34.3% reduction in pain during weeks 2 through 12 ( table 5 ) , and 45%50% of patients were considered to have responded to treatment ( ie , experienced a 30% mean decrease from baseline in pain ) . the proportion of patients who achieved a 50% decrease in pain scores from baseline to weeks 2 through 12 ranged from 28% to 37% . there were no significant differences in pain reduction between the l.m.x.4 and topicaine or betacaine groups , and no trends were observed in nprs scores between the 60- and 90-minute treatment groups ( data not shown ) . analysis of pgic demonstrated that at week 12 , 58%71% of patients considered themselves to have improved ( slightly , much , or very much ) and 35%42% of patients reported being much or very much improved ( table 5 ) . a total of 117 patients were enrolled and received ngx- 4010 treatment : 39 were pretreated with l.m.x.4 , 38 with topicaine , and 40 with betacaine ( figure 1 ) . fourteen patients ( 12% ) terminated the study early ; the number of early- terminating patients was similar among the three groups . six patients terminated early because of unsatisfactory therapeutic response , two from each topical anesthetic group . the average age of patients enrolled in the studies ranged from 58 to 63 years ( table 1 ) . the average duration of pain ranged from 3.8 to 5.3 years , and baseline average pain scores ranged from 5.4 to 5.9 . slightly more than half of the patients were receiving concomitant neuropathic pain treatment consisting of anticonvulsants , non - ssri antidepressants , or opioids at baseline . ngx-4010 was well tolerated regardless of the pretreatment used . the proportion of patients completing at least 90% of the planned ngx-4010 patch application duration was 100% for betacaine , 97% for l.m.x.4 , and 97% for topicaine . one patient pretreated with l.m.x.4 had ngx- 4010 inadvertently removed after 55 minutes instead of the intended 90 minutes , and one patient pretreated with topicaine had ngx-4010 removed after 30 minutes instead of the intended 60 minutes , due to intolerability . the proportion of patients with at least one ae ranged from 50% to 59% ( table 2 ) . aes were primarily capsaicin- related application - site events , which were reported by 30%37% of patients . common application - site events included application - site burning and application - site pain . application - site events were transient , resolved within 1 or 2 days , and were mostly mild or moderate . the proportion of patients with severe application - site events was slightly greater in the topicaine group ( 7 out of 38 , 18% ) compared with the l.m.x.4 ( 4 out of 39 , 10% ) or betacaine ( 5 out of 40 , 13% ) groups and mostly consisted of application - site burning ( data not shown ) . no serious ae was considered related to treatment , and no patients died during the study . on the day of treatment , mean pain now nprs scores decreased following topical anesthetic application and increased following patch application but on average did not or only slightly exceeded pre - anesthetic treatment values ( table 3 ) . no significant differences in the proportion of patients reporting a 33% pain increase from baseline during the first 48 hours were observed between the l.m.x.4 and topicaine or betacaine groups . the proportion of patients using oral analgesics , including opioids , for treatment - related discomfort on days 0 through 5 was slightly greater in the topicaine group ( 66% ) compared with the l.m.x.4 ( 51% ) or betacaine ( 50% ) groups , but the differences were not statistically significant . the majority of patients had a maximum dermal assessment score of 2 or less , indicative of minor dermal irritation ( table 4 ) . maximum scores were typically recorded within 2 hours after patch removal ; no significant differences were observed between the l.m.x.4 and topicaine or betacaine groups . regardless of the pretreatment used , there were no clinically relevant changes in vital signs or any laboratory parameters evaluated across treatment groups . small , transient blood pressure changes were observed during and shortly after patch application and appeared to be associated with treatment - related changes in pain . patients reported a mean 27.2%34.3% reduction in pain during weeks 2 through 12 ( table 5 ) , and 45%50% of patients were considered to have responded to treatment ( ie , experienced a 30% mean decrease from baseline in pain ) . the proportion of patients who achieved a 50% decrease in pain scores from baseline to weeks 2 through 12 ranged from 28% to 37% . there were no significant differences in pain reduction between the l.m.x.4 and topicaine or betacaine groups , and no trends were observed in nprs scores between the 60- and 90-minute treatment groups ( data not shown ) . analysis of pgic demonstrated that at week 12 , 58%71% of patients considered themselves to have improved ( slightly , much , or very much ) and 35%42% of patients reported being much or very much improved ( table 5 ) . treatment with ngx-4010 in conjunction with any of the three topical anesthetics tested was generally well tolerated with a good safety profile . nearly all patients completed at least 90% of the planned ngx-4010 application duration , regardless of the topical anesthetic product applied . as expected , capsaicin - related local application - site reactions were the most common aes and were transient , mostly mild to moderate , and self - limited . application - site events were adequately managed by local cooling or , if needed , by short - acting oral opioid analgesics ( the latter were used for treatment - related discomfort by approximately half of all patients ) . on the day of treatment , the majority of patients had minor dermal irritation irrespective of the topical anesthetic used . in general , ngx-4010 treatment for 60 minutes was better tolerated than treatment for 90 minutes . in each anesthetic group , more patients treated with ngx-4010 for 90 minutes used medication for treatment - related discomfort than those treated for 60 minutes ; patients treated for 90 minutes generally reported larger pain increases during the treatment procedure compared with those treated for 60 minutes ; and maximum dermal assessment scores were generally lower in patients treated for 60 minutes compared with patients treated for 90 minutes ( data not shown ) . the incidence and severity of application - site events and the proportion of patients using oral analgesics for treatment - related discomfort were slightly greater following pretreatment with topicaine than following pretreatment with l.m.x.4 or betacaine . however , differences in medication use were not statistically significant and likely related to the relatively small sample size . since the systemic absorption of capsaicin after application of ngx-4010 is minimal,24 the lack of effect of ngx-4010 on any laboratory parameter evaluated was expected . the analyses of nprs scores and pgic indicate that the selection of topical anesthetic had no influence over the pain relief obtained with ngx-4010 . indeed ngx-4010 resulted in prolonged pain relief for up to 12 weeks following a single application of ngx-4010 in all groups ; other efficacy endpoints also showed similar relief of pain across the different pretreatment groups . mean nprs scores were reduced from baseline by approximately 30% in all groups , which is similar to the degree of pain relief reported previously in phase iii clinical trials of ngx-4010 in patients with phn that utilized a 60-minute pretreatment with l.m.x.4.15,18 limitations of the study included the lack of a control group . in addition , the sample size was insufficient to detect small differences between the topical lidocaine formulations in terms of tolerability and efficacy of ngx-4010 . however , the study was sufficiently powered to detect a difference of 20% in the proportion of subjects completing at least 90% of intended duration between the l.m.x.4-treated group and each of the other topical anesthetic groups . as only a single patient was not able to tolerate the full duration of ngx-4010 application , and changes in mean nprs scores were within the range reported in previous clinical trials in patients with phn,15,18 the results from this study seem to indicate that when used as a pretreatment for ngx-4010 , all of the topical formulations tested are similarly effective and do not impact on the efficacy of ngx-4010 . in conclusion , treatment with ngx-4010 in conjunction with any of the three topical anesthetics tested was generally safe and well tolerated . relief of peripheral neuropathic pain from a single application of ngx-4010 was similar in the three topical anesthetic groups and comparable to the level of pain relief reported in previous phase iii clinical trials of ngx-4010 in patients with phn.15,18 as few differences were seen between the three topical anesthetics tested and each topical anesthetic appeared to be a suitable pretreatment for ngx-4010 , the results of this study suggest that clinicians can select a topical anesthetic formulation for pretreatment according to local clinical practice , product availability , and cost .

backgroundthe objective of this study was to assess the safety , tolerability , and preliminary efficacy of ngx-4010 , a capsaicin 8% patch , following pretreatment with three different topical anesthetics in patients with peripheral neuropathic pain.methodsthis open - label , multicenter study enrolled 117 patients with post - herpetic neuralgia , hiv - associated distal sensory polyneuropathy , or painful diabetic neuropathy . patients received pretreatment with one of three lidocaine 4%-based topical anesthetics ( l.m.x.4 [ ferndale laboratories inc , ferndale , mi ] , topicaine gel [ estela basso , jupiter , fl ] , or betacaine enhanced gel 4 [ tiberius inc , tampa , fl ] ) for 60 minutes followed by a single 60- or 90-minute ngx-4010 application , and were followed for 12 weeks . tolerability and safety measures included pain now numeric pain rating scale ( nprs ) scores , dermal assessments , medication use for treatment - related pain , adverse events ( aes ) , clinical laboratory parameters , physical examinations , and vital signs . the primary efficacy variable was the percentage change in mean nprs scores for average pain for the past 24 hours from baseline to weeks 2 through 12.resultstreatment with ngx-4010 following pretreatment with any of the three topical anesthetics was generally safe and well tolerated . nearly all patients completed 90% of the planned ngx-4010 application duration . the most common treatment - related aes , application - site burning and application - site pain , were transient , mostly mild or moderate , and could be adequately managed by local cooling or short - acting oral opioid analgesics . although slightly more patients used medication for treatment - related discomfort following pretreatment with topicaine compared with l.m.x.4 or betacaine , there were no statistical differences between the topical anesthetics . neuropathic pain reduction from baseline to weeks 2 through 12 was approximately 30% and was similar among the topical anesthetics ; the proportion of responders ranged from 45% to 50%.conclusiontreatment with ngx-4010 following pretreatment with any of the three topical anesthetics was generally safe and well tolerated ; no significant differences in the parameters measured were noted between the pretreatment groups .

Background Methods Patients Procedures Safety Efficacy Results Patients Safety Efficacy Discussion

this results in defunctionalization of trpv1-expressing sensory nerve endings and reduced epidermal nerve fiber density.1114 prolonged relief of neuropathic pain for up to 12 weeks following a single ngx-4010 application has been observed in patients with post - herpetic neuralgia ( phn ) or painful hiv - associated distal sensory polyneuropathy ( hiv - dsp ) in phase ii and iii studies.1520 due to the irritancy of capsaicin , topical application is associated with pain , erythema , and other application - site reactions.1520 because of this , a topical anesthetic is applied to reduce application - site discomfort prior to administration of ngx-4010 . in the above clinical studies , a 4% lidocaine topical anesthetic cream ( l.m.x.4/ela - max4 ; ferndale laboratories inc , ferndale , mi ) was applied for 60 minutes and removed prior to ngx-4010 application . the current open - label study sought to determine whether similar tolerability could be achieved with other commonly available 4% lidocaine formulations and assessed the safety , tolerability , and efficacy of ngx-4010 following pretreatment with either l.m.x.4 or the alternative products ( topicaine gel [ esba laboratories inc , jupiter , fl ] or betacaine enhanced gel 4 [ theraderm inc , tampa , fl ] ) . the study was approved by a central institutional review board ( biomed irb , san diego , ca ) or a local institutional review board at participating sites and conducted in accordance with the ethical principles of the declaration of helsinki , good clinical practice guidelines , and applicable regulatory requirements . patients were at least 18 years old with moderate to severe neuropathic pain ( average numeric pain rating scale [ nprs ] score of 38 inclusive ) for at least 3 months secondary to painful diabetic neuropathy ( pdn ) , phn , or hiv - dsp . exclusion criteria included the following : use of any topically applied pain medication on the painful area within 21 days before study patch application ; history of diabetic foot ulcerations and/or status post - amputation ; any implanted medical device for the treatment of neuropathic pain ; significant ongoing or recurrent pain of another etiology that interfered with judging neuropathic pain ; evidence of another contributing or immunologic cause for the sensory neuropathy ; and neuropathic pain areas located only on the face , above the scalp hairline , or near mucous membranes . patients were randomized to receive pretreatment for 60 minutes with one of three lidocaine 4%-based topical anesthetics : l.m.x.4 , topicaine gel , or betacaine enhanced gel 4 followed by a 60- or 90-minute treatment with ngx-4010 ( qutenza ; neurogesx inc , san mateo , ca ) . safety was assessed by adverse events ( aes ) , which were coded using the medical dictionary for regulatory activities , version 7.0 . the distribution of maximum score between the pooled l.m.x.4 group and the other two pooled anesthetic groups was compared using the cochran mantel nprs scores were recorded immediately prior to anesthetic application ; 30 and 55 minutes after anesthetic application ; 25 , 55 , and 85 ( if applicable ) minutes after patch application ; and 5 , 25 , 55 , 85 , and 115 minutes after patch removal . the change in nprs scores from the pre - anesthetic time point , change in vital signs ( systolic blood pressure , diastolic blood pressure , heart rate , and respiratory rate ) from the pre - patch time point , the proportion of patients with a 33% increase in nprs score from baseline during the first 48 hours after ngx-4010 treatment , and the proportion of patients with < 90% of intended patch application duration were summarized . a cochran mantel haenszel test adjusted for treatment duration ( 60 or 90 minutes ) was used to test for a difference in the proportion of patients with a 33% increase in nprs score from baseline during the first 48 hours between the pooled l.m.x.4 group and the other two pooled anesthetic groups . a chi - squared test was performed to test for a difference in the proportion of patients using medication for treatment - related discomfort between the pooled l.m.x.4 group and the other two pooled anesthetic groups . the sample size was determined based on a chi - squared continuity corrected test to detect a difference of 20% in the proportion of subjects completing at least 90% of intended duration between the l.m.x.4-treated group and each of the other topical anesthetic groups at the 0.05 significance level with 80% power . efficacy was evaluated using nprs scores for average pain for the past 24 hours patient global impression of change ( pgic ; patients reported how they felt after treatment as compared with before treatment on a scale of 3 , indicating very much worse , to + 3 , indicating very much improved , with 0 being no change ) and the investigator - rated clinical global impression of change ( cgic)23 were evaluated at weeks 2 , 6 , and 12 . the primary efficacy endpoint was the percentage change in average pain for the past 24 hours nprs scores from baseline to weeks 2 through 12 . other efficacy measures included : mean absolute changes in nprs scores and the proportion of patients with a 30% or 50% reduction in nprs score from baseline to weeks 2 through 12 ; and the percentage of patients considered improved ( slightly , much , or very much ) on the pgic or cgic at week 12 . an analysis of covariance model with baseline pain score as the covariate was used to test for differences in change from baseline to weeks 2 through 12 between the pooled l.m.x.4 group and each of the other two pooled anesthetic groups . a logistic regression model , with the baseline nprs score as covariate , was performed to test for a difference in the proportion of patients reaching 30% and 50% decreases from baseline between the pooled l.m.x.4 group and each of the other two pooled anesthetic groups . the study was approved by a central institutional review board ( biomed irb , san diego , ca ) or a local institutional review board at participating sites and conducted in accordance with the ethical principles of the declaration of helsinki , good clinical practice guidelines , and applicable regulatory requirements . patients were at least 18 years old with moderate to severe neuropathic pain ( average numeric pain rating scale [ nprs ] score of 38 inclusive ) for at least 3 months secondary to painful diabetic neuropathy ( pdn ) , phn , or hiv - dsp . patients were randomized to receive pretreatment for 60 minutes with one of three lidocaine 4%-based topical anesthetics : l.m.x.4 , topicaine gel , or betacaine enhanced gel 4 followed by a 60- or 90-minute treatment with ngx-4010 ( qutenza ; neurogesx inc , san mateo , ca ) . in addition , patients could take hydrocodone bitartrate/ acetaminophen ( 5 mg/500 mg ) for up to 5 days after patch application for treatment - associated discomfort as needed . safety was assessed by adverse events ( aes ) , which were coded using the medical dictionary for regulatory activities , version 7.0 . treatment - associated erythema , discomfort , and pain on the day of treatment were not captured as aes but reported as dermal assessment scores or nprs scores . the distribution of maximum score between the pooled l.m.x.4 group and the other two pooled anesthetic groups was compared using the cochran mantel nprs scores were recorded immediately prior to anesthetic application ; 30 and 55 minutes after anesthetic application ; 25 , 55 , and 85 ( if applicable ) minutes after patch application ; and 5 , 25 , 55 , 85 , and 115 minutes after patch removal . the change in nprs scores from the pre - anesthetic time point , change in vital signs ( systolic blood pressure , diastolic blood pressure , heart rate , and respiratory rate ) from the pre - patch time point , the proportion of patients with a 33% increase in nprs score from baseline during the first 48 hours after ngx-4010 treatment , and the proportion of patients with < 90% of intended patch application duration were summarized . haenszel test adjusted for treatment duration ( 60 or 90 minutes ) was used to test for a difference in the proportion of patients with a 33% increase in nprs score from baseline during the first 48 hours between the pooled l.m.x.4 group and the other two pooled anesthetic groups . a chi - squared test was performed to test for a difference in the proportion of patients using medication for treatment - related discomfort between the pooled l.m.x.4 group and the other two pooled anesthetic groups . the sample size was determined based on a chi - squared continuity corrected test to detect a difference of 20% in the proportion of subjects completing at least 90% of intended duration between the l.m.x.4-treated group and each of the other topical anesthetic groups at the 0.05 significance level with 80% power . efficacy was evaluated using nprs scores for average pain for the past 24 hours recorded daily at 9 pm in a paper diary throughout the study period . the primary efficacy endpoint was the percentage change in average pain for the past 24 hours nprs scores from baseline to weeks 2 through 12 . other efficacy measures included : mean absolute changes in nprs scores and the proportion of patients with a 30% or 50% reduction in nprs score from baseline to weeks 2 through 12 ; and the percentage of patients considered improved ( slightly , much , or very much ) on the pgic or cgic at week 12 . an analysis of covariance model with baseline pain score as the covariate was used to test for differences in change from baseline to weeks 2 through 12 between the pooled l.m.x.4 group and each of the other two pooled anesthetic groups . a logistic regression model , with the baseline nprs score as covariate , was performed to test for a difference in the proportion of patients reaching 30% and 50% decreases from baseline between the pooled l.m.x.4 group and each of the other two pooled anesthetic groups . a total of 117 patients were enrolled and received ngx- 4010 treatment : 39 were pretreated with l.m.x.4 , 38 with topicaine , and 40 with betacaine ( figure 1 ) . fourteen patients ( 12% ) terminated the study early ; the number of early- terminating patients was similar among the three groups . the proportion of patients completing at least 90% of the planned ngx-4010 patch application duration was 100% for betacaine , 97% for l.m.x.4 , and 97% for topicaine . one patient pretreated with l.m.x.4 had ngx- 4010 inadvertently removed after 55 minutes instead of the intended 90 minutes , and one patient pretreated with topicaine had ngx-4010 removed after 30 minutes instead of the intended 60 minutes , due to intolerability . common application - site events included application - site burning and application - site pain . application - site events were transient , resolved within 1 or 2 days , and were mostly mild or moderate . the proportion of patients with severe application - site events was slightly greater in the topicaine group ( 7 out of 38 , 18% ) compared with the l.m.x.4 ( 4 out of 39 , 10% ) or betacaine ( 5 out of 40 , 13% ) groups and mostly consisted of application - site burning ( data not shown ) . no significant differences in the proportion of patients reporting a 33% pain increase from baseline during the first 48 hours were observed between the l.m.x.4 and topicaine or betacaine groups . the proportion of patients using oral analgesics , including opioids , for treatment - related discomfort on days 0 through 5 was slightly greater in the topicaine group ( 66% ) compared with the l.m.x.4 ( 51% ) or betacaine ( 50% ) groups , but the differences were not statistically significant . maximum scores were typically recorded within 2 hours after patch removal ; no significant differences were observed between the l.m.x.4 and topicaine or betacaine groups . regardless of the pretreatment used , there were no clinically relevant changes in vital signs or any laboratory parameters evaluated across treatment groups . patients reported a mean 27.2%34.3% reduction in pain during weeks 2 through 12 ( table 5 ) , and 45%50% of patients were considered to have responded to treatment ( ie , experienced a 30% mean decrease from baseline in pain ) . the proportion of patients who achieved a 50% decrease in pain scores from baseline to weeks 2 through 12 ranged from 28% to 37% . there were no significant differences in pain reduction between the l.m.x.4 and topicaine or betacaine groups , and no trends were observed in nprs scores between the 60- and 90-minute treatment groups ( data not shown ) . a total of 117 patients were enrolled and received ngx- 4010 treatment : 39 were pretreated with l.m.x.4 , 38 with topicaine , and 40 with betacaine ( figure 1 ) . fourteen patients ( 12% ) terminated the study early ; the number of early- terminating patients was similar among the three groups . slightly more than half of the patients were receiving concomitant neuropathic pain treatment consisting of anticonvulsants , non - ssri antidepressants , or opioids at baseline . the proportion of patients completing at least 90% of the planned ngx-4010 patch application duration was 100% for betacaine , 97% for l.m.x.4 , and 97% for topicaine . one patient pretreated with l.m.x.4 had ngx- 4010 inadvertently removed after 55 minutes instead of the intended 90 minutes , and one patient pretreated with topicaine had ngx-4010 removed after 30 minutes instead of the intended 60 minutes , due to intolerability . common application - site events included application - site burning and application - site pain . application - site events were transient , resolved within 1 or 2 days , and were mostly mild or moderate . the proportion of patients with severe application - site events was slightly greater in the topicaine group ( 7 out of 38 , 18% ) compared with the l.m.x.4 ( 4 out of 39 , 10% ) or betacaine ( 5 out of 40 , 13% ) groups and mostly consisted of application - site burning ( data not shown ) . no significant differences in the proportion of patients reporting a 33% pain increase from baseline during the first 48 hours were observed between the l.m.x.4 and topicaine or betacaine groups . the proportion of patients using oral analgesics , including opioids , for treatment - related discomfort on days 0 through 5 was slightly greater in the topicaine group ( 66% ) compared with the l.m.x.4 ( 51% ) or betacaine ( 50% ) groups , but the differences were not statistically significant . maximum scores were typically recorded within 2 hours after patch removal ; no significant differences were observed between the l.m.x.4 and topicaine or betacaine groups . regardless of the pretreatment used , there were no clinically relevant changes in vital signs or any laboratory parameters evaluated across treatment groups . patients reported a mean 27.2%34.3% reduction in pain during weeks 2 through 12 ( table 5 ) , and 45%50% of patients were considered to have responded to treatment ( ie , experienced a 30% mean decrease from baseline in pain ) . the proportion of patients who achieved a 50% decrease in pain scores from baseline to weeks 2 through 12 ranged from 28% to 37% . there were no significant differences in pain reduction between the l.m.x.4 and topicaine or betacaine groups , and no trends were observed in nprs scores between the 60- and 90-minute treatment groups ( data not shown ) . treatment with ngx-4010 in conjunction with any of the three topical anesthetics tested was generally well tolerated with a good safety profile . nearly all patients completed at least 90% of the planned ngx-4010 application duration , regardless of the topical anesthetic product applied . as expected , capsaicin - related local application - site reactions were the most common aes and were transient , mostly mild to moderate , and self - limited . application - site events were adequately managed by local cooling or , if needed , by short - acting oral opioid analgesics ( the latter were used for treatment - related discomfort by approximately half of all patients ) . in each anesthetic group , more patients treated with ngx-4010 for 90 minutes used medication for treatment - related discomfort than those treated for 60 minutes ; patients treated for 90 minutes generally reported larger pain increases during the treatment procedure compared with those treated for 60 minutes ; and maximum dermal assessment scores were generally lower in patients treated for 60 minutes compared with patients treated for 90 minutes ( data not shown ) . the incidence and severity of application - site events and the proportion of patients using oral analgesics for treatment - related discomfort were slightly greater following pretreatment with topicaine than following pretreatment with l.m.x.4 or betacaine . indeed ngx-4010 resulted in prolonged pain relief for up to 12 weeks following a single application of ngx-4010 in all groups ; other efficacy endpoints also showed similar relief of pain across the different pretreatment groups . mean nprs scores were reduced from baseline by approximately 30% in all groups , which is similar to the degree of pain relief reported previously in phase iii clinical trials of ngx-4010 in patients with phn that utilized a 60-minute pretreatment with l.m.x.4.15,18 limitations of the study included the lack of a control group . in addition , the sample size was insufficient to detect small differences between the topical lidocaine formulations in terms of tolerability and efficacy of ngx-4010 . however , the study was sufficiently powered to detect a difference of 20% in the proportion of subjects completing at least 90% of intended duration between the l.m.x.4-treated group and each of the other topical anesthetic groups . as only a single patient was not able to tolerate the full duration of ngx-4010 application , and changes in mean nprs scores were within the range reported in previous clinical trials in patients with phn,15,18 the results from this study seem to indicate that when used as a pretreatment for ngx-4010 , all of the topical formulations tested are similarly effective and do not impact on the efficacy of ngx-4010 . in conclusion , treatment with ngx-4010 in conjunction with any of the three topical anesthetics tested was generally safe and well tolerated . relief of peripheral neuropathic pain from a single application of ngx-4010 was similar in the three topical anesthetic groups and comparable to the level of pain relief reported in previous phase iii clinical trials of ngx-4010 in patients with phn.15,18 as few differences were seen between the three topical anesthetics tested and each topical anesthetic appeared to be a suitable pretreatment for ngx-4010 , the results of this study suggest that clinicians can select a topical anesthetic formulation for pretreatment according to local clinical practice , product availability , and cost .

in north america , no other solid tumor is as common or causes more deaths in children and adolescents than cancers of the brain [ 16 , 26 , 84 ] , with roughly three quarters of patients presenting at less than 15 years of age . the prognosis for long - term survival is much better in children than in adults , with up to half of pediatric brain tumor patients surviving long term . the main reason for this enhanced survival is that children and adolescents tend to have lower - grade lesions [ 79 , 95 , 101 ] . long - term survival is not without problems , however , with many pediatric brain tumor survivors continuing to suffer from significant morbidity [ 44 , 63 , 76 , 90 , 94 , 108 ] and , sometimes , early death . among the more common long - term sequelae of brain tumors and their treatment in children are seizures , which can be quite disabling and , at times , life - threatening in themselves [ 90 , 3 , 56 , 66 , 67 , 85 , 87 , 104 , 106 , 110 ] . in one study , seizures were the dominant predictor of disability in long - term brain tumor survivors [ 66 , 67 ] . seizures even increase a pediatric brain tumor survivor s risk of suicide into adulthood . of the epileptogenic tumors , low - grade lesions comprise the majority . of these , dysembryoplastic neuroepithelial tumors ( dnets ) present with seizures almost 100 % of the time [ 1 , 65 , 73 , 97 , 104 ] . because these tumors typically are slow - growing and non - invasive , the primary objective of dnet management in most cases is control , if not eradication , of seizures . because tumor - associated seizures tend to be more resistant to anti - epileptic drugs ( aeds ) than idiopathic seizures [ 57 , 98 ] and the long - term use of aeds is not without significant risks in itself [ 22 , 23 , 56 , 66 , 68 , 100 , 107 ] , including wide - ranging adverse effects on cognitive function and development [ 56 , 100 ] , this typically necessitates surgery to resect as much of the tumor as possible [ 21 , 70 , 73 , 78 , 89 , 97 , 104 ] . in addition , several studies have shown that radical removal of an epileptogenic brain tumor is a strong , and likely the strongest , predictor of seizure freedom . on the other hand , especially among younger children , the risks of surgery can not be discounted as , in addition to the risk of perioperative mortality , surgery also can adversely affect neurological development and function . the primary purpose of this paper is to extensively and systematically review and analyze the literature on surgical outcomes in pediatric patients with dnets , specifically looking at perioperative complications and mortality , short- and long - term seizure control , and short- and long - term survival . the main search objective was to identify all papers involving pediatric patients with dnets undergoing surgical resection over the past 20 years ( 19942014 ) , in which the following information was available either for the entire sample or for individual patients ( for papers in which patients with a range of tumor types are represented ) : number of patients with a dnet , ( mean ) age at the time of seizure onset , ( mean ) age at the time of surgery , age range , type of seizure , type of surgery , location of the lesion , number of subjects in which complete resection was achieved , number with immediate postoperative complications including recurrent / persistent seizures , ( mean ) length of follow - up , long - term survival , long - term neurological sequelae , the number with seizures at final follow - up , and final engel rating . the period 1994 to 2014 was selected because it was felt that older papers reported the results of studies with considerably less robust methodology and because most papers prior to 1995 did not report many of the specific variables of interest listed above . all the included papers were identified via an extensive search of the pubmed database , using the following search terms : dysembryoplastic neuroepithelial tumor ( n = 425 ) , dnet ( n = 193 ) , neuroglial tumor ( n = 401 ) , neuroglial tumor and seizure ( n = 1574 ) , and neuroglial tumor and surgery ( n = 871 ) . of these terms , only those with n 2000 were reviewed , because of the lack of specificity of longer lists . a total of 33 papers were identified in which surgery was conducted on children with dnets , and the above - noted data were available in 24 [ 5 , 9 , 10 , 15 , 17 , 21 , 31 , 35 , 36 , 47 , 48 , 51 , 52 , 54 , 55 , 58 , 59 , 70 , 73 , 75 , 80 , 89 , 97 , 105 ] . however , of these , only seven studies had pediatric dnet patients exclusively [ 9 , 30 , 58 , 70 , 73 , 89 , 97 ] , two had both adult and pediatric patients , but individualized data [ 17 , 59 ] , and an additional four papers [ 5 , 15 , 47 , 51 ] had individual data on dnet patients among patients with other lesions that allowed for the extraction of almost all of the variables of interest . analysis consisted of calculating means and percentages , pearson correlation coefficients to identify the strength of correlation between study means of continuous variables , and likelihood ratios and pearson analyses to examine categorical variables . where indicated , a p value of 0.05 or less was considered indicative of a statistically significant intergroup difference or correlation . correlation strength was categorized as weak ( r < 0.40 ) , moderate ( 0.400.69 ) , or strong ( r 0.70 ) as indicated in the review of taylor . table 1 lists the 13 studies identified in which data of interest were available for patients with dysembryoplastic neuroepithelial tumors ( dnets ) , including six papers specific to pediatric dnet tumors for which totals and means are presented [ 9 , 58 , 70 , 73 , 89 , 97 ] and seven papers with data presented for individual dnet patients from which totals and means could be calculated [ 5 , 15 , 17 , 30 , 47 , 51 , 58 ] . these 13 studies encompass 185 patients , of mean age 9.4 years , with individual patients ranging in age from 0.5 to 21 years . all the series were small , one paper reporting a single case and only four having 20 or more patients [ 9 , 58 , 70 , 74 ] . the mean duration of seizures prior to surgical resection of the underlying dnet across the 13 studies was 3.2 years but ranged widely from 4 and 7 months [ 15 , 47 ] to 7 years [ 70 , 97 ] . complex partial seizures accounted for 86.4 % of seizures , ranging from 55.6 % to 100 % [ 5 , 30 , 58 ] . the mean percentage of dnets located within a temporal lobe across the 13 studies was 67.8 % , though this ranged quite broadly from 38.5 % and 42.9 % to 100 % . considering subjects individually , rather than assessing study means , the overall weighted percentage of patients presenting with complex partial seizures was 81.3 % and the percentage with a temporal lobe lesion 63.8 % . gross total resection was achieved in 83.3 % of patients , the percentage lowered by a single study in which gross total resection was achieved in only 11 of 26 . table 1seizure response to surgical resection of dysembryoplastic neuroectodermal tumorsfirst authoryear publishedno . of subjectsmean age ( year)age rangesurgical procedureno . of total resection% total resectionmean fu ( month)no . of seizure free% seizure freeno . of improved% improvedno . of perioperative deathsno . of surgical complicationsbabini2013412518 yearsl??102375.0 % 4100.0 % 00jo201310.50.5 yearsl1100.0 % 981100.0 % 1100.0 % 00spalice2010136.7114 yearsl + e1292.3 % 7813100.0 % 13100.0 % 01bilginer20092910.7321 yearstl ahc??522793.1 % 29100.0 % 0?lee20092212.4318 yearsl l22100.0 % 442090.9 % 22100.0 % 01minkin2008248.9115 yearsl2187.5 % 802083.3 % 24100.0 % 09chan2006311.0814 yearsl , l + e3100.0 % 104266.7 % 3100.0 % 01sandberg2005189.61 month13 yearsl map18100.0 % 1918100.0 % 18100.0 % 01cataltepe20051411.0418 yearsl , l + e1285.7 % 331285.7 % 14100.0 % 0?nolan20042610.0418 years?1142.3 % 521661.5 % 2596.2 % 0?fernandez20031410.1318 yearsl , l + e14100.0 % 87.11285.7 % 1392.9 % 0?lee2000109.3215 yearsl , l + e10100.0 % 40.310100.0 % 10100.0 % 02khajavi1999710.1419 yearsl , l + l685.7 % 33571.4 % 7100.0 % 0?1859.40.521 years13083.3 % 63.315985.9 % 18398.9 % 01311.9 % seizure response to surgical resection of dysembryoplastic neuroectodermal tumors in three of the studies , lesionectomies alone were utilized to resect the lesion , with data on the number of total resections available , accounting for 32 patients . of this number of procedures , 27 yielded a total gross resection ( 84.4 % ) versus 64 of 67 patients in whom lesionectomy was combined with further resection ( 95.5 % ) ; hence , the odds of an incomplete resection ( or ) was 3.49 ( 95 % confidence interval = 0.78 , 15.51 , p = 0.10 ) . over the 13 studies , long - term postoperative freedom from seizures was achieved in 85.9 % of patients , at a mean follow - up of 63.3 months ( 5.25 years ) . the percentage of patients achieving seizure freedom in the 12 years from 1994 through 2006 was 81.6 versus 91.3 % in studies published from 2007 onward ; however , this difference failed to achieve statistical significance ( t = 1.20 , df = 11 , p = 0.26 ) . although a moderate direct correlation was noted between the year of paper publication and the percentage seizure freedom , this also failed to achieve statistical significance ( r = 0.45 , p = 0.23 ) . on the other hand , the percentage of patients achieving seizure freedom in a given study was both moderately and statistically correlated with the percentage of procedures resulting in gross total resection ( r = 0.63 , p = 0.03 ) , but not with mean patient age ( r = -0.45 , p = 0.12 ) or mean duration of follow - up ( r = 0.20 , p = 0.51 ) . no correlation at all was noted between the percentage of subjects achieving seizure freedom and any of the three variables mean duration of seizures prior to surgery ( r = 0.007 , p = 0.98 ) , percentage of patients with complex partial seizures ( r = 0.063 , p = 0.87 ) , or percentage of patients with a temporal lobe lesion ( r = 0.052 , p = 0.087 ) . in the seven studies in which sufficient individual subject data were available , there were 68 gross total resections , resulting in long - term seizure freedom in 62 patients ( 91.2 % ) , and 3 subtotal resections , all having engel stage iii outcomes , so that the likelihood of a poor outcome was more than 13 times higher ( lr = 13.4 ; p < 0.001 ) with a subtotal resection . in those same seven studies , 45 of 75 patients underwent a lesionectomy alone and 30 a lesionectomy plus some additional resection , with or without mapping . of the 45 who underwent lesionectomy alone , 40 ( 88.9 % ) achieve total seizure freedom versus 25 or 30 ( 83.3 % ) among those in whom some additional resection was performed ( pearson = 0.48 , p = 0.49 ) . by age group , postoperative seizure freedom was achieved in 10 of 11 ( 90.9 % ) of children under age 6 , 31 of 35 ( 88.6 % ) children from age 6 up to , but not including 13 , and 24 of 29 ( 82.8 % ) of adolescents of 13 years or older , a seemingly downward trend that was not statistically significant ( = 0.67 , p = 0.71 ) . the mean duration of follow - up over these seven studies with individualized data was 40.3 months . comparing seizure - free rates between patients with a follow - up duration below and above that mean again revealed no significant difference ( 89.1 vs. 82.2 % , = 0.63 , p = 0.43 ) . similarly , neither duration of seizures ( = 2.19 , df = 3 , p = 0.54 ) nor temporal location of the tumor ( = 1.44 , df = 1 , p = 0.23 ) was associated with seizure freedom . since all but one of the patients with seizure - type data had suffered from complex partial seizures , no individualized data analysis for seizure type was performed . an improvement in seizures was documented in 183 of 185 patients across the ten studies ( 98.9 % ) . there were no immediate or late deaths over the course of reported follow - up , and the perioperative complication rate , adjusted for missing data , was just 11.9 % . postoperative tumor recurrence was reported in two patients , including one whose seizures recurred with tumor recurrence and then improved but did not fully abate following a second resection . dnets , which typically become manifest during childhood , adolescence , or young adulthood , represent only a small percentage of cns tumors in either youths or adults . however , these tumors , which typically occur in the temporal lobes , are almost always associated with seizures . together with gangliogliomas and focal cortical dysplasia ( fcd ) consequently , they comprise a disproportionate percentage of tumor - associated epilepsy cases , especially in children [ 1 , 50 , 92 , 93 , 97 , 104 , 109 ] . the reason for the almost ubiquitous presence of seizures with brain tumors like dnets and gangliogliomas versus much lower rates seen with others , like low - grade gliomas , is not entirely understood [ 87 , 88 , 91 , 107 ] , although several conjectures have been made , including differential alterations in regional metabolism and ph ; immunologic activity ; disordered neuronal function ; altered vascular supply and permeability ; the release of altered tumoral amino acids , proteins , and enzymes ; and abnormal protein transport and binding to receptors [ 1 , 14 , 87 , 91 , 93 , 107 , 114 ] . even genetic predispositions for tumor - related seizures have been postulated [ 8 , 107 ] . to date , all that can be said with confidence is that the cause of tumor - induced seizures is almost certainly multifactorial and beyond the mere physical size of the tumor itself . the risks of surgical resection of an epileptogenic but otherwise benign brain tumor , like a dnet , must be weighed against the possible consequences of managing seizures conservatively , due to their potential to inflict significant neurological and cognitive damage , despite favorable rates of survival [ 28 , 66 , 67 ] . in several studies , preoperative cognitive function in pediatric patients with glioneuronal tumors tended to be low average to average and associated with a variety of cognitive deficits , including problems with speech and memory , and delay to meet developmental milestones [ 24 , 29 , 32 , 34 , 82 ] . since dnets tend to be extremely resistant to aed therapy , pharmaceutical control of seizures typically is incomplete [ 9 , 17 , 19 , 58 , 73 , 80 ] . this places patients at risk for continued brain injury and worsening neurocognitive function , prompting many authors to argue for surgical resection of the lesions as soon as possible in most cases [ 10 , 11 , 19 , 34 , 37 , 47 , 51 , 99 , 113 , 115 ] . among the various specific arguments given for early surgical resection of glioneuronal tumors , including dnets , are the optimization of seizure control [ 21 , 75 , 89 , 105 , 113 , 115 , 116 ] ; the optimization of brain development [ 10 , 11 , 17 , 19 ] ; avoiding or at least minimizing the long - term risks of aeds [ 22 , 53 , 66 , 68 , 96 , 107 ] , especially in children and those who may require chemotherapy to control tumor growth ; and reducing the risk , albeit low , of later , catastrophic malignant change [ 2 , 20 , 41 , 61 , 71 , 72 , 81 ] . brain surgery is certainly not without its own potential consequences and risks , however , including the prospect of postoperative mortality and worsening seizures and of immediate postoperative neurological deficits and cognitive decline . in their retrospective review of 223 patients 19 years old and under who underwent a combined 229 surgical resections of non - epileptogenic brain tumors , hardesty et al . identified an incidence of new , postoperative seizures of 7.4 % . on the other hand , almost all were single events that resolved without the need for long - term anti - epileptic drugs ( aeds ) . supratentorial tumors , patient age less than 2 years , and the presence of significant postoperative hyponatremia were independent risk factors for new seizures . presumably , then , these same risk factors of young age and postoperative serum sodium imbalance would at least slightly predispose patients with epileptogenic tumors to having continued or worsened seizures postoperatively . however , worsened seizures postoperatively were reported uncommonly in our review of 13 published studies / series , and the vast majority was transient and controlled prior to hospital discharge . moreover , steinbok et al . , in their retrospective analysis of 116 pediatric patients under age 3 drawn from eight centers across canada ( mean age at first surgery 15.8 months ; range 135 months ) , identified only one surgical death . the most common surgical complications over 151 operations were infection ( 17 ) and aseptic meningitis ( 13 ) . moreover , more than 1 year postoperatively , 72 ( 67.3 % ) were seizure free and more than 90 % significantly improved . in addition , cognitive development improved in 55.3 % postoperatively . in the medical literature , the rate of seizure freedom in series with either adults alone or adults plus children has varied widely for dnets , from as low as 52.4 and 53.3 % [ 33 , 83 ] to as high as 90 and 100 % [ 89 , 58 ] . in the current review , we chose to look exclusively at dnets operated upon during the pediatric years , with one patient aged 21 and all others 19 or younger , to as young as 5 months of age . overall , in this age group , long - term freedom from seizures was achieved in almost 86 % ( 85.9 % ) and some improvement in 99 % ( all but two of 185 patients ) . moreover , there were no deaths , and the rate of postoperative complications , the vast majority transient neurological deficits , was only 12 % . the only variable either correlated with seizure freedom rate at a study mean level or associated with seizure freedom at an individual level was degree of tumor resection , with subtotal resections virtually always associated with either the persistence or recurrence of seizures . patient age was not associated , and neither was whether or not lesionectomy alone or lesionectomy plus some additional resection was performed . these results are congruent with those of a recently published retrospective analysis of 29 children undergoing resection of glioneuronal tumors , in which the rate of seizure freedom 12 months after surgery was 94 % in those in whom gross total resection was achieved versus just 54 % in those in which it was not ( p < 0.05 ) ; unfortunately , only 13 of the patients were determined to have a dnet , versus 16 with a ganglioglioma , limiting comparisons against our own results . with even more limitations , our results are also consistent with a larger series of 332 patients ( mean age 39.3 years , range 1695 ) with low - grade gliomas who underwent operative resections for a variety of tumors , in whom seizure control again was far more likely to be achieved after gross total resection than after subtotal resection or biopsy alone ( odds ratio 16 , 95 % confidence interval 2.2124 , p = 0.0064 ) . one further characteristic that hampers these afore - mentioned studies to some degree is the disproportionate number of complete to incomplete resections . the same is not true of the one series of 26 pediatric patients with dnets reported by nolan et al . , in which the distribution of gross complete to incomplete resections was fairly evenly split ( 12 complete , 14 incomplete resections ) ; in this series , all nine children who had no detectable tumor on postoperative imaging were seizure free at 12 months , with only one relapsing at final follow - up , versus an approximately 50 % rate of seizure freedom in the remainder ( p = 0.02 ) . given that completeness of tumor resection seems to be a determinant of seizure outcomes , the question arises : is it better to do more than just a simple lesionectomy , either via brain mapping to detect epileptogenic foci apart from the tumor itself , or more extensive resections ? over the years , attempts have repeatedly been made to optimize the resection of epileptogenic lesions , both by better delineating their margins and by enhancing the identification of extra - tumoral epileptogenic tissue , using intraoperative tools like electrocorticography ( ecog ) to identify potential seizure - inducing tissue irregularities like fcd [ 4 , 7 , 28 , 35 , 46 , 58 , 75 , 89 , 111 , 112 ] . this has led to considerable speculation with respect to the relative benefits and safety of performing epilepsy surgery rather than just lesionectomies in patients with tumor - induced seizures , even though surgeons have been utilizing additional surgical steps like lobectomy , amygdalohippocampectomy , and , in extreme cases , hemispherotomy for decades [ 5 , 9 , 10 , 15 , 35 , 45 , 48 , 51 , 55 , 75 , 77 , 80 , 89 , 97 , 103 , 105 ] . to date , almost no direct empirical comparisons have been undertaken . in perhaps the most methodologically sound study , retrospectively compared 34 patients who underwent ecog - aided epilepsy surgery and 33 patients who had undergone simple lesionectomy without ecog , all between the ages of 3 months and 16 years , in vancouver , canada . one year following surgery , roughly 80 % of patients in each group were seizure free . however , long - term data trended toward improved seizure freedom in patients in the ecog group , with 79 versus 61 % patients still seizure free at a mean 5.8 years of follow - up ( p = 0.08 ) . the investigators also noted no increase in neurological morbidity among patients who had undergone the more extensive ecog - guided cortical resection and that these patients were less likely to require repeat epilepsy surgery . in another smaller retrospective analysis reported by chan et al . , whereas 10 of 12 pediatric dnet patients undergoing a temporal lobectomy achieved seizure freedom , such freedom was achieved in only two of six who had a lesionectomy alone , a difference that , using pearson analysis , is statistically significant ( = 4.5 , p = 0.03 ) despite the small numbers . these two studies aside , how effective such tools and approaches are in terms of seizure outcomes , especially in children , remains largely unstudied and , hence , unclear . the question therefore should no longer be whether or not surgery is indicated in children with an epileptogenic neuroglial tumor like dnet or ganglioglioma , or even at what age such surgery begins to be safe [ 99 , 69 ] , but how surgery should be performed and how aggressive one should be to remove all tumor and/or epileptogenic tissues . in some children , because of the location of the tumor , both in terms of accessibility and proximity to high - function areas of the brain , the child s overall health status , and perhaps other issues as well , total resection is infeasible . however , even in patients in whom only partial resection was achieved , long - term seizure - free rates have exceeded 50 % [ 73 , 80 ] . more importantly , in this review of 185 pediatric dnet cases spanning 13 studies and two decades , only two patients failed to improve , and there were no perioperative deaths . in addition , in patients in whom seizure control is initially attained but then lost , repeat surgery appears to be of value . for example , among 106 children ( mean age 13.5 years at surgery ) who underwent temporal lobe resections for either low - grade tumor or vascular - anomaly - induced epilepsy at the hospital for sick children in toronto , canada , between 1983 and 2003 , 12 ultimately required a second temporal lobe procedure for intractable recurrent seizures ; of these , seven returned to a seizure - free state . our analysis has admitted limitations , starting with the non - random nature of patient selection which , technically , prohibits the use of certain statistical tests . note also that the wide range in sample sizes generates weighting issues , in that a report with a single patient was treated statistically the same as series with more than 20 . it was for this reason that we attempted to identify as much individual subject data as possible . in fact , this resulted in almost 100 cases being available for analysis , among whom the same association between completeness of resection and long - term seizure freedom was apparent , albeit merely approaching statistical significance . moreover , we do not claim that our results are empirically definitive ; they merely illustrate the inadequacy of current series , all too small to allow for most of the statistical manipulations that we have attempted , and the need for further research , preferably across multiple centers to allow for more adequate patient numbers . from this review of 13 studies on dnet resections in children and adolescents , it is clear that surgical resection of the lesion is effective at improving seizures in almost all patients and at achieving long - term seizure freedom in the vast majority . surgical resection of dnets also appears to be very safe in children , in terms of both mortality and long - term surgery - related morbidity . however , data are lacking on whether this translates into more extensive procedures like brain mapping and partial lobectomy being any more effective than simple lesionectomy alone . further prospective research , preferably involving multiple centers to generate more adequate subject numbers , is clearly indicated .

purposein children and adolescents , dysembryoplastic neuroepithelial tumors ( dnets ) of the brain present with seizures almost 100 % of the time , potentially creating significant long - term morbidity and disability despite the generally indolent course of the lesion . these tumors also tend to be quite resistant to anti - epileptic drugs which , themselves , can be associated with long - term side effects and resultant disability . many clinicians advocate early surgical resection of these lesions , but how effective this approach is , and how aggressive tumor removal should be , continues to be debated.methodswe performed a systematic review of the relevant literature to identify all reports of dnet resections in pediatric patients published over the past 20 years . in all , over 3000 medline abstracts were reviewed , ultimately resulting in 13 studies with 185 pediatric dnet patients to review.resultssurgical resection of the lesion was effective at improving seizures in over 98 % of patients and at achieving long - term seizure freedom in 86 % . surgical resection of dnets also appeared to be quite safe , with no reported perioperative deaths and an overall rate of postoperative complications of 12 % ; the vast majority of these complications were transient.conclusionstotal gross resection of the lesion was the only factor statistically correlated with long - term seizure freedom ( r = 0.63 , p = 0.03 ) . however , data remain lacking regarding whether this translates into more extensive procedures like brain mapping and partial lobectomies being any more effective than simple lesionectomies alone . further research is clearly needed to address this and other crucial questions .

Introduction Search methods Search results and analysis Discussion Conclusions

in north america , no other solid tumor is as common or causes more deaths in children and adolescents than cancers of the brain [ 16 , 26 , 84 ] , with roughly three quarters of patients presenting at less than 15 years of age . the prognosis for long - term survival is much better in children than in adults , with up to half of pediatric brain tumor patients surviving long term . the main reason for this enhanced survival is that children and adolescents tend to have lower - grade lesions [ 79 , 95 , 101 ] . long - term survival is not without problems , however , with many pediatric brain tumor survivors continuing to suffer from significant morbidity [ 44 , 63 , 76 , 90 , 94 , 108 ] and , sometimes , early death . among the more common long - term sequelae of brain tumors and their treatment in children are seizures , which can be quite disabling and , at times , life - threatening in themselves [ 90 , 3 , 56 , 66 , 67 , 85 , 87 , 104 , 106 , 110 ] . in one study , seizures were the dominant predictor of disability in long - term brain tumor survivors [ 66 , 67 ] . of these , dysembryoplastic neuroepithelial tumors ( dnets ) present with seizures almost 100 % of the time [ 1 , 65 , 73 , 97 , 104 ] . because these tumors typically are slow - growing and non - invasive , the primary objective of dnet management in most cases is control , if not eradication , of seizures . because tumor - associated seizures tend to be more resistant to anti - epileptic drugs ( aeds ) than idiopathic seizures [ 57 , 98 ] and the long - term use of aeds is not without significant risks in itself [ 22 , 23 , 56 , 66 , 68 , 100 , 107 ] , including wide - ranging adverse effects on cognitive function and development [ 56 , 100 ] , this typically necessitates surgery to resect as much of the tumor as possible [ 21 , 70 , 73 , 78 , 89 , 97 , 104 ] . in addition , several studies have shown that radical removal of an epileptogenic brain tumor is a strong , and likely the strongest , predictor of seizure freedom . the primary purpose of this paper is to extensively and systematically review and analyze the literature on surgical outcomes in pediatric patients with dnets , specifically looking at perioperative complications and mortality , short- and long - term seizure control , and short- and long - term survival . the main search objective was to identify all papers involving pediatric patients with dnets undergoing surgical resection over the past 20 years ( 19942014 ) , in which the following information was available either for the entire sample or for individual patients ( for papers in which patients with a range of tumor types are represented ) : number of patients with a dnet , ( mean ) age at the time of seizure onset , ( mean ) age at the time of surgery , age range , type of seizure , type of surgery , location of the lesion , number of subjects in which complete resection was achieved , number with immediate postoperative complications including recurrent / persistent seizures , ( mean ) length of follow - up , long - term survival , long - term neurological sequelae , the number with seizures at final follow - up , and final engel rating . the period 1994 to 2014 was selected because it was felt that older papers reported the results of studies with considerably less robust methodology and because most papers prior to 1995 did not report many of the specific variables of interest listed above . all the included papers were identified via an extensive search of the pubmed database , using the following search terms : dysembryoplastic neuroepithelial tumor ( n = 425 ) , dnet ( n = 193 ) , neuroglial tumor ( n = 401 ) , neuroglial tumor and seizure ( n = 1574 ) , and neuroglial tumor and surgery ( n = 871 ) . of these terms , only those with n 2000 were reviewed , because of the lack of specificity of longer lists . however , of these , only seven studies had pediatric dnet patients exclusively [ 9 , 30 , 58 , 70 , 73 , 89 , 97 ] , two had both adult and pediatric patients , but individualized data [ 17 , 59 ] , and an additional four papers [ 5 , 15 , 47 , 51 ] had individual data on dnet patients among patients with other lesions that allowed for the extraction of almost all of the variables of interest . correlation strength was categorized as weak ( r < 0.40 ) , moderate ( 0.400.69 ) , or strong ( r 0.70 ) as indicated in the review of taylor . table 1 lists the 13 studies identified in which data of interest were available for patients with dysembryoplastic neuroepithelial tumors ( dnets ) , including six papers specific to pediatric dnet tumors for which totals and means are presented [ 9 , 58 , 70 , 73 , 89 , 97 ] and seven papers with data presented for individual dnet patients from which totals and means could be calculated [ 5 , 15 , 17 , 30 , 47 , 51 , 58 ] . these 13 studies encompass 185 patients , of mean age 9.4 years , with individual patients ranging in age from 0.5 to 21 years . the mean duration of seizures prior to surgical resection of the underlying dnet across the 13 studies was 3.2 years but ranged widely from 4 and 7 months [ 15 , 47 ] to 7 years [ 70 , 97 ] . the mean percentage of dnets located within a temporal lobe across the 13 studies was 67.8 % , though this ranged quite broadly from 38.5 % and 42.9 % to 100 % . gross total resection was achieved in 83.3 % of patients , the percentage lowered by a single study in which gross total resection was achieved in only 11 of 26 . ?522793.1 % 29100.0 % 0?lee20092212.4318 yearsl l22100.0 % 442090.9 % 22100.0 % 01minkin2008248.9115 yearsl2187.5 % 802083.3 % 24100.0 % 09chan2006311.0814 yearsl , l + e3100.0 % 104266.7 % 3100.0 % 01sandberg2005189.61 month13 yearsl map18100.0 % 1918100.0 % 18100.0 % 01cataltepe20051411.0418 yearsl , l + e1285.7 % 331285.7 % 14100.0 % 0?nolan20042610.0418 years?1142.3 % 521661.5 % 2596.2 % 0?fernandez20031410.1318 yearsl , l + e14100.0 % 87.11285.7 % 1392.9 % 0?lee2000109.3215 yearsl , l + e10100.0 % 40.310100.0 % 10100.0 % 02khajavi1999710.1419 yearsl , l + l685.7 % 33571.4 % 7100.0 % 0?1859.40.521 years13083.3 % 63.315985.9 % 18398.9 % 01311.9 % seizure response to surgical resection of dysembryoplastic neuroectodermal tumors in three of the studies , lesionectomies alone were utilized to resect the lesion , with data on the number of total resections available , accounting for 32 patients . of this number of procedures , 27 yielded a total gross resection ( 84.4 % ) versus 64 of 67 patients in whom lesionectomy was combined with further resection ( 95.5 % ) ; hence , the odds of an incomplete resection ( or ) was 3.49 ( 95 % confidence interval = 0.78 , 15.51 , p = 0.10 ) . over the 13 studies , long - term postoperative freedom from seizures was achieved in 85.9 % of patients , at a mean follow - up of 63.3 months ( 5.25 years ) . the percentage of patients achieving seizure freedom in the 12 years from 1994 through 2006 was 81.6 versus 91.3 % in studies published from 2007 onward ; however , this difference failed to achieve statistical significance ( t = 1.20 , df = 11 , p = 0.26 ) . although a moderate direct correlation was noted between the year of paper publication and the percentage seizure freedom , this also failed to achieve statistical significance ( r = 0.45 , p = 0.23 ) . on the other hand , the percentage of patients achieving seizure freedom in a given study was both moderately and statistically correlated with the percentage of procedures resulting in gross total resection ( r = 0.63 , p = 0.03 ) , but not with mean patient age ( r = -0.45 , p = 0.12 ) or mean duration of follow - up ( r = 0.20 , p = 0.51 ) . no correlation at all was noted between the percentage of subjects achieving seizure freedom and any of the three variables mean duration of seizures prior to surgery ( r = 0.007 , p = 0.98 ) , percentage of patients with complex partial seizures ( r = 0.063 , p = 0.87 ) , or percentage of patients with a temporal lobe lesion ( r = 0.052 , p = 0.087 ) . in the seven studies in which sufficient individual subject data were available , there were 68 gross total resections , resulting in long - term seizure freedom in 62 patients ( 91.2 % ) , and 3 subtotal resections , all having engel stage iii outcomes , so that the likelihood of a poor outcome was more than 13 times higher ( lr = 13.4 ; p < 0.001 ) with a subtotal resection . of the 45 who underwent lesionectomy alone , 40 ( 88.9 % ) achieve total seizure freedom versus 25 or 30 ( 83.3 % ) among those in whom some additional resection was performed ( pearson = 0.48 , p = 0.49 ) . by age group , postoperative seizure freedom was achieved in 10 of 11 ( 90.9 % ) of children under age 6 , 31 of 35 ( 88.6 % ) children from age 6 up to , but not including 13 , and 24 of 29 ( 82.8 % ) of adolescents of 13 years or older , a seemingly downward trend that was not statistically significant ( = 0.67 , p = 0.71 ) . comparing seizure - free rates between patients with a follow - up duration below and above that mean again revealed no significant difference ( 89.1 vs. 82.2 % , = 0.63 , p = 0.43 ) . similarly , neither duration of seizures ( = 2.19 , df = 3 , p = 0.54 ) nor temporal location of the tumor ( = 1.44 , df = 1 , p = 0.23 ) was associated with seizure freedom . there were no immediate or late deaths over the course of reported follow - up , and the perioperative complication rate , adjusted for missing data , was just 11.9 % . however , these tumors , which typically occur in the temporal lobes , are almost always associated with seizures . to date , all that can be said with confidence is that the cause of tumor - induced seizures is almost certainly multifactorial and beyond the mere physical size of the tumor itself . in several studies , preoperative cognitive function in pediatric patients with glioneuronal tumors tended to be low average to average and associated with a variety of cognitive deficits , including problems with speech and memory , and delay to meet developmental milestones [ 24 , 29 , 32 , 34 , 82 ] . since dnets tend to be extremely resistant to aed therapy , pharmaceutical control of seizures typically is incomplete [ 9 , 17 , 19 , 58 , 73 , 80 ] . this places patients at risk for continued brain injury and worsening neurocognitive function , prompting many authors to argue for surgical resection of the lesions as soon as possible in most cases [ 10 , 11 , 19 , 34 , 37 , 47 , 51 , 99 , 113 , 115 ] . among the various specific arguments given for early surgical resection of glioneuronal tumors , including dnets , are the optimization of seizure control [ 21 , 75 , 89 , 105 , 113 , 115 , 116 ] ; the optimization of brain development [ 10 , 11 , 17 , 19 ] ; avoiding or at least minimizing the long - term risks of aeds [ 22 , 53 , 66 , 68 , 96 , 107 ] , especially in children and those who may require chemotherapy to control tumor growth ; and reducing the risk , albeit low , of later , catastrophic malignant change [ 2 , 20 , 41 , 61 , 71 , 72 , 81 ] . brain surgery is certainly not without its own potential consequences and risks , however , including the prospect of postoperative mortality and worsening seizures and of immediate postoperative neurological deficits and cognitive decline . on the other hand , almost all were single events that resolved without the need for long - term anti - epileptic drugs ( aeds ) . however , worsened seizures postoperatively were reported uncommonly in our review of 13 published studies / series , and the vast majority was transient and controlled prior to hospital discharge . in the medical literature , the rate of seizure freedom in series with either adults alone or adults plus children has varied widely for dnets , from as low as 52.4 and 53.3 % [ 33 , 83 ] to as high as 90 and 100 % [ 89 , 58 ] . overall , in this age group , long - term freedom from seizures was achieved in almost 86 % ( 85.9 % ) and some improvement in 99 % ( all but two of 185 patients ) . moreover , there were no deaths , and the rate of postoperative complications , the vast majority transient neurological deficits , was only 12 % . the only variable either correlated with seizure freedom rate at a study mean level or associated with seizure freedom at an individual level was degree of tumor resection , with subtotal resections virtually always associated with either the persistence or recurrence of seizures . these results are congruent with those of a recently published retrospective analysis of 29 children undergoing resection of glioneuronal tumors , in which the rate of seizure freedom 12 months after surgery was 94 % in those in whom gross total resection was achieved versus just 54 % in those in which it was not ( p < 0.05 ) ; unfortunately , only 13 of the patients were determined to have a dnet , versus 16 with a ganglioglioma , limiting comparisons against our own results . with even more limitations , our results are also consistent with a larger series of 332 patients ( mean age 39.3 years , range 1695 ) with low - grade gliomas who underwent operative resections for a variety of tumors , in whom seizure control again was far more likely to be achieved after gross total resection than after subtotal resection or biopsy alone ( odds ratio 16 , 95 % confidence interval 2.2124 , p = 0.0064 ) . , in which the distribution of gross complete to incomplete resections was fairly evenly split ( 12 complete , 14 incomplete resections ) ; in this series , all nine children who had no detectable tumor on postoperative imaging were seizure free at 12 months , with only one relapsing at final follow - up , versus an approximately 50 % rate of seizure freedom in the remainder ( p = 0.02 ) . given that completeness of tumor resection seems to be a determinant of seizure outcomes , the question arises : is it better to do more than just a simple lesionectomy , either via brain mapping to detect epileptogenic foci apart from the tumor itself , or more extensive resections ? over the years , attempts have repeatedly been made to optimize the resection of epileptogenic lesions , both by better delineating their margins and by enhancing the identification of extra - tumoral epileptogenic tissue , using intraoperative tools like electrocorticography ( ecog ) to identify potential seizure - inducing tissue irregularities like fcd [ 4 , 7 , 28 , 35 , 46 , 58 , 75 , 89 , 111 , 112 ] . one year following surgery , roughly 80 % of patients in each group were seizure free . however , long - term data trended toward improved seizure freedom in patients in the ecog group , with 79 versus 61 % patients still seizure free at a mean 5.8 years of follow - up ( p = 0.08 ) . , whereas 10 of 12 pediatric dnet patients undergoing a temporal lobectomy achieved seizure freedom , such freedom was achieved in only two of six who had a lesionectomy alone , a difference that , using pearson analysis , is statistically significant ( = 4.5 , p = 0.03 ) despite the small numbers . the question therefore should no longer be whether or not surgery is indicated in children with an epileptogenic neuroglial tumor like dnet or ganglioglioma , or even at what age such surgery begins to be safe [ 99 , 69 ] , but how surgery should be performed and how aggressive one should be to remove all tumor and/or epileptogenic tissues . in some children , because of the location of the tumor , both in terms of accessibility and proximity to high - function areas of the brain , the child s overall health status , and perhaps other issues as well , total resection is infeasible . however , even in patients in whom only partial resection was achieved , long - term seizure - free rates have exceeded 50 % [ 73 , 80 ] . more importantly , in this review of 185 pediatric dnet cases spanning 13 studies and two decades , only two patients failed to improve , and there were no perioperative deaths . in fact , this resulted in almost 100 cases being available for analysis , among whom the same association between completeness of resection and long - term seizure freedom was apparent , albeit merely approaching statistical significance . moreover , we do not claim that our results are empirically definitive ; they merely illustrate the inadequacy of current series , all too small to allow for most of the statistical manipulations that we have attempted , and the need for further research , preferably across multiple centers to allow for more adequate patient numbers . from this review of 13 studies on dnet resections in children and adolescents , it is clear that surgical resection of the lesion is effective at improving seizures in almost all patients and at achieving long - term seizure freedom in the vast majority . surgical resection of dnets also appears to be very safe in children , in terms of both mortality and long - term surgery - related morbidity . however , data are lacking on whether this translates into more extensive procedures like brain mapping and partial lobectomy being any more effective than simple lesionectomy alone .

urethral injury results from various acquired and congenital abnormalities , including trauma , infection , cancer , and hypospadias . the reconstruction of urethral injuries remains a great challenge for urologists , owing to the difficulties of a successful urethroplasty that achieves desirable outcomes without complications such as fistula and stricture . although diverse remedies such as direct suture , transurethral resection , and urethral substitution with different tissues or xenografts have been applied to maintain urethra continuity during reconstructive surgery , few are highly effective or reliable , especially in the management of a long urethral defect [ 35 ] . amniotic membrane ( am ) , a biocompatible material that has been intensively investigated in ophthalmology and dermatology reconstructive surgery , shows great potential for urethral reconstruction . the basic structure of am consists of 3 layers : epithelial layer , basement layer , and avascular stromal layer . it has been demonstrated that am exhibits excellent properties of reducing inflammation , scarring , and the risk of rejection , as well as facilitating the migration , localization , and proliferation of epithelial cells , which largely depend on the function of the basement layer . amniotic membranes have been applied successfully in the reconstruction of long ureteral strictures in humans . however , the use of human am is relatively infrequent due to the shortage of tissue sources . am xenografts prepared from non - human donors can dramatically expand am sources for clinical use , but few studies have achieved satisfactory results using am xenotransplantation in urethral reconstruction , probably due to anti - xenoresponses . to overcome this limitation , we separated the basement layer of am to obtain denuded human amniotic scaffold ( dhas ) , then engrafted primary rabbit urethral epithelial cells on the surface of dhas to minimize potential xenograft rejection and maximize the biocompatibility of human am . both dhas and cell - seeded dhas were then applied as urethroplastic materials in rabbit models of urethral injury to fully explore the feasibility and efficacy of tissue - engineered dhas in urethral substitution application . all animal experiments were approved by the ethics committee and conducted in accordance with guidelines of the fourth military medical university for animal experiments . twenty male new zealand white rabbits weighing 2.53.5 kilograms each were purchased from the experimental animal center of the fourth military medical university and housed individually in a temperature - controlled cage with 5055% humidity and a 12-h light dark cycle , with free access to standard commercial feed and tap water . eight new zealand rabbits underwent subcutaneous implantation of dhas ( n=4 ) and human am ( n=4 ) . others were randomly segregated into 2 groups for urethral reconstruction surgery : the experimental group receiving tissue - engineered dhas ( n=6 ) and the control group receiving intact am patches ( n=6 ) . human am the dhas was prepared and preserved using a method described elsewhere . in brief , the human placenta was obtained immediately after delivery with negative serologic tests for human immunodeficiency virus , human hepatitis type b and c , and syphilis . under sterile conditions , the am was washed with 0.01 m phosphate buffer solution ( pbs , invitrogen ) containing antibiotic - antimycotic liquid solution ( penicillin 50 g / ml , streptomycin 50 g / ml , neomycin 100 g / ml , and amphotericin b 2.5 g / ml , sigma , st . part of the human am was then deprived of amniotic epithelial cells to obtain dhas by mixed digestive solution ( 0.25% trypsin + 0.02% ethylene diamine tetraacetic acid ; sigma , st . finally , the prepared dhas was rewashed 3 times with pbs . scanning electron microscopy ( sem ) and hematoxylin - eosin ( he ) and immunofluorescence staining were performed to characterize the surface structure and histology of dhas . for sem analysis , after the sample was spattered with an ultrathin gold layer , the surface structure of dhas was observed under a sem s-3400n ( hitachi , tokyo , japan ) at an accelerating voltage of 5 kv for imaging . for histological analysis , 6-m polylysine - coated frozen sections were obtained from dhas embedded in optimal cutting temperature ( oct ) compound ( leica , nussloch , germany ) and subjected to hematoxylin - eosin staining . for immunofluorescence assay , 6-m polylysine - coated frozen sections were fixed with cold acetone for 15 min and blocked with 10% goat serum for 30 min . sections were then incubated at room temperature for 1 h with primary antibodies : rabbit anti - human collagen i , collagen iii , and collagen iv polyclonal antibody , mouse anti - human fibronectin , vascular endothelial growth factor monoclonal antibody ( 1:500 , santa cruz , ca ) . appropriate nonspecific mouse or rabbit igg ( dako , kyoto , japan ) at the same concentration was used as control . after washing 3 times over a period of 15 min in pbs containing 0.15% triton x-100 ( sigma , usa ) , sections were incubated at room temperature for 1 h with a fluorescent secondary antibody ( rhodamine red - x goat anti - mouse or rabbit igg ; 1:500 , invitrogen ) . sections were then rewashed 3 times with pbs and mounted using anti - fade mounting medium containing propidium iodide ( vectashield ; vector , burlingame , ca ) . finally , sections were observed under an olympus bx51 fluorescence microscope ( olympus , tokyo , japan ) . anesthetization and disinfection of male new zealand white rabbits , mucous membrane ( 43 mm ) was harvested from the posterior wall of the urethra and rinsed repeatedly with d - hanks solution containing 50 g / ml penicillin , 50 g / ml streptomycin , and 2.5 g / ml amphotericin b ( life technologies inc . , gaithersburg , md ) followed by disintegration using 2.24 u / ml dispase ii for 30 min ( gibco - invitrogen , carlsbad , ca ) . then the epithelium layer was carefully separated from the mucous membrane and cut into pieces . cell suspension was collected and cultured in dulbecco s modified eagle medium ; nutrient mix f-12 medium ( containing n2 , 15% fetal bovine serum , hydrocortisone , l - glutamin , and -mercaptoethanol ; invitrogen , grand island , ny ) under a humidified environment containing 5% co2 at 37c . cell culture with epithelium origin was confirmed by immunofluorescence with cytokeratin 18 . for the following inoculation , briefly , 1 ml of cell suspension ( 10 cells / ml ) was dripped on the surface of dhas placed in a 6-cm petri dish . then the cell - seeded sheet was cultured for 2 weeks in the previously mentioned medium and environment to obtain tissue - engineered dhas . the histocompatibility was investigated by the implantation of dhas and cell - seeded dhas subcutaneously into 2 groups of 4 male new zealand white rabbits each weighing 2.53.5 kg . the rabbit that underwent intraperitoneal anesthesia with ketamine ( dose : 5 mg / kg ) and xylazine ( dose : 8 mg / kg ) was placed on the operating table in prone position . after shaving , disinfection with povidone - iodine scrub and covering the rest of its body with sterile drapes , 1-cm vertical incisions were made at both dorsal parts spaced 3 cm away from the middle line of the vertebrae , with a deep reaching aponeurotic fascia . prepared materials ( 11 cm ) were then implanted into both lacunas , followed by the suture of incisions and sterilization . xenografts were obtained 1 , 2 , 4 , and 8 weeks after implantation surgery in rabbits . the grafts were fixed in 4% paraformaldehyde and then underwent he staining for histological analyses . immunohistochemical analysis of cd4 and cd8 cells was performed to assess the intensity of immune response using goat anti - rabbit cd4 , cd8 monoclonal antibodies ( 1:500 ) and hrp conjugated mouse anti - goat igg ( 1:500 , biolegend , san diego , ca ) . semi - quantification of the percentage of cd4 and cd8 cells were assessed by image pro plus 6.0 software . the surgical procedures of urethral injury model and urethroplasty have been described in detail elsewhere . anesthetization and local preparation of the lower abdomen , a transurethral fr6 catheter was inserted into the bladder through the urethral orifice . under an operating microscope with 2.5 magnification , a 510 mm defect that exposed the catheter was made on the ventral part of the urethra 1 cm away from the distal urethral meatus with the penis on stretch . human am graft or tissue - engineered human amniotic scaffold was covered onto the defect , which was closed by interrupted suturing with absorbable sterile vicryl 6 - 0 suture . the urethral catheter was fixed to the glans of the penis by silk 4 - 0 with a short 1-cm protrusion to avoid dislocation caused by movement of the rabbit . daily antibiotic injections ( penicillin 50 000 unit / kg ) were started at the time of surgery and continued for 1 week . rabbits were closely observed for abnormal mental status , growth , diet , defecation , and the development of infection and fistula . at intervals of 2 weeks and again at 3 months after surgery , implants in the rabbits were collected for histological examinations . all animal experiments were approved by the ethics committee and conducted in accordance with guidelines of the fourth military medical university for animal experiments . twenty male new zealand white rabbits weighing 2.53.5 kilograms each were purchased from the experimental animal center of the fourth military medical university and housed individually in a temperature - controlled cage with 5055% humidity and a 12-h light dark cycle , with free access to standard commercial feed and tap water . eight new zealand rabbits underwent subcutaneous implantation of dhas ( n=4 ) and human am ( n=4 ) . others were randomly segregated into 2 groups for urethral reconstruction surgery : the experimental group receiving tissue - engineered dhas ( n=6 ) and the control group receiving intact am patches ( n=6 ) . human am was obtained with informed consent from mothers before delivery . the dhas was prepared and preserved using a method described elsewhere . in brief , the human placenta was obtained immediately after delivery with negative serologic tests for human immunodeficiency virus , human hepatitis type b and c , and syphilis . under sterile conditions , the am was washed with 0.01 m phosphate buffer solution ( pbs , invitrogen ) containing antibiotic - antimycotic liquid solution ( penicillin 50 g / ml , streptomycin 50 g / ml , neomycin 100 g / ml , and amphotericin b 2.5 g / ml , sigma , st . part of the human am was then deprived of amniotic epithelial cells to obtain dhas by mixed digestive solution ( 0.25% trypsin + 0.02% ethylene diamine tetraacetic acid ; sigma , st . scanning electron microscopy ( sem ) and hematoxylin - eosin ( he ) and immunofluorescence staining were performed to characterize the surface structure and histology of dhas . for sem analysis , after the sample was spattered with an ultrathin gold layer , the surface structure of dhas was observed under a sem s-3400n ( hitachi , tokyo , japan ) at an accelerating voltage of 5 kv for imaging . for histological analysis , 6-m polylysine - coated frozen sections were obtained from dhas embedded in optimal cutting temperature ( oct ) compound ( leica , nussloch , germany ) and subjected to hematoxylin - eosin staining . for immunofluorescence assay , 6-m polylysine - coated frozen sections were fixed with cold acetone for 15 min and blocked with 10% goat serum for 30 min . sections were then incubated at room temperature for 1 h with primary antibodies : rabbit anti - human collagen i , collagen iii , and collagen iv polyclonal antibody , mouse anti - human fibronectin , vascular endothelial growth factor monoclonal antibody ( 1:500 , santa cruz , ca ) . appropriate nonspecific mouse or rabbit igg ( dako , kyoto , japan ) at the same concentration was used as control . after washing 3 times over a period of 15 min in pbs containing 0.15% triton x-100 ( sigma , usa ) , sections were incubated at room temperature for 1 h with a fluorescent secondary antibody ( rhodamine red - x goat anti - mouse or rabbit igg ; 1:500 , invitrogen ) . sections were then rewashed 3 times with pbs and mounted using anti - fade mounting medium containing propidium iodide ( vectashield ; vector , burlingame , ca ) . finally , sections were observed under an olympus bx51 fluorescence microscope ( olympus , tokyo , japan ) . anesthetization and disinfection of male new zealand white rabbits , mucous membrane ( 43 mm ) was harvested from the posterior wall of the urethra and rinsed repeatedly with d - hanks solution containing 50 g / ml penicillin , 50 g / ml streptomycin , and 2.5 g / ml amphotericin b ( life technologies inc . , gaithersburg , md ) followed by disintegration using 2.24 u / ml dispase ii for 30 min ( gibco - invitrogen , carlsbad , ca ) . then the epithelium layer was carefully separated from the mucous membrane and cut into pieces . cell suspension was collected and cultured in dulbecco s modified eagle medium ; nutrient mix f-12 medium ( containing n2 , 15% fetal bovine serum , hydrocortisone , l - glutamin , and -mercaptoethanol ; invitrogen , grand island , ny ) under a humidified environment containing 5% co2 at 37c . cell culture with epithelium origin was confirmed by immunofluorescence with cytokeratin 18 . for the following inoculation , briefly , 1 ml of cell suspension ( 10 cells / ml ) was dripped on the surface of dhas placed in a 6-cm petri dish . then the cell - seeded sheet was cultured for 2 weeks in the previously mentioned medium and environment to obtain tissue - engineered dhas . the histocompatibility was investigated by the implantation of dhas and cell - seeded dhas subcutaneously into 2 groups of 4 male new zealand white rabbits each weighing 2.53.5 kg . the rabbit that underwent intraperitoneal anesthesia with ketamine ( dose : 5 mg / kg ) and xylazine ( dose : 8 mg / kg ) was placed on the operating table in prone position . after shaving , disinfection with povidone - iodine scrub and covering the rest of its body with sterile drapes , 1-cm vertical incisions were made at both dorsal parts spaced 3 cm away from the middle line of the vertebrae , with a deep reaching aponeurotic fascia . prepared materials ( 11 cm ) were then implanted into both lacunas , followed by the suture of incisions and sterilization . finally , the rabbits were caged alone until regaining consciousness . xenografts were obtained 1 , 2 , 4 , and 8 weeks after implantation surgery in rabbits . the grafts were fixed in 4% paraformaldehyde and then underwent he staining for histological analyses . immunohistochemical analysis of cd4 and cd8 cells was performed to assess the intensity of immune response using goat anti - rabbit cd4 , cd8 monoclonal antibodies ( 1:500 ) and hrp conjugated mouse anti - goat igg ( 1:500 , biolegend , san diego , ca ) . semi - quantification of the percentage of cd4 and cd8 cells were assessed by image pro plus 6.0 software . the surgical procedures of urethral injury model and urethroplasty have been described in detail elsewhere . briefly , after i.p . anesthetization and local preparation of the lower abdomen , a transurethral fr6 catheter was inserted into the bladder through the urethral orifice . under an operating microscope with 2.5 magnification , a 510 mm defect that exposed the catheter was made on the ventral part of the urethra 1 cm away from the distal urethral meatus with the penis on stretch . human am graft or tissue - engineered human amniotic scaffold was covered onto the defect , which was closed by interrupted suturing with absorbable sterile vicryl 6 - 0 suture . the urethral catheter was fixed to the glans of the penis by silk 4 - 0 with a short 1-cm protrusion to avoid dislocation caused by movement of the rabbit . daily antibiotic injections ( penicillin 50 000 unit / kg ) were started at the time of surgery and continued for 1 week . rabbits were closely observed for abnormal mental status , growth , diet , defecation , and the development of infection and fistula . at intervals of 2 weeks and again at 3 months after surgery , implants in the rabbits were collected for histological examinations . trypsinization with mixed digestive solution successfully removed the epithelial layer of the amniotic membrane , as revealed by sem and he staining ( figure 1a , 1b ) . the expression patterns of extracellular matrix molecules and growth factor in dhas were characterized by immunofluorescence imaging ( figure 1c1i ) . ubiquitous expressions of collagen ( types i , iii , and iv ) , fibronectin , and vascular endothelial growth factor ( vegf ) were demonstrated , although fibronectin and vegf expressions were lower compared to collagen expression . the immunostaining using cytokeratin 18 confirmed the epithelial origin of harvested cells ( figure 2a ) . in the first few days of co - culture with rabbit urethral epithelial cells and dhas , slow growth of urethral epithelial cells was observed . by the seventh day after 2 weeks of co - culture , a confluent layer of urethral epithelial cells was found on the entire surface of the dhas sheet ( figure 2b ) . to investigate the histocompatibility of cell - seeded dhas , the prepared dhas and cell - seeded dhas were implanted subcutaneously to posterior portions of rabbits . in the follow - up studies with duration up to 8 weeks , no serious inflammation or rejection was observed in the cell - seeded dhas implantation group , as indicated by he staining ( figure 3 ) , as well as the scant infiltration of cd4 and cd8 cells revealed through immunohistochemical analysis ( figure 4 ) . apparent accumulation of cd4 and cd8 cells was found in the group that underwent am implantation . there was a significant statistical difference ( p<0.05 ) in cd4 and cd8 infiltrations between these 2 groups ( figure 4e ) . urethral injury was induced and then urethroplasty was performed successfully in all rabbits ( figure 5a ) . one rabbit developed serious infection and another was found with fistula in the group that received dhas patches . neither infection nor fistula was observed in the group with cell - seeded dhas implantation . two weeks after surgery , cell - seeded dhas was intact without obvious inflammatory cell infiltration , as confirmed by immunohistochemical evaluation of cd4 cell and cd8 cell infiltrations ( figure 5b ) . the anastomosis repair between the defective and normal urethral tissue was revealed by he staining in the cell - seeded dhas group ( figure 5c ) , in contrast to few repairs in the control group using dhas ( figure 5d ) . three months after surgery , the urethral defect was completely repaired in the cell - seeded dhas group . trypsinization with mixed digestive solution successfully removed the epithelial layer of the amniotic membrane , as revealed by sem and he staining ( figure 1a , 1b ) . the expression patterns of extracellular matrix molecules and growth factor in dhas were characterized by immunofluorescence imaging ( figure 1c1i ) . ubiquitous expressions of collagen ( types i , iii , and iv ) , fibronectin , and vascular endothelial growth factor ( vegf ) were demonstrated , although fibronectin and vegf expressions were lower compared to collagen expression . the immunostaining using cytokeratin 18 confirmed the epithelial origin of harvested cells ( figure 2a ) . in the first few days of co - culture with rabbit urethral epithelial cells and dhas , slow growth of urethral epithelial cells was observed . by the seventh day after 2 weeks of co - culture , a confluent layer of urethral epithelial cells was found on the entire surface of the dhas sheet ( figure 2b ) . to investigate the histocompatibility of cell - seeded dhas , the prepared dhas and cell - seeded dhas were implanted subcutaneously to posterior portions of rabbits . in the follow - up studies with duration up to 8 weeks , no serious inflammation or rejection was observed in the cell - seeded dhas implantation group , as indicated by he staining ( figure 3 ) , as well as the scant infiltration of cd4 and cd8 cells revealed through immunohistochemical analysis ( figure 4 ) . apparent accumulation of cd4 and cd8 cells was found in the group that underwent am implantation . there was a significant statistical difference ( p<0.05 ) in cd4 and cd8 infiltrations between these 2 groups ( figure 4e ) . urethral injury was induced and then urethroplasty was performed successfully in all rabbits ( figure 5a ) . one rabbit developed serious infection and another was found with fistula in the group that received dhas patches . neither infection nor fistula was observed in the group with cell - seeded dhas implantation . two weeks after surgery , cell - seeded dhas was intact without obvious inflammatory cell infiltration , as confirmed by immunohistochemical evaluation of cd4 cell and cd8 cell infiltrations ( figure 5b ) . the anastomosis repair between the defective and normal urethral tissue was revealed by he staining in the cell - seeded dhas group ( figure 5c ) , in contrast to few repairs in the control group using dhas ( figure 5d ) . three months after surgery , the urethral defect was completely repaired in the cell - seeded dhas group . urethral injury that originates from either congenital abnormality or acquired disease negatively affects the quality of life for patients . autologous tissues , such as buccal mucosa , intestine and small intestine submucosa , and biodegradable materials are widely accepted as patches for urethroplasty to close the long defects of the urethra . nonetheless , additional surgeries to obtain these substitutes add complexity to the operation and put patients at risk of various complications such as serious infection , intestinal obstruction , urethral stricture , metabolic disturbance , and chronic renal failure . moreover , the presence of bowel in the urethra elevates the risk of fistula formation , lithiasis , and cancers . therefore , it remains a critical challenge to develop novel biomaterials for effective urethra reconstruction . human amniotic membrane has been accepted as a reliable , safe , and available material for tissue repair with relatively low immunogenicity . the first documented application of am in urethroplasty involved female urethral reconstruction with am grafts in year 2000 , which was considered easy , quick , and effective for reconstructive surgery . successfully used cryopreserved human amniotic membrane for the reconstruction of extensive ureteral wall defects in 11 patients with rare complications . applications of amniotic membrane as xenograft for urethroplasty were also described in a rabbit model of urethral injury . one in 20 rabbits experienced infection , followed by fistula formation , and 2 urethral strictures . in chen s study , the role of intact human amniotic membrane in repairing ureteric defect was explored in a rabbit urethral injury model . fresh human amnions were clipped into pieces and sutured around the wound , using epidural tubes as a stent . twenty - two out of 36 rabbits successfully developed a functional urethra without significant contraction 3 months after surgery . however , the potential xenorejection remains a critical issue in using am xenografts as patches for the management of urethral injury . the basement membrane of the amniotic membrane contains collagens , laminin , fibronectin , multiple growth factors , and high molecular weight hyaluronan with immunosuppressive properties that can facilitate the adhesion , migration , and proliferation of epithelial cells , promote epithelial differentiation , and prevent epithelial apoptosis . natural inhibitors of metalloproteinases in the matrix of am stabilize the expression of matrix metalloproteinases in the inflammatory environment , playing a crucial role in regulation of the healing process . therefore , tissue - engineered dhas has been developed and used as an optimal xenograft for rabbit urethral reconstruction . the modified dhas presents favorable biocompatibility , likely due to the absence of human epithelial cells that exhibit pro - inflammatory capability . these findings corroborate with previous reports revealing that denuded am is a more amenable substrate for the cultivation of corneal epithelial cells than intact am , human peritoneum , or omentum . in comparison with other allografts and xenografts such as buccal mucosa , bladder mucosa , and intestine , tissue - engineered dhas is readily available and safe for urethroplasty , with the advantages of unsophisticated surgical techniques required for its application , thereby reducing the operation time , mitigating postoperative patient care , and , most importantly , lowering the risk of serious postoperative complications . cell - seeded dhas displays good biocompatibility and may be an ideal implant for urethral reconstruction with low immunogenicity . the use of dhas seeded with epithelial cells from the recipients may effectively mitigate potential immune response . the major drawback in this experimental study is that the dhas was seeded with rabbit urethral epithelium cells and tested in rabbits , so we are not able to initiate a human - based investigation . moreover , a longer period of postoperative observation might be required to uncover latent adverse effects and complications . confirmation on a larger scale with longer follow - up is necessary to facilitate future investigations of tissue - engineered dhas as a satisfactory implant for human urethral reconstruction .

backgroundmitigating urethral injury remains a great challenge for urologists due to lack of ideal biomaterials for urethroplasty . the application of amniotic membrane ( am ) over other synthetic materials makes it a better potential source for urethral reconstruction . we separated the basement layer of am to obtain denuded human amniotic scaffold ( dhas ) and then inoculated primary rabbit urethral epithelial cells on the surface of dhas to determine whether this strategy minimizes potential rejection and maximizes the biocompatibility of human am.material/methodsafter the successful acquisition of dhas from am , cell - seeded dhas were prepared and characterized . both cell - seeded dhas and acellular dhas were subcutaneously implanted . immune responses were compared by histological evaluation and cd4 + cell and cd8 + cell infiltrations . then they were applied as urethroplastic materials in the rabbit models of urethral injury to fully explore the feasibility and efficacy of tissue - engineered dhas xenografts in urethral substitution application.resultsmild inflammatory infiltration was observed in cell - seeded dhas grafts , as revealed by fewer accumulations of cd4 + cells and cd8 + cells ( or neutrophils or other immune cells ) . urethral defects of rabbits in the urethroplastic group with dhas implantation ( n=6 ) were completely resolved in 1 month , while there were 1 infection and 1 fistula in the control group with acellular dhas patches ( n=6 ) . histopathological analysis revealed mild immune response in the cell - seeded dhas group ( p<0.05).conclusionstissue - engineered dhas minimizes potential rejection and maximizes the biocompatibility of am , which makes it a potential ideal xenograft for urethral reconstruction .

Background Material and Methods Animals and experimental groups Preparation of denuded human amniotic scaffold Characterizations of denuded human amniotic scaffold Inoculation of primary rabbit urethral epithelium cells to dHAS Histocompatibility comparison of dHAS and cell-seeded dHAS Urethral reconstruction using cell-seeded dHAS in a rabbit urethral injury model Statistical analysis Results Preparation of dHAS Inoculation of rabbit urethral epithelial cells to dHAS Histocompatibility studies of dHAS Application of cell-seeded dHAS for urethroplasty Discussion Conclusions

urethral injury results from various acquired and congenital abnormalities , including trauma , infection , cancer , and hypospadias . the reconstruction of urethral injuries remains a great challenge for urologists , owing to the difficulties of a successful urethroplasty that achieves desirable outcomes without complications such as fistula and stricture . although diverse remedies such as direct suture , transurethral resection , and urethral substitution with different tissues or xenografts have been applied to maintain urethra continuity during reconstructive surgery , few are highly effective or reliable , especially in the management of a long urethral defect [ 35 ] . amniotic membrane ( am ) , a biocompatible material that has been intensively investigated in ophthalmology and dermatology reconstructive surgery , shows great potential for urethral reconstruction . the basic structure of am consists of 3 layers : epithelial layer , basement layer , and avascular stromal layer . it has been demonstrated that am exhibits excellent properties of reducing inflammation , scarring , and the risk of rejection , as well as facilitating the migration , localization , and proliferation of epithelial cells , which largely depend on the function of the basement layer . amniotic membranes have been applied successfully in the reconstruction of long ureteral strictures in humans . however , the use of human am is relatively infrequent due to the shortage of tissue sources . am xenografts prepared from non - human donors can dramatically expand am sources for clinical use , but few studies have achieved satisfactory results using am xenotransplantation in urethral reconstruction , probably due to anti - xenoresponses . to overcome this limitation , we separated the basement layer of am to obtain denuded human amniotic scaffold ( dhas ) , then engrafted primary rabbit urethral epithelial cells on the surface of dhas to minimize potential xenograft rejection and maximize the biocompatibility of human am . both dhas and cell - seeded dhas were then applied as urethroplastic materials in rabbit models of urethral injury to fully explore the feasibility and efficacy of tissue - engineered dhas in urethral substitution application . eight new zealand rabbits underwent subcutaneous implantation of dhas ( n=4 ) and human am ( n=4 ) . others were randomly segregated into 2 groups for urethral reconstruction surgery : the experimental group receiving tissue - engineered dhas ( n=6 ) and the control group receiving intact am patches ( n=6 ) . human am the dhas was prepared and preserved using a method described elsewhere . part of the human am was then deprived of amniotic epithelial cells to obtain dhas by mixed digestive solution ( 0.25% trypsin + 0.02% ethylene diamine tetraacetic acid ; sigma , st . scanning electron microscopy ( sem ) and hematoxylin - eosin ( he ) and immunofluorescence staining were performed to characterize the surface structure and histology of dhas . for sem analysis , after the sample was spattered with an ultrathin gold layer , the surface structure of dhas was observed under a sem s-3400n ( hitachi , tokyo , japan ) at an accelerating voltage of 5 kv for imaging . for histological analysis , 6-m polylysine - coated frozen sections were obtained from dhas embedded in optimal cutting temperature ( oct ) compound ( leica , nussloch , germany ) and subjected to hematoxylin - eosin staining . anesthetization and disinfection of male new zealand white rabbits , mucous membrane ( 43 mm ) was harvested from the posterior wall of the urethra and rinsed repeatedly with d - hanks solution containing 50 g / ml penicillin , 50 g / ml streptomycin , and 2.5 g / ml amphotericin b ( life technologies inc . for the following inoculation , briefly , 1 ml of cell suspension ( 10 cells / ml ) was dripped on the surface of dhas placed in a 6-cm petri dish . then the cell - seeded sheet was cultured for 2 weeks in the previously mentioned medium and environment to obtain tissue - engineered dhas . the histocompatibility was investigated by the implantation of dhas and cell - seeded dhas subcutaneously into 2 groups of 4 male new zealand white rabbits each weighing 2.53.5 kg . the rabbit that underwent intraperitoneal anesthesia with ketamine ( dose : 5 mg / kg ) and xylazine ( dose : 8 mg / kg ) was placed on the operating table in prone position . prepared materials ( 11 cm ) were then implanted into both lacunas , followed by the suture of incisions and sterilization . the grafts were fixed in 4% paraformaldehyde and then underwent he staining for histological analyses . immunohistochemical analysis of cd4 and cd8 cells was performed to assess the intensity of immune response using goat anti - rabbit cd4 , cd8 monoclonal antibodies ( 1:500 ) and hrp conjugated mouse anti - goat igg ( 1:500 , biolegend , san diego , ca ) . semi - quantification of the percentage of cd4 and cd8 cells were assessed by image pro plus 6.0 software . the surgical procedures of urethral injury model and urethroplasty have been described in detail elsewhere . under an operating microscope with 2.5 magnification , a 510 mm defect that exposed the catheter was made on the ventral part of the urethra 1 cm away from the distal urethral meatus with the penis on stretch . human am graft or tissue - engineered human amniotic scaffold was covered onto the defect , which was closed by interrupted suturing with absorbable sterile vicryl 6 - 0 suture . the urethral catheter was fixed to the glans of the penis by silk 4 - 0 with a short 1-cm protrusion to avoid dislocation caused by movement of the rabbit . daily antibiotic injections ( penicillin 50 000 unit / kg ) were started at the time of surgery and continued for 1 week . rabbits were closely observed for abnormal mental status , growth , diet , defecation , and the development of infection and fistula . at intervals of 2 weeks and again at 3 months after surgery , implants in the rabbits were collected for histological examinations . eight new zealand rabbits underwent subcutaneous implantation of dhas ( n=4 ) and human am ( n=4 ) . others were randomly segregated into 2 groups for urethral reconstruction surgery : the experimental group receiving tissue - engineered dhas ( n=6 ) and the control group receiving intact am patches ( n=6 ) . human am was obtained with informed consent from mothers before delivery . the dhas was prepared and preserved using a method described elsewhere . part of the human am was then deprived of amniotic epithelial cells to obtain dhas by mixed digestive solution ( 0.25% trypsin + 0.02% ethylene diamine tetraacetic acid ; sigma , st . scanning electron microscopy ( sem ) and hematoxylin - eosin ( he ) and immunofluorescence staining were performed to characterize the surface structure and histology of dhas . for sem analysis , after the sample was spattered with an ultrathin gold layer , the surface structure of dhas was observed under a sem s-3400n ( hitachi , tokyo , japan ) at an accelerating voltage of 5 kv for imaging . for histological analysis , 6-m polylysine - coated frozen sections were obtained from dhas embedded in optimal cutting temperature ( oct ) compound ( leica , nussloch , germany ) and subjected to hematoxylin - eosin staining . anesthetization and disinfection of male new zealand white rabbits , mucous membrane ( 43 mm ) was harvested from the posterior wall of the urethra and rinsed repeatedly with d - hanks solution containing 50 g / ml penicillin , 50 g / ml streptomycin , and 2.5 g / ml amphotericin b ( life technologies inc . for the following inoculation , briefly , 1 ml of cell suspension ( 10 cells / ml ) was dripped on the surface of dhas placed in a 6-cm petri dish . then the cell - seeded sheet was cultured for 2 weeks in the previously mentioned medium and environment to obtain tissue - engineered dhas . the histocompatibility was investigated by the implantation of dhas and cell - seeded dhas subcutaneously into 2 groups of 4 male new zealand white rabbits each weighing 2.53.5 kg . the rabbit that underwent intraperitoneal anesthesia with ketamine ( dose : 5 mg / kg ) and xylazine ( dose : 8 mg / kg ) was placed on the operating table in prone position . prepared materials ( 11 cm ) were then implanted into both lacunas , followed by the suture of incisions and sterilization . the grafts were fixed in 4% paraformaldehyde and then underwent he staining for histological analyses . immunohistochemical analysis of cd4 and cd8 cells was performed to assess the intensity of immune response using goat anti - rabbit cd4 , cd8 monoclonal antibodies ( 1:500 ) and hrp conjugated mouse anti - goat igg ( 1:500 , biolegend , san diego , ca ) . semi - quantification of the percentage of cd4 and cd8 cells were assessed by image pro plus 6.0 software . the surgical procedures of urethral injury model and urethroplasty have been described in detail elsewhere . under an operating microscope with 2.5 magnification , a 510 mm defect that exposed the catheter was made on the ventral part of the urethra 1 cm away from the distal urethral meatus with the penis on stretch . human am graft or tissue - engineered human amniotic scaffold was covered onto the defect , which was closed by interrupted suturing with absorbable sterile vicryl 6 - 0 suture . the urethral catheter was fixed to the glans of the penis by silk 4 - 0 with a short 1-cm protrusion to avoid dislocation caused by movement of the rabbit . rabbits were closely observed for abnormal mental status , growth , diet , defecation , and the development of infection and fistula . at intervals of 2 weeks and again at 3 months after surgery , implants in the rabbits were collected for histological examinations . trypsinization with mixed digestive solution successfully removed the epithelial layer of the amniotic membrane , as revealed by sem and he staining ( figure 1a , 1b ) . the expression patterns of extracellular matrix molecules and growth factor in dhas were characterized by immunofluorescence imaging ( figure 1c1i ) . ubiquitous expressions of collagen ( types i , iii , and iv ) , fibronectin , and vascular endothelial growth factor ( vegf ) were demonstrated , although fibronectin and vegf expressions were lower compared to collagen expression . the immunostaining using cytokeratin 18 confirmed the epithelial origin of harvested cells ( figure 2a ) . in the first few days of co - culture with rabbit urethral epithelial cells and dhas , slow growth of urethral epithelial cells was observed . by the seventh day after 2 weeks of co - culture , a confluent layer of urethral epithelial cells was found on the entire surface of the dhas sheet ( figure 2b ) . to investigate the histocompatibility of cell - seeded dhas , the prepared dhas and cell - seeded dhas were implanted subcutaneously to posterior portions of rabbits . in the follow - up studies with duration up to 8 weeks , no serious inflammation or rejection was observed in the cell - seeded dhas implantation group , as indicated by he staining ( figure 3 ) , as well as the scant infiltration of cd4 and cd8 cells revealed through immunohistochemical analysis ( figure 4 ) . apparent accumulation of cd4 and cd8 cells was found in the group that underwent am implantation . there was a significant statistical difference ( p<0.05 ) in cd4 and cd8 infiltrations between these 2 groups ( figure 4e ) . urethral injury was induced and then urethroplasty was performed successfully in all rabbits ( figure 5a ) . one rabbit developed serious infection and another was found with fistula in the group that received dhas patches . neither infection nor fistula was observed in the group with cell - seeded dhas implantation . two weeks after surgery , cell - seeded dhas was intact without obvious inflammatory cell infiltration , as confirmed by immunohistochemical evaluation of cd4 cell and cd8 cell infiltrations ( figure 5b ) . the anastomosis repair between the defective and normal urethral tissue was revealed by he staining in the cell - seeded dhas group ( figure 5c ) , in contrast to few repairs in the control group using dhas ( figure 5d ) . three months after surgery , the urethral defect was completely repaired in the cell - seeded dhas group . trypsinization with mixed digestive solution successfully removed the epithelial layer of the amniotic membrane , as revealed by sem and he staining ( figure 1a , 1b ) . the expression patterns of extracellular matrix molecules and growth factor in dhas were characterized by immunofluorescence imaging ( figure 1c1i ) . ubiquitous expressions of collagen ( types i , iii , and iv ) , fibronectin , and vascular endothelial growth factor ( vegf ) were demonstrated , although fibronectin and vegf expressions were lower compared to collagen expression . the immunostaining using cytokeratin 18 confirmed the epithelial origin of harvested cells ( figure 2a ) . in the first few days of co - culture with rabbit urethral epithelial cells and dhas , slow growth of urethral epithelial cells was observed . by the seventh day after 2 weeks of co - culture , a confluent layer of urethral epithelial cells was found on the entire surface of the dhas sheet ( figure 2b ) . to investigate the histocompatibility of cell - seeded dhas , the prepared dhas and cell - seeded dhas were implanted subcutaneously to posterior portions of rabbits . in the follow - up studies with duration up to 8 weeks , no serious inflammation or rejection was observed in the cell - seeded dhas implantation group , as indicated by he staining ( figure 3 ) , as well as the scant infiltration of cd4 and cd8 cells revealed through immunohistochemical analysis ( figure 4 ) . apparent accumulation of cd4 and cd8 cells was found in the group that underwent am implantation . there was a significant statistical difference ( p<0.05 ) in cd4 and cd8 infiltrations between these 2 groups ( figure 4e ) . urethral injury was induced and then urethroplasty was performed successfully in all rabbits ( figure 5a ) . one rabbit developed serious infection and another was found with fistula in the group that received dhas patches . neither infection nor fistula was observed in the group with cell - seeded dhas implantation . two weeks after surgery , cell - seeded dhas was intact without obvious inflammatory cell infiltration , as confirmed by immunohistochemical evaluation of cd4 cell and cd8 cell infiltrations ( figure 5b ) . the anastomosis repair between the defective and normal urethral tissue was revealed by he staining in the cell - seeded dhas group ( figure 5c ) , in contrast to few repairs in the control group using dhas ( figure 5d ) . three months after surgery , the urethral defect was completely repaired in the cell - seeded dhas group . urethral injury that originates from either congenital abnormality or acquired disease negatively affects the quality of life for patients . autologous tissues , such as buccal mucosa , intestine and small intestine submucosa , and biodegradable materials are widely accepted as patches for urethroplasty to close the long defects of the urethra . nonetheless , additional surgeries to obtain these substitutes add complexity to the operation and put patients at risk of various complications such as serious infection , intestinal obstruction , urethral stricture , metabolic disturbance , and chronic renal failure . moreover , the presence of bowel in the urethra elevates the risk of fistula formation , lithiasis , and cancers . therefore , it remains a critical challenge to develop novel biomaterials for effective urethra reconstruction . human amniotic membrane has been accepted as a reliable , safe , and available material for tissue repair with relatively low immunogenicity . the first documented application of am in urethroplasty involved female urethral reconstruction with am grafts in year 2000 , which was considered easy , quick , and effective for reconstructive surgery . successfully used cryopreserved human amniotic membrane for the reconstruction of extensive ureteral wall defects in 11 patients with rare complications . applications of amniotic membrane as xenograft for urethroplasty were also described in a rabbit model of urethral injury . in chen s study , the role of intact human amniotic membrane in repairing ureteric defect was explored in a rabbit urethral injury model . however , the potential xenorejection remains a critical issue in using am xenografts as patches for the management of urethral injury . the basement membrane of the amniotic membrane contains collagens , laminin , fibronectin , multiple growth factors , and high molecular weight hyaluronan with immunosuppressive properties that can facilitate the adhesion , migration , and proliferation of epithelial cells , promote epithelial differentiation , and prevent epithelial apoptosis . natural inhibitors of metalloproteinases in the matrix of am stabilize the expression of matrix metalloproteinases in the inflammatory environment , playing a crucial role in regulation of the healing process . therefore , tissue - engineered dhas has been developed and used as an optimal xenograft for rabbit urethral reconstruction . the modified dhas presents favorable biocompatibility , likely due to the absence of human epithelial cells that exhibit pro - inflammatory capability . these findings corroborate with previous reports revealing that denuded am is a more amenable substrate for the cultivation of corneal epithelial cells than intact am , human peritoneum , or omentum . in comparison with other allografts and xenografts such as buccal mucosa , bladder mucosa , and intestine , tissue - engineered dhas is readily available and safe for urethroplasty , with the advantages of unsophisticated surgical techniques required for its application , thereby reducing the operation time , mitigating postoperative patient care , and , most importantly , lowering the risk of serious postoperative complications . cell - seeded dhas displays good biocompatibility and may be an ideal implant for urethral reconstruction with low immunogenicity . the use of dhas seeded with epithelial cells from the recipients may effectively mitigate potential immune response . the major drawback in this experimental study is that the dhas was seeded with rabbit urethral epithelium cells and tested in rabbits , so we are not able to initiate a human - based investigation . confirmation on a larger scale with longer follow - up is necessary to facilitate future investigations of tissue - engineered dhas as a satisfactory implant for human urethral reconstruction .

multicomponent reactions ( mcrs ) have appeared as an imperative means for the construction of diverse and complex organic molecules . they have intrinsic advantages over two component reactions in several aspects including the simplicity of a one - pot procedures and possible structural variation . the synthetic competence comes from several tandem bond formation reactions in mcrs , which save time , energy , and raw material . betti reaction is a modified type of mannich reaction which has subsequently become vital in synthetic chemistry because of c c bond formation under mild experimental conditions . interest in the chemistry of betti reaction derivatives was also strengthened as it was found to possess various catalytic and biological applications [ 35 ] . nonsteroidal anti - inflammatory drugs ( nsaids ) are the most clinically important medicine used for the treatment of inflammation - related diseases like arthritis , asthma , and cardiovascular diseases . however , the long - term administration of nsaid may induce gastrointestinal ulcers , bleeding , and renal disorders due to their nonselective inhibition of both constitutive ( cox-1 ) and inducible ( cox-2 ) isoforms of the cyclooxygenase enzymes [ 79 ] . therefore , new anti - inflammatory drugs lacking those effects are being searched all over the world as alternatives to nsaids . due to the emerging need of improved and highly selective inhibitors of cox-2 , various heterocyclic compounds are synthesized amongst pyrazole compounds and their derivatives are some of them . 4-aminoantipyrine is known for the variety of its clinical applications such as anti - inflammatory , analgesic , antipyretic [ 11 , 12 ] , and several chemotherapeutic agents . it is evident from the reported literatures that compounds possessing pyrazole nuclei showed significant anthelmintic as well as antimicrobial activities [ 1416 ] . the molecular manipulation of a promising lead compound is still a major line of approach for the discovery of new drugs . molecular rearrangement involves the efforts to combine separate groups having similar activity in one compound by eliminating or substituting new moiety to a parent lead compound . hence , an attempt has been made in this study to condense 4-aminoantipyrine in a betti reaction to formulate novel biologically potent moieties using fluorite [ 1719 ] as an excellent catalyst . fluorite ( also called fluorspar ) is a natural occurring mineral composed of calcium fluoride ( caf2 ) . fluorite acts as a mild acid in the dehydration reaction and increases the reaction rate without affecting the yield of desired products . the paper deals with the synthesis of 4-aminoantipyrine derivatives via three - component betti reaction and its assessment for biological applications , namely , anti - inflammatory and anthelmintic . we have also investigated the biological applications of these derivatives using online cheminformatics molinspiration software . a comparison between experimental and theoretical predictions of the biological activity has enabled us to identify alternative combined pharmacophore sites structures . the main interesting task of this work is to develop robust prediction models for inhibitory properties ( solubility , bioavailability , etc . ) to interpret the calculated / predicted results for the design of specific new compounds . all the reagents and solvents are of analytical grade purchased from a commercial source and used directly . fluorite was purchased in the form of crystalline block from an indian supplier and hammered into pieces of 13 mm in size before use . the purity of compounds was checked routinely by tlc ( 0.5 mm thickness ) using silica gel - g coated al - plates ( merck ) and spots were visualized by exposing the dry plates in iodine vapours . ir spectra ( max in cm ) were recorded on a schimadzu - ir prestige 21 spectrometer using kbr technique ; h nmr spectra and c nmr spectra of the synthesized compounds were recorded on a bruker - avance ii 400 ( 400 mhz ) and varian - gemini ( 100 mhz ) spectrometer using dmso - d6 solvent and tms as an internal standard . mass spectra were recorded on a micromass q - t of high resolution mass spectrometer . the elemental analysis ( c , h , n , and s ) of compounds was performed on carlo erba-1108 elemental analyzer . all the experimental protocols were approved by the institutional animal ethics committee ( iaec ) of sharad pawar college of pharmacy , nagpur , india ( approval number : spcp/2013/595 ) . the experiments and the care of the laboratory animals were according to current ethical guidelines by the committee for the purpose of control and supervision on experiments on animals ( cpcsea ) , ministry of environment and forests , government of india , new delhi . diclofenac was used as a reference drug at 10 mg / kg and all the synthesized compounds were administered at 150 mg / kg of body weight . after one hour of the oral administration of synthesized drugs and standard drug , freshly prepared 0.1 ml carrageenin ( 1% carrageenin in 0.9% nacl ) was injected into the left hind limb of each rat under the subplantar aponeurosis . paw volume was recorded at the interval of 0 , 1 , 2 , 3 , and 4 h after carrageenin injection . results were expressed as an increase in paw volume in comparison with the control group . the results were expressed as mean s.e.m and data were statistically analyzed by one - way analysis of variance ( anova ) and p < 0.05 was considered as significant . indian earthworms of the genus and species pheretima posthuma ( family : megascolecidae ) were used for this study . the earthworms that are 35 cm in length and 0.1 - 0.2 cm in width were used for all experimental protocols . the test compounds 4(a h ) and albendazole were dissolved in minimum quantity of 2% dimethyl sulfoxide ( dmso ) and the volume was adjusted to 10 ml with saline water for making the concentration of 12.5 , 25 , 50 , 100 , and 150 mg / ml . the anthelmintic activity was determined in six observations . paralysis was said to occur when the worms do not revive even in saline water . death was concluded when the worms lost their motility followed by fading away of their body color . the results were expressed as mean s.e.m and data were statistically analyzed by one - way analysis of variance ( anova ) and p < 0.05 was considered as significant . a mixture of 4-aminoantipyrine ( 0.01 mol ) 1 , substituted aromatic aldehyde ( 0.01 mol ) 2 , and 8-hydroxyquinoline ( 0.01 mol ) 3 was dissolved in 10 ml of 95% ethanol in one pot and was magnetically stirred at room temperature in presence of fluorite ( 2% weight with respect to all reactants ) ( scheme 1 ) . the completion of the reaction was monitored by tlc by using mixture of ethyl acetate and hexane as mobile phase . the crude product was purified by recrystallization from hot ethanol to get the pure product . yield : 95% ; ir ( kbr , cm ) : 3420 ( oh ) , 3325 ( nh ) , 3010 ( ar h ) , 2970 ( ch3 ) , 2872 ( ch ) , h nmr ( dmso - d6 , ppm ) : 2.54 ( s , 3h , ch3 ) , 2.83 ( s , 1h , nh ) , 3.26 ( s , 3h , ch3 ) , 5.33 ( s , 1h , oh ) , 5.45 ( s , 1h , ch ) , 6.717.14 ( m , 5h , ar ) , 7.187.40 ( m , 4h , ar ) , 7.458.21 ( m , 5h , ar ) . c nmr ( dmso - d6 , ppm ) : 144.4 ( c1 ) , 124.5 ( c2 ) , 149.9 ( c3 ) , 120.4 ( c4 ) , 131.2 ( c5 ) , 134.7 ( c6 ) , 153.2 ( c1 ) , 126.4 ( c2 ) , 135.6 ( c3 ) , 127.5 ( c4 ) , 124.7 ( c5 ) , 129.6 ( c6 ) , 122.4 ( c7 ) , 148.2 ( c8 ) , 136.8 ( c9 ) , 53.0 ( ch ) , 128.5 ( c1 ) , 135.6 ( c2 ) , 161.6 ( c3 ) , 117.4 ( c4 ) , 33.8 ( c5 ) , 133.5 ( c6 ) , 120.6 ( c7 ) , 127.4 ( c8 ) , 121.2 ( c9 ) , 127.9 ( c10 ) , 120.9 ( c11 ) . ( found ) : c , 71.35 ( 71.30 ) ; h , 4.77(4.76 ) ; n , 15.12 ( 15.09 ) . yield : 88% ; ir ( kbr , cm ) : 3422 ( oh ) , 3328 ( nh str . ) , 3025 ( ar h ) , 2972 ( ch3 ) , 2874 ( ch ) , 1690 ( c = o ) , 1590 ( c = n ) , 1543 ( no2 ) . h nmr ( dmso - d6 , ppm ) : 2.53 ( s , 3h , ch3 ) , 2.81 ( s , 1h , nh ) , 3.21 ( s , 3h , ch3 ) , 5.31 ( s , 1h , oh ) , 5.42 ( s , 1h , ch ) , 6.747.16 ( m , 5h , ar ) , 7.207.33 ( m , 4h , ar ) , 7.428.22 ( m , 5h , ar ) . c nmr ( dmso - d6 , ppm ) : 143.1 ( c1 ) , 124.7 ( c2 ) , 150.1 ( c3 ) , 120.3 ( c4 ) , 131.0 ( c5 ) , 134.5 ( c6 ) , 151.6 ( c1 ) , 126.8 ( c2 ) , 134.7 ( c3 ) , 127.1 ( c4 ) , 124.5 ( c5 ) , 129.9 ( c6 ) , 121.9 ( c7 ) , 148.7 ( c8 ) , 135.5 ( c9 ) , 47.6 ( ch ) , 128.1 ( c1 ) , 135.5 ( c2 ) , 161.7 ( c3 ) , 117.2 ( c4 ) , 33.2 ( c5 ) , 133.5 ( c6 ) , 120.5 ( c7 ) , 127.5 ( c8 ) , 120.5 ( c9 ) , 127.7 ( c10 ) , 120.3 ( c11 ) . ( found ) : c , 73.53 ( 73.56 ) ; h , 4.87(4.86 ) ; n , 15.10 ( 15.12 ) . yield : 92% ; ir ( kbr , cm ) : 3426 ( oh ) , 3327 ( nh ) , 3028 ( ar h ) , 2971 ( ch3 ) , 2870 ( ch ) , 1693 ( c = o ) , 1593 ( c = n ) , 1247 ( och3 ) . h nmr ( dmso - d6 , ppm ) : 2.51 ( s , 3h , ch3 ) , 2.82 ( s , 1h , nh ) , 3.23 ( s , 3h , ch3 ) , 3.73 ( s , 3h , och3 ) , 5.35 ( s , 1h , oh ) , 5.41 ( s , 1h , ch ) , 6.726.81 ( m , 5h , ar ) , 6.957.18 ( d , 4h , ar ) , 7.268.18 ( m , 5h , ar ) . c nmr ( dmso - d6 , ppm ) : 143.3 ( c1 ) , 124.5 ( c2 ) , 151.2 ( c3 ) , 120.1 ( c4 ) , 131.5 ( c5 ) , 134.6 ( c6 ) , 150.1 ( c1 ) , 125.5 ( c2 ) , 135.5 ( c3 ) , 126.6 ( c4 ) , 124.3 ( c5 ) , 121.3 ( c6 ) , 121.7 ( c7 ) , 147.7 ( c8 ) , 135.3 ( c9 ) , 52.1 ( ch ) , 128.5 ( c1 ) , 135.3 ( c2 ) , 161.0 ( c3 ) , 116.8 ( c4 ) , 33.6 ( c5 ) , 132.5 ( c6 ) , 121.2 ( c7 ) , 126.4 ( c8 ) , 121.2 ( c9 ) , 128.1 ( c10 ) , 121.4 ( c11 ) . ( found ) : c , 70.03 ( 70.05 ) ; h , 5.71(5.76 ) ; n , 13.90 ( 13.92 ) . yield : 83% ; ir ( kbr , cm ) : 3428 ( oh ) , 3334 ( nh ) , 3015 ( ar h ) , 2973 ( ch3 ) , 2872 ( ch ) , 1695 ( c = o ) , 1591 ( c = h nmr ( dmso - d6 , ppm ) : 2.52 ( s , 3h , ch3 ) , 2.84 ( s , 1h , nh ) , 2.88 ( s , 6h , n(ch3)2 ) , 3.25 ( s , 3h , ch3 ) , 5.32 ( s , 1h , oh ) , 5.43 ( s , 1h , ch ) , 6.736.88 ( m , 5h , ar ) , 6.987.20 ( d , 4h , ar ) , 7.258.23 ( m , 5h , ar ) . c nmr ( dmso - d6 , ppm ) : 144.6 ( c1 ) , 125.0 ( c2 ) , 149.7 ( c3 ) , 119.3 ( c4 ) , 131.3 ( c5 ) , 134.5 ( c6 ) , 153.4 ( c1 ) , 126.4 ( c2 ) , 135.6 ( c3 ) , 126.8 ( c4 ) , 124.9 ( c5 ) , 128.7 ( c6 ) , 122.3 ( c7 ) , 148.3 ( c8 ) , 136.7 ( c9 ) , 53.0 ( ch ) , 128.7 ( c1 ) , 135.8 ( c2 ) , 161.2 ( c3 ) , 116.7 ( c4 ) , 33.4 ( c5 ) , 132.3 ( c6 ) , 120.3 ( c7 ) , 126.6 ( c8 ) , 121.5 ( c9 ) , 128.3 ( c10 ) , 120.6 ( c11 ) . ( found ) : c , 73.53 ( 73.56 ) ; h , 4.77(4.76 ) ; n , 13.01 ( 13.05 ) . yield : 91% ; ir ( kbr , cm ) : 3423 ( oh ) , 3332 ( nh ) , 3020 ( ar h ) , 2970 ( ch3 ) , 2871 ( ch ) , 1690 ( c = o ) , 1590 ( c = h nmr ( dmso - d6 , ppm ) : 2.53 ( s , 3h , ch3 ) , 2.81 ( s , 1h , nh ) , 3.24 ( s , 3h , ch3 ) , 5.08 ( s , 1h , oh ) , 5.31 ( s , 1h , oh ) , 5.44 ( s , 1h , ch ) , 6.716.84 ( m , 5h , ar ) , 6.987.16 ( d , 4h , ar ) , 7.278.18 ( m , 5h , ar ) . c nmr ( dmso - d6 , ppm ) : 144.4 ( c1 ) , 124.4 ( c2 ) , 149.8 ( c3 ) , 119.2 ( c4 ) , 130.6 ( c5 ) , 134.7 ( c6 ) , 152.5 ( c1 ) , 125.3 ( c2 ) , 134.9 ( c3 ) , 127.4 ( c4 ) , 124.4 ( c5 ) , 128.6 ( c6 ) , 122.4 ( c7 ) , 148.2 ( c8 ) , 135.9 ( c9 ) , 53.0 ( ch ) , 128.9 ( c1 ) , 135.3 ( c2 ) , 161.6 ( c3 ) , 117.1 ( c4 ) , 33.1 ( c5 ) , 133.3 ( c6 ) , 120.6 ( c7 ) , 127.2 ( c8 ) , 120.3 ( c9 ) , 127.5 ( c10 ) , 120.8 ( c11 ) . ( found ) : c , 71.03 ( 71.06 ) ; h , 5.37(5.24 ) ; n , 12.34 ( 12.32 ) . yield : 87% ; ir ( kbr , cm ) : 3422 ( oh ) , 3330 ( nh ) , 3021 ( ar h ) , 2974 ( ch3 ) , 2874 ( ch ) , 1695 ( c = o ) , 1593 ( c = n ) . h nmr ( dmso - d6 , ppm ) : 2.54 ( s , 3h , ch3 ) , 2.83 ( s , 1h , nh ) , 3.21 ( s , 3h , ch3 ) , 4.55 ( s , 1h , oh ) , 5.31 ( s , 1h , oh ) , 5.43 ( s , 1h , ch ) , 6.727.12 ( m , 5h , ar ) , 7.147.20 ( m , 4h , ar ) , 7.258.22 ( m , 5h , ar ) . c nmr ( dmso - d6 , ppm ) : 143.9 ( c1 ) , 124.5 ( c2 ) , 149.9 ( c3 ) , 120.4 ( c4 ) , 130.8 ( c5 ) , 134.2 ( c6 ) , 151.6 ( c1 ) , 126.9 ( c2 ) , 135.5 ( c3 ) , 128.1 ( c4 ) , 124.7 ( c5 ) , 129.6 ( c6 ) , 121.4 ( c7 ) , 147.2 ( c8 ) , 136.8 ( c9 ) , 45.5 ( ch ) , 128.7 ( c1 ) , 134.6 ( c2 ) , 161.5 ( c3 ) , 117.3 ( c4 ) , 33.0 ( c5 ) , 133.6 ( c6 ) , 121.7 ( c7 ) , 127.4 ( c8 ) , 120.8 ( c9 ) , 127.7 ( c10 ) , 120.9 ( c11 ) . ( found ) : c , 71.62 ( 71.66 ) ; h , 5.57(5.52 ) ; n , 12.12 ( 12.11 ) . yield : 90% ; ir ( kbr , cm ) : 3427 ( oh ) , 3328 ( nh ) , 3018 ( ar h ) , 2972 ( ch3 ) , 2870 ( ch ) , 1692 ( c = o ) , 1592 ( c = h nmr ( dmso - d6 , ppm ) : 2.51 ( s , 3h , ch3 ) , 2.84 ( s , 1h , nh ) , 3.26 ( s , 3h , ch3 ) , 5.35 ( s , 1h , oh ) , 5.45 ( s , 1h , ch ) , 6.736.88 ( m , 5h , ar ) , 7.007.15 ( d , 4h , ar ) , 7.238.21 ( m , 5h , ar ) . c nmr ( dmso - d6 , ppm ) : 144.4 ( c1 ) , 125.1 ( c2 ) , 149.7 ( c3 ) , 120.1 ( c4 ) , 131.3 ( c5 ) , 134.7 ( c6 ) , 153.2 ( c1 ) , 126.4 ( c2 ) , 134.8 ( c3 ) , 127.3 ( c4 ) , 124.2 ( c5 ) , 128.5 ( c6 ) , 122.3 ( c7 ) , 147.3 ( c8 ) , 136.7 ( c9 ) , 52.1 ( ch ) , 128.6 ( c1 ) , 135.5 ( c2 ) , 161.8 ( c3 ) , 116.9 ( c4 ) , 33.4 ( c5 ) , 132.7 ( c6 ) , 121.2 ( c7 ) , 127.8 ( c8 ) , 121.7 ( c9 ) , 128.2 ( c10 ) , 121.5 ( c11 ) . ( found ) : c , 63.53 ( 63.56 ) ; h , 4.97(4.92 ) ; n , 13.90 ( 13.92 ) . yield : 86% ; ir ( kbr , cm ) : 3425 ( oh ) , 3325 ( nh ) , 3022 ( ar h ) , 2971 ( ch3 ) , 2871 ( ch ) , 1690 ( c = o ) , 1593 ( c = h nmr ( dmso - d6 , ppm ) : 2.53 ( s , 3h , ch3 ) , 2.82 ( s , 1h , nh ) , 3.22 ( s , 3h , ch3 ) , 5.33 ( s , 1h , oh ) , 5.42 ( s , 1h , ch ) , 6.717.11 ( m , 5h , ar ) , 7.187.22 ( m , 4h , ar ) , 7.268.23 ( m , 5h , ar ) . c nmr ( dmso - d6 , ppm ) : 144.7 ( c1 ) , 124.5 ( c2 ) , 149.6 ( c3 ) , 119.4 ( c4 ) , 131.2 ( c5 ) , 134.5 ( c6 ) , 150.0 ( c1 ) , 125.8 ( c2 ) , 135.5 ( c3 ) , 127.5 ( c4 ) , 124.5 ( c5 ) , 129.2 ( c6 ) , 121.4 ( c7 ) , 148.2 ( c8 ) , 136.4 ( c9 ) , 47.5 ( ch ) , 128.9 ( c1 ) , 135.6 ( c2 ) , 161.6 ( c3 ) , 116.4 ( c4 ) , 33.8 ( c5 ) , 132.2 ( c6 ) , 120.6 ( c7 ) , 127.7 ( c8 ) , 121.3 ( c9 ) , 128.5 ( c10 ) , 121.2 ( c11 ) . ( found ) : c , 69.83 ( 69.86 ) ; h , 4.87(4.86 ) ; n , 12.90 ( 12.92 ) . in order to carry out the synthesis in a more efficient way that minimizes time and the amount of catalyst , a model reaction ( scheme 1 ) was magnetically stirred at room temperature using a naturally occurring mineral , fluorite , as a catalyst . the product was isolated by simple and usual workup with 9295% of yield in simply 1015 min . the catalyst was reused in at least eight reactions with no reduction in its efficiency . the solid state ir spectra of these compounds reveal a characteristic aromatic stretch between 3010 and 3022 cm . sharp carbonyl ( c = o ) stretching vibrations for pyrazolone were seen around 16901695 cm . the presence of secondary amine n h in the skeleton was confirmed from the stretching frequencies between 3325 and 3334 cm . the stretching vibrations for phenolic o h were present between 3420 and 3428 cm . all other peaks in the spectra are in well agreement with the contents of functionalities in the synthesized molecules . the h nmr data of all compounds for the presence of aromatic protons reveal multiplets peak between 6.71 and 8.23 ppm . the spectral data showed a characteristic singlet around 2.812.84 for the presence of n h in the skeleton . a singlet for three protons around 3.213.26 ppm indicates the presence of ch3 on the ring . the c nmr spectrum of all the isolated compounds shows aliphatic ch signals between 45.5 and 53.0 ppm . the other signals and peaks of h nmr , c nmr , and ir are in complete agreement with the assigned structures . the mass spectra of these compounds displayed a molecular ion peak at appropriate m / z values , which were corresponding well with the respected molecular formulas . the second phase of oedema is due to the release of prostaglandins , protease , and lysosome . subcutaneous injection of carrageenin into the rat paw produces inflammation resulting from plasma extravasations , increased tissue water , and plasma protein exudation along with neutrophil extravasations , all due to the metabolism of arachidonic acid . the second phase begins at the end of first phase and remains through third hour up to five hours . in carrageenin administered animals the severe swelling was found to increase upto second hour and then started decreasing till fourth hour . the group treated by standard drug showed decreased paw oedema significantly throughout the period of study . the in vivo study reveals that compounds 4c , 4d , 4f , and 4h are significantly potent anti - inflammatory agents . it is noteworthy from table 1 that all the synthesized compounds except 4a and 4b were found to possess potential anti - inflammatory activity when compared with the reference drug . anthelmintic activities of all prototypes were tested in this bioassay at various concentrations of 12.5 , 25 , 50 , 100 , and 150 mg / ml described in table 2 . all the investigational compounds 4(a h ) acquired the anthelmintic activity at minimal dose of 12.5 mg / ml . compounds 4a , 4b , 4e , and 4 g had shown their significant activity for time taken to paralysis and death when compared to the reference drug , albendazole . compounds 4d and 4f showed their moderate significant action for time taken to paralysis while compound 4a exhibited their highly significant action for time taken to paralysis and death and which is almost equipotent action with respect to reference drug , albendazole . all the synthesized compounds were screened theoretically by using online molinspiration software program . octanol - water partition coefficient ( mi log p ) calculation is used in rational drug design as a measure of molecular hydrophobicity which affects drug - receptor interaction . the polar surface area ( psa ) of a molecule is the surface sum over all polar atoms , primarily oxygen and nitrogen including their attached hydrogens . a topological polar surface area ( tpsa ) calculation is based on summation of tabulated surface contributions of polar fragments ( i.e. , bonding pattern ) . lipophilicity ( log p value ) and polar surface area ( psa ) values are the properties of the prediction of oral bioavailability of drug molecules [ 25 , 26 ] . therefore we have calculated these values for compounds 4(a h ) and compared them with the values obtained for reference drugs , diclofenac and albendazole . lipinski 's rule of 5 is a thumb rule to evaluate drug - likeness ( a chemical compound ) . the rule states that most drug - like molecules have logp 5 , molecular weight 500 , number of hydrogen bond acceptors 10 , and number of hydrogen bond donors 5 . molecules violating more than one of these rules may have problems with bioavailability . for all the compounds , the calculated log p values are less than 5 . the lowest degree of lipophilicity among all the compounds was exhibited by compounds 4a , 4b , 4e , and 4f which are an indication for good water solubility . in conclusion , a rapid and efficient synthesis of 4-aminoantipyrine derivatives via betti reaction has been achieved . higher yields were obtained in a less reaction time following a simple and usual workup . the in vivo and in vitro screening results revealed that these derivatives possess potential anti - inflammatory and anthelmintic activity , respectively . also the molinspiration calculations justify that the derivatives do not violate lipinski 's rule of 5 ; hence , a favourable bioavailability based on drug likeness is indicated .

the present work deals with the synthesis and evaluation of biological activities of 4-aminoantipyrine derivatives derived from a three - component betti reaction . the synthesis was initiated by the condensation of aromatic aldehyde , 4-aminoantipyrine , and 8-hydroxyquinoline in presence of fluorite as catalyst in a simple one - step protocol . the reactions were stirred at room temperature for 1015 min achieving 9295% yield . the structures of synthesized derivatives were established on the basis of spectroscopic and elemental analysis . all derivatives 4(a h ) were screened in vivo and in vitro for anti - inflammatory and anthelmintic activity against a reference drug , diclofenac and albendazole , respectively . the screening results show that compounds 4c , 4d , 4f , and 4h were found to possess potential anti - inflammatory activity while compounds 4a , 4b , 4e , and 4 g are potent anthelmintic agents when compared with reference drugs , respectively . the bioactivity of these derivatives has also been evaluated with respect to lipinski 's rule of five using molinspiration cheminformatics software .

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

multicomponent reactions ( mcrs ) have appeared as an imperative means for the construction of diverse and complex organic molecules . they have intrinsic advantages over two component reactions in several aspects including the simplicity of a one - pot procedures and possible structural variation . the synthetic competence comes from several tandem bond formation reactions in mcrs , which save time , energy , and raw material . betti reaction is a modified type of mannich reaction which has subsequently become vital in synthetic chemistry because of c c bond formation under mild experimental conditions . interest in the chemistry of betti reaction derivatives was also strengthened as it was found to possess various catalytic and biological applications [ 35 ] . nonsteroidal anti - inflammatory drugs ( nsaids ) are the most clinically important medicine used for the treatment of inflammation - related diseases like arthritis , asthma , and cardiovascular diseases . however , the long - term administration of nsaid may induce gastrointestinal ulcers , bleeding , and renal disorders due to their nonselective inhibition of both constitutive ( cox-1 ) and inducible ( cox-2 ) isoforms of the cyclooxygenase enzymes [ 79 ] . therefore , new anti - inflammatory drugs lacking those effects are being searched all over the world as alternatives to nsaids . 4-aminoantipyrine is known for the variety of its clinical applications such as anti - inflammatory , analgesic , antipyretic [ 11 , 12 ] , and several chemotherapeutic agents . it is evident from the reported literatures that compounds possessing pyrazole nuclei showed significant anthelmintic as well as antimicrobial activities [ 1416 ] . the molecular manipulation of a promising lead compound is still a major line of approach for the discovery of new drugs . hence , an attempt has been made in this study to condense 4-aminoantipyrine in a betti reaction to formulate novel biologically potent moieties using fluorite [ 1719 ] as an excellent catalyst . fluorite ( also called fluorspar ) is a natural occurring mineral composed of calcium fluoride ( caf2 ) . fluorite acts as a mild acid in the dehydration reaction and increases the reaction rate without affecting the yield of desired products . the paper deals with the synthesis of 4-aminoantipyrine derivatives via three - component betti reaction and its assessment for biological applications , namely , anti - inflammatory and anthelmintic . we have also investigated the biological applications of these derivatives using online cheminformatics molinspiration software . a comparison between experimental and theoretical predictions of the biological activity has enabled us to identify alternative combined pharmacophore sites structures . all the reagents and solvents are of analytical grade purchased from a commercial source and used directly . ir spectra ( max in cm ) were recorded on a schimadzu - ir prestige 21 spectrometer using kbr technique ; h nmr spectra and c nmr spectra of the synthesized compounds were recorded on a bruker - avance ii 400 ( 400 mhz ) and varian - gemini ( 100 mhz ) spectrometer using dmso - d6 solvent and tms as an internal standard . the elemental analysis ( c , h , n , and s ) of compounds was performed on carlo erba-1108 elemental analyzer . all the experimental protocols were approved by the institutional animal ethics committee ( iaec ) of sharad pawar college of pharmacy , nagpur , india ( approval number : spcp/2013/595 ) . the experiments and the care of the laboratory animals were according to current ethical guidelines by the committee for the purpose of control and supervision on experiments on animals ( cpcsea ) , ministry of environment and forests , government of india , new delhi . diclofenac was used as a reference drug at 10 mg / kg and all the synthesized compounds were administered at 150 mg / kg of body weight . after one hour of the oral administration of synthesized drugs and standard drug , freshly prepared 0.1 ml carrageenin ( 1% carrageenin in 0.9% nacl ) was injected into the left hind limb of each rat under the subplantar aponeurosis . paw volume was recorded at the interval of 0 , 1 , 2 , 3 , and 4 h after carrageenin injection . results were expressed as an increase in paw volume in comparison with the control group . the results were expressed as mean s.e.m and data were statistically analyzed by one - way analysis of variance ( anova ) and p < 0.05 was considered as significant . indian earthworms of the genus and species pheretima posthuma ( family : megascolecidae ) were used for this study . the test compounds 4(a h ) and albendazole were dissolved in minimum quantity of 2% dimethyl sulfoxide ( dmso ) and the volume was adjusted to 10 ml with saline water for making the concentration of 12.5 , 25 , 50 , 100 , and 150 mg / ml . the anthelmintic activity was determined in six observations . paralysis was said to occur when the worms do not revive even in saline water . the results were expressed as mean s.e.m and data were statistically analyzed by one - way analysis of variance ( anova ) and p < 0.05 was considered as significant . a mixture of 4-aminoantipyrine ( 0.01 mol ) 1 , substituted aromatic aldehyde ( 0.01 mol ) 2 , and 8-hydroxyquinoline ( 0.01 mol ) 3 was dissolved in 10 ml of 95% ethanol in one pot and was magnetically stirred at room temperature in presence of fluorite ( 2% weight with respect to all reactants ) ( scheme 1 ) . the crude product was purified by recrystallization from hot ethanol to get the pure product . yield : 95% ; ir ( kbr , cm ) : 3420 ( oh ) , 3325 ( nh ) , 3010 ( ar h ) , 2970 ( ch3 ) , 2872 ( ch ) , h nmr ( dmso - d6 , ppm ) : 2.54 ( s , 3h , ch3 ) , 2.83 ( s , 1h , nh ) , 3.26 ( s , 3h , ch3 ) , 5.33 ( s , 1h , oh ) , 5.45 ( s , 1h , ch ) , 6.717.14 ( m , 5h , ar ) , 7.187.40 ( m , 4h , ar ) , 7.458.21 ( m , 5h , ar ) . ( found ) : c , 71.35 ( 71.30 ) ; h , 4.77(4.76 ) ; n , 15.12 ( 15.09 ) . , 3025 ( ar h ) , 2972 ( ch3 ) , 2874 ( ch ) , 1690 ( c = o ) , 1590 ( c = n ) , 1543 ( no2 ) . h nmr ( dmso - d6 , ppm ) : 2.53 ( s , 3h , ch3 ) , 2.81 ( s , 1h , nh ) , 3.21 ( s , 3h , ch3 ) , 5.31 ( s , 1h , oh ) , 5.42 ( s , 1h , ch ) , 6.747.16 ( m , 5h , ar ) , 7.207.33 ( m , 4h , ar ) , 7.428.22 ( m , 5h , ar ) . ( found ) : c , 73.53 ( 73.56 ) ; h , 4.87(4.86 ) ; n , 15.10 ( 15.12 ) . yield : 92% ; ir ( kbr , cm ) : 3426 ( oh ) , 3327 ( nh ) , 3028 ( ar h ) , 2971 ( ch3 ) , 2870 ( ch ) , 1693 ( c = o ) , 1593 ( c = n ) , 1247 ( och3 ) . ( found ) : c , 70.03 ( 70.05 ) ; h , 5.71(5.76 ) ; n , 13.90 ( 13.92 ) . yield : 83% ; ir ( kbr , cm ) : 3428 ( oh ) , 3334 ( nh ) , 3015 ( ar h ) , 2973 ( ch3 ) , 2872 ( ch ) , 1695 ( c = o ) , 1591 ( c = h nmr ( dmso - d6 , ppm ) : 2.52 ( s , 3h , ch3 ) , 2.84 ( s , 1h , nh ) , 2.88 ( s , 6h , n(ch3)2 ) , 3.25 ( s , 3h , ch3 ) , 5.32 ( s , 1h , oh ) , 5.43 ( s , 1h , ch ) , 6.736.88 ( m , 5h , ar ) , 6.987.20 ( d , 4h , ar ) , 7.258.23 ( m , 5h , ar ) . c nmr ( dmso - d6 , ppm ) : 144.6 ( c1 ) , 125.0 ( c2 ) , 149.7 ( c3 ) , 119.3 ( c4 ) , 131.3 ( c5 ) , 134.5 ( c6 ) , 153.4 ( c1 ) , 126.4 ( c2 ) , 135.6 ( c3 ) , 126.8 ( c4 ) , 124.9 ( c5 ) , 128.7 ( c6 ) , 122.3 ( c7 ) , 148.3 ( c8 ) , 136.7 ( c9 ) , 53.0 ( ch ) , 128.7 ( c1 ) , 135.8 ( c2 ) , 161.2 ( c3 ) , 116.7 ( c4 ) , 33.4 ( c5 ) , 132.3 ( c6 ) , 120.3 ( c7 ) , 126.6 ( c8 ) , 121.5 ( c9 ) , 128.3 ( c10 ) , 120.6 ( c11 ) . yield : 91% ; ir ( kbr , cm ) : 3423 ( oh ) , 3332 ( nh ) , 3020 ( ar h ) , 2970 ( ch3 ) , 2871 ( ch ) , 1690 ( c = o ) , 1590 ( c = h nmr ( dmso - d6 , ppm ) : 2.53 ( s , 3h , ch3 ) , 2.81 ( s , 1h , nh ) , 3.24 ( s , 3h , ch3 ) , 5.08 ( s , 1h , oh ) , 5.31 ( s , 1h , oh ) , 5.44 ( s , 1h , ch ) , 6.716.84 ( m , 5h , ar ) , 6.987.16 ( d , 4h , ar ) , 7.278.18 ( m , 5h , ar ) . yield : 87% ; ir ( kbr , cm ) : 3422 ( oh ) , 3330 ( nh ) , 3021 ( ar h ) , 2974 ( ch3 ) , 2874 ( ch ) , 1695 ( c = o ) , 1593 ( c = n ) . ( found ) : c , 71.62 ( 71.66 ) ; h , 5.57(5.52 ) ; n , 12.12 ( 12.11 ) . yield : 90% ; ir ( kbr , cm ) : 3427 ( oh ) , 3328 ( nh ) , 3018 ( ar h ) , 2972 ( ch3 ) , 2870 ( ch ) , 1692 ( c = o ) , 1592 ( c = h nmr ( dmso - d6 , ppm ) : 2.51 ( s , 3h , ch3 ) , 2.84 ( s , 1h , nh ) , 3.26 ( s , 3h , ch3 ) , 5.35 ( s , 1h , oh ) , 5.45 ( s , 1h , ch ) , 6.736.88 ( m , 5h , ar ) , 7.007.15 ( d , 4h , ar ) , 7.238.21 ( m , 5h , ar ) . c nmr ( dmso - d6 , ppm ) : 144.4 ( c1 ) , 125.1 ( c2 ) , 149.7 ( c3 ) , 120.1 ( c4 ) , 131.3 ( c5 ) , 134.7 ( c6 ) , 153.2 ( c1 ) , 126.4 ( c2 ) , 134.8 ( c3 ) , 127.3 ( c4 ) , 124.2 ( c5 ) , 128.5 ( c6 ) , 122.3 ( c7 ) , 147.3 ( c8 ) , 136.7 ( c9 ) , 52.1 ( ch ) , 128.6 ( c1 ) , 135.5 ( c2 ) , 161.8 ( c3 ) , 116.9 ( c4 ) , 33.4 ( c5 ) , 132.7 ( c6 ) , 121.2 ( c7 ) , 127.8 ( c8 ) , 121.7 ( c9 ) , 128.2 ( c10 ) , 121.5 ( c11 ) . yield : 86% ; ir ( kbr , cm ) : 3425 ( oh ) , 3325 ( nh ) , 3022 ( ar h ) , 2971 ( ch3 ) , 2871 ( ch ) , 1690 ( c = o ) , 1593 ( c = h nmr ( dmso - d6 , ppm ) : 2.53 ( s , 3h , ch3 ) , 2.82 ( s , 1h , nh ) , 3.22 ( s , 3h , ch3 ) , 5.33 ( s , 1h , oh ) , 5.42 ( s , 1h , ch ) , 6.717.11 ( m , 5h , ar ) , 7.187.22 ( m , 4h , ar ) , 7.268.23 ( m , 5h , ar ) . c nmr ( dmso - d6 , ppm ) : 144.7 ( c1 ) , 124.5 ( c2 ) , 149.6 ( c3 ) , 119.4 ( c4 ) , 131.2 ( c5 ) , 134.5 ( c6 ) , 150.0 ( c1 ) , 125.8 ( c2 ) , 135.5 ( c3 ) , 127.5 ( c4 ) , 124.5 ( c5 ) , 129.2 ( c6 ) , 121.4 ( c7 ) , 148.2 ( c8 ) , 136.4 ( c9 ) , 47.5 ( ch ) , 128.9 ( c1 ) , 135.6 ( c2 ) , 161.6 ( c3 ) , 116.4 ( c4 ) , 33.8 ( c5 ) , 132.2 ( c6 ) , 120.6 ( c7 ) , 127.7 ( c8 ) , 121.3 ( c9 ) , 128.5 ( c10 ) , 121.2 ( c11 ) . in order to carry out the synthesis in a more efficient way that minimizes time and the amount of catalyst , a model reaction ( scheme 1 ) was magnetically stirred at room temperature using a naturally occurring mineral , fluorite , as a catalyst . the product was isolated by simple and usual workup with 9295% of yield in simply 1015 min . the solid state ir spectra of these compounds reveal a characteristic aromatic stretch between 3010 and 3022 cm . the presence of secondary amine n h in the skeleton was confirmed from the stretching frequencies between 3325 and 3334 cm . all other peaks in the spectra are in well agreement with the contents of functionalities in the synthesized molecules . the h nmr data of all compounds for the presence of aromatic protons reveal multiplets peak between 6.71 and 8.23 ppm . the spectral data showed a characteristic singlet around 2.812.84 for the presence of n h in the skeleton . a singlet for three protons around 3.213.26 ppm indicates the presence of ch3 on the ring . the other signals and peaks of h nmr , c nmr , and ir are in complete agreement with the assigned structures . the mass spectra of these compounds displayed a molecular ion peak at appropriate m / z values , which were corresponding well with the respected molecular formulas . the second phase of oedema is due to the release of prostaglandins , protease , and lysosome . subcutaneous injection of carrageenin into the rat paw produces inflammation resulting from plasma extravasations , increased tissue water , and plasma protein exudation along with neutrophil extravasations , all due to the metabolism of arachidonic acid . the second phase begins at the end of first phase and remains through third hour up to five hours . in carrageenin administered animals the severe swelling was found to increase upto second hour and then started decreasing till fourth hour . the in vivo study reveals that compounds 4c , 4d , 4f , and 4h are significantly potent anti - inflammatory agents . it is noteworthy from table 1 that all the synthesized compounds except 4a and 4b were found to possess potential anti - inflammatory activity when compared with the reference drug . anthelmintic activities of all prototypes were tested in this bioassay at various concentrations of 12.5 , 25 , 50 , 100 , and 150 mg / ml described in table 2 . all the investigational compounds 4(a h ) acquired the anthelmintic activity at minimal dose of 12.5 mg / ml . compounds 4a , 4b , 4e , and 4 g had shown their significant activity for time taken to paralysis and death when compared to the reference drug , albendazole . compounds 4d and 4f showed their moderate significant action for time taken to paralysis while compound 4a exhibited their highly significant action for time taken to paralysis and death and which is almost equipotent action with respect to reference drug , albendazole . all the synthesized compounds were screened theoretically by using online molinspiration software program . octanol - water partition coefficient ( mi log p ) calculation is used in rational drug design as a measure of molecular hydrophobicity which affects drug - receptor interaction . the polar surface area ( psa ) of a molecule is the surface sum over all polar atoms , primarily oxygen and nitrogen including their attached hydrogens . a topological polar surface area ( tpsa ) calculation is based on summation of tabulated surface contributions of polar fragments ( i.e. , bonding pattern ) . therefore we have calculated these values for compounds 4(a h ) and compared them with the values obtained for reference drugs , diclofenac and albendazole . lipinski 's rule of 5 is a thumb rule to evaluate drug - likeness ( a chemical compound ) . the rule states that most drug - like molecules have logp 5 , molecular weight 500 , number of hydrogen bond acceptors 10 , and number of hydrogen bond donors 5 . molecules violating more than one of these rules may have problems with bioavailability . the lowest degree of lipophilicity among all the compounds was exhibited by compounds 4a , 4b , 4e , and 4f which are an indication for good water solubility . in conclusion , a rapid and efficient synthesis of 4-aminoantipyrine derivatives via betti reaction has been achieved . higher yields were obtained in a less reaction time following a simple and usual workup . the in vivo and in vitro screening results revealed that these derivatives possess potential anti - inflammatory and anthelmintic activity , respectively . also the molinspiration calculations justify that the derivatives do not violate lipinski 's rule of 5 ; hence , a favourable bioavailability based on drug likeness is indicated .

fragment - based drug discovery ( fbdd ) has emerged as a powerful approach to discover drug leads by exploring greater chemical diversity space with smaller libraries . the major challenge , however , is to detect weak binding interactions between drug - like fragments and their protein targets . disulfide tethering was developed as one solution to this problem . in this approach , disulfide - containing fragments are covalently trapped on the protein surface via the reversible formation of disulfide bonds . the advantages of this method include screening the fragments as mixtures rather than as separate entities . screening fragments as mixtures increases the throughput capability of the assay and reduces the number of false positives by introducing competition between the fragments . another technique relies on the use of an -cyanoacrylamide moiety attached to drug - like fragments that react reversibly with noncatalytic cysteines present at the binding site of the protein of interest . whether it is possible to design a robust system where the protein can select the best binder from a mixture of electrophilic fragments under irreversible conditions to identify novel leads such an approach would be particularly powerful because the identified fragments can subsequently retain their electrophilic tether while being elaborated into a covalent drug . however , one concern with such an approach is the danger of selecting the most reactive fragment rather than the fragment with the most specific binding affinity to the protein target . if the electrophilic fragments are too reactive , cysteines or other nucleophilic residues present on the protein surface can undergo nonspecific covalent modifications by the fragments irrespective of their binding affinity . alternatively , hyper - reactive cysteines or other nucleophilic residues can nonspecifically react with even moderately electrophilic fragments , leading to nonspecific covalent modifications of the protein . in addition , no systematic studies have been done to investigate the kinetic reactivity of cysteine reactive electrophiles attached to a large number ( 50 ) drug - like fragments in order to outline general principles and design rules for irreversible tethering . while this work was in progress , nonoo , et al . reported the first irreversible tethering method using a small 10-member acrylamide library , which included known reversible thymidylate synthase inhibitor scaffolds . however , a hyper - reactive acrylamide in their library had to be discarded , and no systematic studies have been done further to investigate the reactivity of and outline design rules for drug - like libraries for irreversible tethering . moreover , there are still no reports of irreversible fragment screening of an unbiased library to identify novel and selective binding fragments . therefore , whether it is possible to rationally design an electrophilic library of drug - like fragments for irreversible tethering is still a concern . this report addresses this concern and shows that the proper selection of a cysteine reactive electrophile yields a chemical system that can select weakly bound electrophilic fragments from a mixture and covalently trap the best binders at the highly reactive catalytic cysteine of the model cysteine protease papain . the discovered fragments behave as weak and irreversible inhibitors of papain and have novel nonpeptidic structures . the reported method serves as an entry point to discover nonpeptidic inhibitors of other cysteine proteases , which are promising drug targets to treat parasitic infections . to find an electrophile which is suitable for irreversible tethering , we explored the cysteine reactivity profiles of four michael acceptors : acrylamides 1 , vinylsulfonamides 2 , aminomethyl methyl acrylates 3 , methyl vinylsulfones 4 ( figure 1a , b ) . ( a ) general scheme of nmr rate studies . ( b ) chemical structures of the electrophiles 14 tested for suitability for irreversible tethering and their pseudo - first - order reaction rates with n - acetylcysteine methylester at pd 8.0 as measured by nmr spectroscopy . to test how the cysteine reactivity of these electrophiles would be affected by the structure of attached drug - like fragments , we installed acrylamide and vinylsulfonamide electrophiles on aniline , p - meo - aniline , and p - no2-aniline to yield electrophiles 1a c and 2a c . the methyl acrylate and vinylsulfone electrophiles in 3 and 4 were covalently attached to derivatives of benzoic acid , p - meo - benzoic acid , and p - no2-benzoic acid to yield 3a c and 4a c . we envisioned that the different mesomeric and inductive effects of the och3 , h , and no2 moieties would cause changes in the reactivity of electrophiles 14 toward cysteine , and these changes would be representative of fluctuations in the reactivity of drug - like fragments toward cysteines . the electrophile that displayed the least fluctuation in reactivity toward cysteine would be the most optimal electrophile to use for irreversible tethering . we therefore measured the pseudo - first - order reaction rates for each of the compounds 14 with n - acetylcysteine methyl ester using nmr spectroscopy ( figure 1b ) . interestingly , we found that acrylamides 1a c displayed a 2044-fold difference in reactivity , with the no2 derivative being the most reactive . because many drug - like fragments contain an amino group attached directly to electron - deficient aromatic rings , we envisioned that similar to compounds 1a c there could be large fluctuations in the reactivity of such an acrylamide library toward thiols , which would make this library problematic to use . indeed , as we mentioned previously , in the first publication detailing irreversible tethering method using acrylamides one fragment had to be discarded due to its hyperreactivity . vinylsulfonamides 2a c displayed only an 8-fold difference in reactivity toward n - acetylcysteine methyl ester . this result was encouraging , yet we sought electrophiles with an even more narrow range of reactivities . to our delight , both the 3a c and 4a c series displayed much more balanced reactivity toward cysteine , with only 1.6- and 1.4-fold differences , respectively , in the reactivity between the least reactive and the most reactive electrophiles . we chose acrylates 3 for further studies because they were 10-fold less reactive than vinylsulfones 4 and therefore less prone to nonspecific covalent modifications of nucleophilic amino acid side chains in proteins . in addition , acrylates are established electrophiles present in irreversible inhibitors of cysteine proteases with activities in vitro biochemical and cell - based assays . importantly , in vitro kinact / ki values of acrylate cysteine protease inhibitors vary dramatically ( up to 170-fold in the case of falcipain inhibitors ) with changes in the structure of the peptide - derived directing group . this indicates that useful levels of kinetic discrimination can be achieved upon structural changes of the directing group despite the high reactivity of the catalytic cysteine in cysteine proteases . moreover , the acrylate functionality has been shown to have good pharmacokinetic properties and is present in an orally bioavailable inhibitor of human rhinovirus 3c protease . these considerations further confirmed to us that acrylate 3 is a good starting point for validating irreversible tethering . because known acrylate inhibitors are mostly peptidic in nature , we sought to discover novel nonpeptidic inhibitors with irreversible tethering . we further validated the utility of electrophile 3 as a thiol - reactive tether by making a library of 100 structurally diverse drug - like fragments 6105 containing this electrophile . the library was constructed with an hbtu amide coupling with commercially available carboxylic acid fragments ( figure 2a ) . the acids were selected with rule of three criteria and a subsequent diversity analysis . we measured the reaction rates for the first 50 fragments to confirm that this library would have balanced cysteine reactivity and could be used for irreversible tethering ( figure 2b ) . as we expected , these 50 fragments displayed a narrow range of chemical reactivities similar to 3a c . overall , we observed only a 2.4-fold difference in the reactivity between the least reactive ( k1 3.327 10 s ) and the most reactive ( k1 7.951 10 s ) fragment ( figure 2b , supporting information ( si ) table s1 ) . pseudo - first - order nmr rate plots of the reaction of compounds 655 with n - acetyl cysteine methyl ester . , we asked if we could use this library to discover specific covalent enzyme inhibitors with novel structures . as a model protein we chose the cysteine protease papain . we reasoned that the presence of a highly reactive active site cysteine in papain would serve as a stringent specificity test for the proposed irreversible tethering method . we hypothesized that if the designed chemical system displays specificity in the presence of the highly reactive catalytic cysteine of papain , this system could also be used to discover ligands for less reactive noncatalytic cysteines . in addition , papain is the founding member of a large family of cysteine proteases , so if the developed system produced inhibitors of papain , it could serve as an entry point to discover inhibitors of other medically relevant cysteine proteases . for our initial screening , we used a simple ms assay similar to the original disulfide tethering screening conditions . papain ( 10 m ) was incubated for 1 h with 10 reaction mixtures that each contained 10 electrophilic fragments ( 100 m each ) ( si table s2 ) . each fragment in the reaction mixture had a unique molecular weight ( at least 5 da difference from the closest fragment ) to ensure that whole protein esi - ms could identify candidate hits unambiguously . hits were defined as any compounds which labeled papain more than 50% . remarkably , under these reaction conditions , we observed strong monolabeling of papain by three electrophilic fragments in three separate reaction mixtures : 6 , 7 , and 8 ( figure 3 ) . such selectivity is impressive , given a 9-fold excess of other cysteine reactive electrophiles over compounds 6 , 7 , and 8 . moreover , we did not detect significant covalent modification of papain with the other seven reaction mixtures ( si figure s1 ) . this is despite the fact that these reaction mixtures contain a 100-fold excess of cysteine reactive electrophiles relative to the highly reactive catalytic cysteine of papain . furthermore , compounds 6 , 7 , and 8 labeled papain even though the corresponding reaction mixtures contained fragments that were equally or even more reactive toward n - acetylcysteine methyl ester . this observation further suggests that in our system the chemical structure of the drug - like fragment rather than its reactivity determines the covalent labeling of papain . representative ms spectra of four reaction mixtures containing 10 electrophilic fragments each screened against papain . papain ( 10 m ) was incubated with a mixture of 10 electrophilic fragments ( 100 m each ) for 1 h , followed by gel filtration and esi - ms of the intact protein . additionally , compounds 68 demonstrated robust labeling of papain in the presence of 10 mm glutathione ( 1000-fold excess relative to papain ) , confirming that compounds 68 covalently label papain due to their specific binding to papain and not simply due to their greater thiol reactivity ( si figure s2 ) . we were unable to directly confirm labeling of the catalytic cysteine because the catalytic cysteine peptide was not detectable by esi - ms or maldi - tof upon digestion with trypsin , chymotrypsin , or glu - c proteases . however , preincubation of papain with compounds 68 , followed by treatment with 106 , a known papain inhibitor which reacts with its catalytic cysteine , did not cause dilabeling of papain ( si figure s3a ) . additionally , pretreatment of papain with 106 also blocked subsequent labeling by compounds 68 ( si figure s3b ) . these results suggest that compounds 68 and inhibitor 106 most likely react with the same nucleophilic residue of papain . compounds 68 labeled papain in a 1:1 stoichiometry at both 100 m and 1 mm concentrations , confirming the specificity of these electrophiles for cysteine ( si figure s4 ) . moreover , the observed covalent labeling of papain was irreversible because the covalent adducts were stable to dialysis . we subsequently tested compounds 68 in an enzymatic assay to confirm that they inhibited papain in the concentration and time dependent manner that is characteristic of irreversible inhibitors . using assay conditions previously described for papain , we determined kinact / ki values for compounds 68 ( figure 4 , si figure s6 ) . notably , compound 7 was as potent at inhibiting papain as a known moderate peptidic inhibitor 107 , but compounds 68 were less potent inhibitors than the known strong peptidic papain inhibitor 106 . this result is expected because irreversible tethering is designed to detect weak binding interactions between the drug - like fragments and the protein target to identify initial hits . compounds 68 were all more potent inhibitors than the weak peptidic papain inhibitor 108 . a negative control molecule 19 , which did not label papain in our screen , was 10-fold less potent at inhibiting papain than the least potent inhibitor 6 and 33-fold less potent than the most potent inhibitor 7 . remarkably , compounds 68 do not have a peptidic character in comparison to traditional cysteine protease inhibitors , including known papain inhibitors ( figure 4 ) . this result is significant because the proposed method can serve as an entry point to discover other types of nonpeptidic inhibitors for medically relevant cysteine proteases , avoiding the known undesirable pharmacological properties of peptidic inhibitors . second - order inhibition plots and kinact / ki values for papain inhibitor compounds 68 and known papain inhibitors 106108 . note : testing of compound 7 at higher concentrations was limited by poor solubility . to further test the specificity of the developed irreversible tethering system , we conducted a counter - screen of the same set of 100 compounds ( 10 mixtures of 10 compounds each ) against three other enzymes : human rhinovirus 3c protease , the deubiquitinase usp08 , and the e2 ubiquitin - conjugating enzyme ubch7 . human rhinovirus 3c protease is a cysteine protease , an antiviral drug target , and there are known orally bioavailable acrylate inhibitors for this protease . recent reports have indicated that targeting usp08 is a promising approach to overcome gefitinib resistance in lung cancer , while ubch7 on the other hand regulates the entrance into and progression through the s - phase of the cell cycle . as a source of hrv3c protease for our experiments we used gst - tagged hrv3c protease . we have found that hrv3c protease was labeled by compound 22 ( 35% labeling ) as well as compounds 32 and 98 ( 20% labeling ) under the same reaction conditions ( si figure s7 ) . none of the three papain hits and remaining electrophilic fragments reacted with hrv3c protease under these reaction conditions , indicating that these hits are selective binders . although the three hrv3c hits did not label their target as strongly as the papain hits did , they could eventually be optimized into potent inhibitors of this clinically important cysteine protease . for ubch7 and usp08 , we found that none of compounds 6105 covalently modified these enzymes ( si figures s8 , s9 ) under the same reaction conditions . when we increased the incubation time with usp08 to 4 h , we found two compounds that weakly labeled 30% of usp08 . one was compound 6 , while another was a unique compound ( 9 ) ( si figure s10 ) . the other two papain inhibitors 7 and 8 did not label usp08 even after 4 h , showing that our system is well behaved and can identify selective binders . in summary , we have rationally designed a chemical system for screening mixtures of electrophilic fragments against the catalytic cysteine of a protein of interest , which eliminates the concern that such an approach would only select the most reactive fragment or otherwise be nonspecific due to the high reactivity of the catalytic cysteine . using this method , we identified specific , nonpeptidic covalent inhibitors of the cysteine protease papain , which contain novel chemical scaffolds . this is the first example of a successful screen of an unbiased library of electrophilic compounds under irreversible conditions which led to the discovery of specific and novel inhibitor structures for the enzyme of interest . , electrophilic fragments 6105 are prepared in one step from commercially available materials using a robust amide bond formation reaction . moreover , the synthesized electrophilic fragments elicit a predictable and narrow range of chemical reactivities toward thiols and do not react with other nucleophilic residues such as histidine or lysine . one hundred compounds can be screened in one day without the use of special robotic equipment . moreover , mixtures of electrophilic fragments can be stored as dmso stocks , transported , and used to screen fragments against novel protein targets . the developed irreversible tethering method displays a high hit rate ( 3% for papain and hrv3c protease ) , and the discovered papain inhibitors have weak potency in enzymatic assays . our failure to discover strong inhibitors of usp08 and ubch7 is most likely not due to the limitations of the method but rather due to the limited sampling of chemical space because only 100 fragments were prepared and tested . because usp08 and ubch7 do not have classical hydrophobic binding pockets like the p2 substrate pocket of papain , it is likely that a larger library will be required to find adequate binders . while the developed approach can be used to tether weakly bound fragments to the highly reactive catalytic cysteine of papain , it remains to be seen whether the same approach can be used to tether weakly bound fragments to noncatalytic cysteines on protein surfaces . protein interaction inhibitors by targeting catalytic and noncatalytic cysteines will be reported in the near future . using the discovery studio package with pipeline pilot from accelrys , 94275 commercially available carboxylic acids were identified from the chembridge , chemdiv , maybridge , nci , and sigma - aldrich libraries using smarts query strings . of these , 62000 were removed because they contained reactive functional groups ( e.g. , acyl halides ) or were unsuitable leads ( e.g. , nitro compounds ) . criteria which were modified to increase the number of resulting compounds : molecular weight ( mw ) 350 da , alogp 3 , hydrogen - bond acceptors 3 , hydrogen - bond donors 3 , rotatable bonds 3 , and polar surface area 80 . a principal component analysis and neighborhood algorithm was applied to the 1522 remaining compounds to produce 281 fragments with a 0.75 diversity index . then 100 of these compounds were initially selected based on affordability and the ease of future analogue synthesis the carboxylic acid fragment ( 0.2 mmol ) was dissolved in dimethylformamide ( 0.2 m , 1 ml ) , then 5 ( 46 mg , 0.2 mmol ) , hbtu ( 73.8 mg , 0.16 mmol ) , and hobt ( 29.8 mg , 0.22 mmol ) were added , followed by etn(i - pr)2 ( 100.7 l , 0.6 mmol ) . the reaction was stirred at 23 c for 16 h. tlc at 16 h showed conversion to product . the reaction was quenched with h2o ( 5 ml ) and extracted three times with ch2cl2 ( 5 ml ) . the combined organic layers were washed with 1 m hcl ( 10 ml ) , saturated aqueous nahco3 ( 10 ml ) , and saturated aqueous nacl ( 10 ml ) . purified by flash column chromatography with a ch3oh / ch2cl2 , ch3oh gradient 05% to yield compounds 6108 . for initial library creation , compounds were characterized by h nmr and low resolution ms . all compounds tested in enzymatic assays were also characterized by c nmr and 95% purity was confirmed by hplc . n - acetyl cysteine methyl ester was dissolved in 2:1 deuterated pbs : dmso - d6 ( 78 mm ) with 10 mm ch2cl2 as an internal standard . the electrophile ( 10 mm ) was then added immediately prior to acquiring nmr spectra . h spectra were taken every 30 s for 30 min ( or every 4 s for 5 min for highly reactive compounds 1c and 2a c ) . the integrals of the vinyl peaks were used to determine the concentration of the electrophile over time . the natural logarithm of the concentration of the electrophile vs time was then plotted using graphpad prism software . the linear slope of this plot was used to determine the pseudo - first - order rate constant . deuterated pbs recipe : 20 mm na3po4 , 50 mm nacl in d2o was adjusted to pd 8 with dcl solution . papain ( sigma p4762 , 10 m ) , ubch7 ( recombinantly expressed , 10 m ) , gst-264 hrv3c protease ( recombinantly expressed , 10 m ) , or usp08 ( recombinantly expressed , 10 m ) in 50 mm hepes , 150 mm nacl , and 0.1 mm edta ph 7.5 was treated with a mixture of 10 fragments ( si table s2 ) ( 10 mm dmso stock solutions , final concentrations : 100 m of each fragment , and 1% dmso ) . the reaction mixture was incubated for 1 h or 4 h at 23 c before being passed through zeba gel filtration columns ( thermo , 7k mwco ) to remove unreacted fragments . the protein solution was then immediately analyzed by whole protein lc / esi - ms . accurate - mass data were obtained on an agilent 6210a lc - tof mass spectrometer in positive ion mode using electrospray ionization . samples were chromatographed on the lc - tof instrument using a poroshell 120 ec - c18 hplc column ( 2.1 mm 50 mm , 2.7 m ) , an agilent series 1200 hplc binary pump , and an agilent series 1200 autoinjector . the hplc column was held at 45 c , and the autosampler was held at 8 c . mobile phase a was a solution of 0.1% formic acid in water : acetonitrile ( 19:1 ) . the gradient used was 0% b for 2 min , ramping linearly to 90% b from 2 to 5 min , holding at 90% b from 5 to 7 min , and then returning to 0% b at 7.1 min . the column was allowed to equilibrate for 2.7 min before the next injection was initiated . the eluent from the column was diverted to waste for the first 2 min . the spectra were acquired from 301 to 3200 da using a gas temperature of 340 c , a gas flow of 7 l / min , and the nebulizer gas at 35 psi . the following voltages were used : capillary 4200 v , fragmentor 230 v , skimmer 64 v , and octapole rf peak 250 v. spectra were acquired at a rate of 1 spectra / s . maximum entropy deconvolutions were performed with a mass step of 1 , s / n threshold of 30 , average mass at 90% of peak height , and 5 charge states minimum . papain ( 4.8 m ) in 50 mm na3po4 and 2 mm edta was preactivated with 1 mm dtt for 30 min . activated papain ( 3.84 m ) in 4:1 mixture of 50 mm na3po4 and 2 mm edta at ph 6.2 and acetronitrile was then preincubated for 1 h with varying concentrations of the electrophilic fragment . every 10 min , 10 l of the reaction mixture was added to a well of 96-well plate containing 100 l of 4:1 mixture of 50 mm na3po4/2 mm edta / ph 6.2:acetronitrile with 400 m cbz - gly - onp . p - nitrophenol product formation was monitored by absorbance at 340 nm ( : 6800 m cm ) with a biotek synergy 4 plate reader . product concentration vs time was plotted with graphpad prism software , and the initial slope was calculated to determine enzymatic activity ( e ) . the values of kinact / ki for each inhibitor were then determined according to the method of kitz and wilson . briefly , the slopes of the plots of ln(100 einhibited / euninhibited ) vs time were used to determine the pseudo - first - order inhibition constant kobs for a given concentration of a given inhibitor . the slope of the plot of kobs vs [ inhibitor ] was then used to determine the second - order inhibition constant kinact / ki ( because [ i ] ki , the plots were linear at the concentrations tested ) .

a novel fragment - based drug discovery approach is reported which irreversibly tethers drug - like fragments to catalytic cysteines . we attached an electrophile to 100 fragments without significant alterations in the reactivity of the electrophile . a mass spectrometry assay discovered three nonpeptidic inhibitors of the cysteine protease papain . the identified compounds display the characteristics of irreversible inhibitors . the irreversible tethering system also displays specificity : the three identified papain inhibitors did not covalently react with ubch7 , usp08 , or gst - tagged human rhinovirus 3c protease .

Introduction Results Discussion and Conclusion Experimental Section

fragment - based drug discovery ( fbdd ) has emerged as a powerful approach to discover drug leads by exploring greater chemical diversity space with smaller libraries . the major challenge , however , is to detect weak binding interactions between drug - like fragments and their protein targets . screening fragments as mixtures increases the throughput capability of the assay and reduces the number of false positives by introducing competition between the fragments . another technique relies on the use of an -cyanoacrylamide moiety attached to drug - like fragments that react reversibly with noncatalytic cysteines present at the binding site of the protein of interest . whether it is possible to design a robust system where the protein can select the best binder from a mixture of electrophilic fragments under irreversible conditions to identify novel leads such an approach would be particularly powerful because the identified fragments can subsequently retain their electrophilic tether while being elaborated into a covalent drug . however , one concern with such an approach is the danger of selecting the most reactive fragment rather than the fragment with the most specific binding affinity to the protein target . if the electrophilic fragments are too reactive , cysteines or other nucleophilic residues present on the protein surface can undergo nonspecific covalent modifications by the fragments irrespective of their binding affinity . alternatively , hyper - reactive cysteines or other nucleophilic residues can nonspecifically react with even moderately electrophilic fragments , leading to nonspecific covalent modifications of the protein . in addition , no systematic studies have been done to investigate the kinetic reactivity of cysteine reactive electrophiles attached to a large number ( 50 ) drug - like fragments in order to outline general principles and design rules for irreversible tethering . reported the first irreversible tethering method using a small 10-member acrylamide library , which included known reversible thymidylate synthase inhibitor scaffolds . however , a hyper - reactive acrylamide in their library had to be discarded , and no systematic studies have been done further to investigate the reactivity of and outline design rules for drug - like libraries for irreversible tethering . moreover , there are still no reports of irreversible fragment screening of an unbiased library to identify novel and selective binding fragments . therefore , whether it is possible to rationally design an electrophilic library of drug - like fragments for irreversible tethering is still a concern . this report addresses this concern and shows that the proper selection of a cysteine reactive electrophile yields a chemical system that can select weakly bound electrophilic fragments from a mixture and covalently trap the best binders at the highly reactive catalytic cysteine of the model cysteine protease papain . the discovered fragments behave as weak and irreversible inhibitors of papain and have novel nonpeptidic structures . the reported method serves as an entry point to discover nonpeptidic inhibitors of other cysteine proteases , which are promising drug targets to treat parasitic infections . to find an electrophile which is suitable for irreversible tethering , we explored the cysteine reactivity profiles of four michael acceptors : acrylamides 1 , vinylsulfonamides 2 , aminomethyl methyl acrylates 3 , methyl vinylsulfones 4 ( figure 1a , b ) . ( a ) general scheme of nmr rate studies . ( b ) chemical structures of the electrophiles 14 tested for suitability for irreversible tethering and their pseudo - first - order reaction rates with n - acetylcysteine methylester at pd 8.0 as measured by nmr spectroscopy . to test how the cysteine reactivity of these electrophiles would be affected by the structure of attached drug - like fragments , we installed acrylamide and vinylsulfonamide electrophiles on aniline , p - meo - aniline , and p - no2-aniline to yield electrophiles 1a c and 2a c . we envisioned that the different mesomeric and inductive effects of the och3 , h , and no2 moieties would cause changes in the reactivity of electrophiles 14 toward cysteine , and these changes would be representative of fluctuations in the reactivity of drug - like fragments toward cysteines . the electrophile that displayed the least fluctuation in reactivity toward cysteine would be the most optimal electrophile to use for irreversible tethering . we therefore measured the pseudo - first - order reaction rates for each of the compounds 14 with n - acetylcysteine methyl ester using nmr spectroscopy ( figure 1b ) . because many drug - like fragments contain an amino group attached directly to electron - deficient aromatic rings , we envisioned that similar to compounds 1a c there could be large fluctuations in the reactivity of such an acrylamide library toward thiols , which would make this library problematic to use . indeed , as we mentioned previously , in the first publication detailing irreversible tethering method using acrylamides one fragment had to be discarded due to its hyperreactivity . to our delight , both the 3a c and 4a c series displayed much more balanced reactivity toward cysteine , with only 1.6- and 1.4-fold differences , respectively , in the reactivity between the least reactive and the most reactive electrophiles . we chose acrylates 3 for further studies because they were 10-fold less reactive than vinylsulfones 4 and therefore less prone to nonspecific covalent modifications of nucleophilic amino acid side chains in proteins . in addition , acrylates are established electrophiles present in irreversible inhibitors of cysteine proteases with activities in vitro biochemical and cell - based assays . importantly , in vitro kinact / ki values of acrylate cysteine protease inhibitors vary dramatically ( up to 170-fold in the case of falcipain inhibitors ) with changes in the structure of the peptide - derived directing group . this indicates that useful levels of kinetic discrimination can be achieved upon structural changes of the directing group despite the high reactivity of the catalytic cysteine in cysteine proteases . moreover , the acrylate functionality has been shown to have good pharmacokinetic properties and is present in an orally bioavailable inhibitor of human rhinovirus 3c protease . these considerations further confirmed to us that acrylate 3 is a good starting point for validating irreversible tethering . because known acrylate inhibitors are mostly peptidic in nature , we sought to discover novel nonpeptidic inhibitors with irreversible tethering . we further validated the utility of electrophile 3 as a thiol - reactive tether by making a library of 100 structurally diverse drug - like fragments 6105 containing this electrophile . we measured the reaction rates for the first 50 fragments to confirm that this library would have balanced cysteine reactivity and could be used for irreversible tethering ( figure 2b ) . overall , we observed only a 2.4-fold difference in the reactivity between the least reactive ( k1 3.327 10 s ) and the most reactive ( k1 7.951 10 s ) fragment ( figure 2b , supporting information ( si ) table s1 ) . pseudo - first - order nmr rate plots of the reaction of compounds 655 with n - acetyl cysteine methyl ester . as a model protein we chose the cysteine protease papain . we reasoned that the presence of a highly reactive active site cysteine in papain would serve as a stringent specificity test for the proposed irreversible tethering method . we hypothesized that if the designed chemical system displays specificity in the presence of the highly reactive catalytic cysteine of papain , this system could also be used to discover ligands for less reactive noncatalytic cysteines . in addition , papain is the founding member of a large family of cysteine proteases , so if the developed system produced inhibitors of papain , it could serve as an entry point to discover inhibitors of other medically relevant cysteine proteases . each fragment in the reaction mixture had a unique molecular weight ( at least 5 da difference from the closest fragment ) to ensure that whole protein esi - ms could identify candidate hits unambiguously . such selectivity is impressive , given a 9-fold excess of other cysteine reactive electrophiles over compounds 6 , 7 , and 8 . moreover , we did not detect significant covalent modification of papain with the other seven reaction mixtures ( si figure s1 ) . this observation further suggests that in our system the chemical structure of the drug - like fragment rather than its reactivity determines the covalent labeling of papain . papain ( 10 m ) was incubated with a mixture of 10 electrophilic fragments ( 100 m each ) for 1 h , followed by gel filtration and esi - ms of the intact protein . additionally , compounds 68 demonstrated robust labeling of papain in the presence of 10 mm glutathione ( 1000-fold excess relative to papain ) , confirming that compounds 68 covalently label papain due to their specific binding to papain and not simply due to their greater thiol reactivity ( si figure s2 ) . we were unable to directly confirm labeling of the catalytic cysteine because the catalytic cysteine peptide was not detectable by esi - ms or maldi - tof upon digestion with trypsin , chymotrypsin , or glu - c proteases . however , preincubation of papain with compounds 68 , followed by treatment with 106 , a known papain inhibitor which reacts with its catalytic cysteine , did not cause dilabeling of papain ( si figure s3a ) . these results suggest that compounds 68 and inhibitor 106 most likely react with the same nucleophilic residue of papain . moreover , the observed covalent labeling of papain was irreversible because the covalent adducts were stable to dialysis . we subsequently tested compounds 68 in an enzymatic assay to confirm that they inhibited papain in the concentration and time dependent manner that is characteristic of irreversible inhibitors . this result is expected because irreversible tethering is designed to detect weak binding interactions between the drug - like fragments and the protein target to identify initial hits . compounds 68 were all more potent inhibitors than the weak peptidic papain inhibitor 108 . a negative control molecule 19 , which did not label papain in our screen , was 10-fold less potent at inhibiting papain than the least potent inhibitor 6 and 33-fold less potent than the most potent inhibitor 7 . remarkably , compounds 68 do not have a peptidic character in comparison to traditional cysteine protease inhibitors , including known papain inhibitors ( figure 4 ) . this result is significant because the proposed method can serve as an entry point to discover other types of nonpeptidic inhibitors for medically relevant cysteine proteases , avoiding the known undesirable pharmacological properties of peptidic inhibitors . second - order inhibition plots and kinact / ki values for papain inhibitor compounds 68 and known papain inhibitors 106108 . to further test the specificity of the developed irreversible tethering system , we conducted a counter - screen of the same set of 100 compounds ( 10 mixtures of 10 compounds each ) against three other enzymes : human rhinovirus 3c protease , the deubiquitinase usp08 , and the e2 ubiquitin - conjugating enzyme ubch7 . human rhinovirus 3c protease is a cysteine protease , an antiviral drug target , and there are known orally bioavailable acrylate inhibitors for this protease . recent reports have indicated that targeting usp08 is a promising approach to overcome gefitinib resistance in lung cancer , while ubch7 on the other hand regulates the entrance into and progression through the s - phase of the cell cycle . as a source of hrv3c protease for our experiments we used gst - tagged hrv3c protease . none of the three papain hits and remaining electrophilic fragments reacted with hrv3c protease under these reaction conditions , indicating that these hits are selective binders . although the three hrv3c hits did not label their target as strongly as the papain hits did , they could eventually be optimized into potent inhibitors of this clinically important cysteine protease . for ubch7 and usp08 , we found that none of compounds 6105 covalently modified these enzymes ( si figures s8 , s9 ) under the same reaction conditions . the other two papain inhibitors 7 and 8 did not label usp08 even after 4 h , showing that our system is well behaved and can identify selective binders . in summary , we have rationally designed a chemical system for screening mixtures of electrophilic fragments against the catalytic cysteine of a protein of interest , which eliminates the concern that such an approach would only select the most reactive fragment or otherwise be nonspecific due to the high reactivity of the catalytic cysteine . using this method , we identified specific , nonpeptidic covalent inhibitors of the cysteine protease papain , which contain novel chemical scaffolds . this is the first example of a successful screen of an unbiased library of electrophilic compounds under irreversible conditions which led to the discovery of specific and novel inhibitor structures for the enzyme of interest . moreover , the synthesized electrophilic fragments elicit a predictable and narrow range of chemical reactivities toward thiols and do not react with other nucleophilic residues such as histidine or lysine . the developed irreversible tethering method displays a high hit rate ( 3% for papain and hrv3c protease ) , and the discovered papain inhibitors have weak potency in enzymatic assays . our failure to discover strong inhibitors of usp08 and ubch7 is most likely not due to the limitations of the method but rather due to the limited sampling of chemical space because only 100 fragments were prepared and tested . while the developed approach can be used to tether weakly bound fragments to the highly reactive catalytic cysteine of papain , it remains to be seen whether the same approach can be used to tether weakly bound fragments to noncatalytic cysteines on protein surfaces . protein interaction inhibitors by targeting catalytic and noncatalytic cysteines will be reported in the near future . using the discovery studio package with pipeline pilot from accelrys , 94275 commercially available carboxylic acids were identified from the chembridge , chemdiv , maybridge , nci , and sigma - aldrich libraries using smarts query strings . all compounds tested in enzymatic assays were also characterized by c nmr and 95% purity was confirmed by hplc . the electrophile ( 10 mm ) was then added immediately prior to acquiring nmr spectra . the integrals of the vinyl peaks were used to determine the concentration of the electrophile over time . the natural logarithm of the concentration of the electrophile vs time was then plotted using graphpad prism software . deuterated pbs recipe : 20 mm na3po4 , 50 mm nacl in d2o was adjusted to pd 8 with dcl solution . papain ( sigma p4762 , 10 m ) , ubch7 ( recombinantly expressed , 10 m ) , gst-264 hrv3c protease ( recombinantly expressed , 10 m ) , or usp08 ( recombinantly expressed , 10 m ) in 50 mm hepes , 150 mm nacl , and 0.1 mm edta ph 7.5 was treated with a mixture of 10 fragments ( si table s2 ) ( 10 mm dmso stock solutions , final concentrations : 100 m of each fragment , and 1% dmso ) . accurate - mass data were obtained on an agilent 6210a lc - tof mass spectrometer in positive ion mode using electrospray ionization . the hplc column was held at 45 c , and the autosampler was held at 8 c . the gradient used was 0% b for 2 min , ramping linearly to 90% b from 2 to 5 min , holding at 90% b from 5 to 7 min , and then returning to 0% b at 7.1 min . maximum entropy deconvolutions were performed with a mass step of 1 , s / n threshold of 30 , average mass at 90% of peak height , and 5 charge states minimum . activated papain ( 3.84 m ) in 4:1 mixture of 50 mm na3po4 and 2 mm edta at ph 6.2 and acetronitrile was then preincubated for 1 h with varying concentrations of the electrophilic fragment . every 10 min , 10 l of the reaction mixture was added to a well of 96-well plate containing 100 l of 4:1 mixture of 50 mm na3po4/2 mm edta / ph 6.2:acetronitrile with 400 m cbz - gly - onp . briefly , the slopes of the plots of ln(100 einhibited / euninhibited ) vs time were used to determine the pseudo - first - order inhibition constant kobs for a given concentration of a given inhibitor . the slope of the plot of kobs vs [ inhibitor ] was then used to determine the second - order inhibition constant kinact / ki ( because [ i ] ki , the plots were linear at the concentrations tested ) .

since watson and crick proposed its three - dimensional structure , much effort has been spent to understand its structure and dynamics . crick double - helix from separated strands is a biologically very important process observed during replication , translation , or dna repair . also , recently , the rapidly growing field of computer simulations of biological systems has developed models and tools that can provide some insight into this important process . all - atom molecular dynamics ( md ) methods offer the deepest insight into the time evolution of dna at the atomic level . the charmm and amber simulation packages have been used to study the duplex dynamics of dna , mainly in the vicinity of equilibrium . the highlights of near - equilibrium all - atom dna molecular dynamics simulations include the systematic study of nearest - neighbor effects on various short dna sequences performed by the ascona b - dna consortium , and the microsecond simulations of the drew - dickerson dodecamer or the b dna element . although all - atom md simulations have proven to be very useful for studying near - equilibrium dynamics , their very high computational cost , related to the large number of degrees of freedom and high - frequency oscillations , forcing a short time - step of integration , currently render them unsuitable for simulating long time - scale nonequilibrium processes , such as formation of duplex dna from separate strands . only recently , those enhanced sampling methods , such as replica exchange , facilitated simulations of formation of very short ( 4 base pair ( bp ) ) duplexes of dna . the combination of metadynamics and replica exchange , called be - meta method , was applied to slightly larger system , dna hexamer , but the more - important biological process of formation of long helices remains prohibitively expensive for all - atom md simulations and has recently become a target for coarse - graining methodology . coarse - grained models can be divided into three categories : statistical models , continuum models , and reduced interaction - center models . in the first category , the early work of zimm and rice and poland and scheraga neglected all structural and dynamical information and used only the free - energy gain per base - pair formation for computation of the partition function , which , in turn , was transformed into a thermodynamic picture of the process of helix formation . the continuum models approximate dsdna as a continuum elastic rod and by design can not be applied to the dna formation process . the last category covers all models in which groups of atoms are replaced by a reduced number of interaction centers connected by elastic and/or rigid virtual bonds . a properly designed coarse - graining procedure should reduce the number of interaction centers , leading to a significant speedup of computations , but simultaneously , the simplified potential energy function should include the most important interactions that determine the behavior of the real system . there are two common approaches to the problem of parametrization of a coarse - grained model . in the top - down parametrization method , selected parameters are adjusted to reproduce large - scale properties of the investigated systems . in parametrization method , interactions are directly determined from either all - atom molecular dynamics simulations or quantum chemistry computations applied to some model systems . in the past decade , many coarse - grained models of dna have been developed , but only several of them managed to form double - stranded dna from separated complementary chains . a three - bead - per - nucleotide model of de pablo and co - workers was successfully used to study dna melting and the renaturation processes . the model was designed around the potential energy function , which effectively depends on the single parameter , which was optimized to reproduce experimental melting curves . the base base interactions were approximated by a go-like potential . the model of ouldridge et al . successfully addresses the phenomena of single - strand stacking , duplex hybridization , and dna hairpin formation processes . the model was also applied to study dna holliday junctions self - assembly and dna nanotweezers . although both models successfully fold double - stranded dna from separate complementary chains , they rely on base base nonbonded potentials , which either distinguish between native and non - native pairs or nearest - neighbors and all remaining pairs . such an approach should speed up the in silico folding process , because many unwanted free energy local minima are eliminated , but in the real physical system , the nonbonded potential energy of two interacting bases does not depend on their sequential position in the polynucleotide chain but rather depends on their relative position and orientation in space . the third model named nares-2p , which does not incorporate any go-like potentials and successfully folds double - stranded dna from separate strands was recently published by our group . nares-2p model was developed in parallel to the rigid - body model presented in this paper . nares-2p is a lower - resolution model with two interaction centers per nucleotide ( located on phosphate groups and bases ) , and it seems to be a minimal physics - based model capable of folding of a double - helix from separate strands . the rigid - body model presented here has more interaction centers and more internal degrees of freedom than nares-2p , and consequently , it is computationally more demanding . although the conformational space of rigid - body model is larger than in case of nares-2p , it is still capable of locating double - helical structures in simulated annealing process started from separate strands . the advantage of the dipolar - bead model over recently published nares-2p is its higher resolution . another model , which is capable of folding dsdna from separate strands , was published recently by cragnolini et al . the degree of coarse - graining ( 67 interaction sites per nucleotide ) is similar to that of our dipolar - bead model . however , as opposed to our dipolar - bead model , which places more emphasis on the representation of nucleic - acid bases , the model of cragnolini et al . elaborates on the nucleotide backbone , which is represented by 5 sites per nucleotide than on the nucleic - acid bases , which are represented by 1 or 2 interaction sites , depending on the kind of base ; it also utilizes a classical bonded potential and a many - body nonbonded potential responsible for correct watson crick hydrogen - bonding and was parametrized in a top - down fashion . this model does not incorporate any go-like potentials and does not treat the interactions between nearest - neighbor nucleotides differently than those between the other pairs of nucleotides . in this paper , we present a physics - based , middle - resolution model of dna , which is capable of folding the double - helical structure from separate complementary strands . the model was parametrized in the bottom - up fashion , although some adjustment of parameters was necessary to obtain correct balance of key interactions . the non - bonded pairwise potentials do not depend on the sequential position of interacting bases in the polynucleotide chain because the native and non - native , nearest - neighbors , and all other pairs rely on the same potential energy function , which includes a lennard - jones and an electrostatic term . the full , physics - based ( pb model ) version of model keeps stable canonical b dna double helices in a long room - temperature simulations and folds short dna molecules . the reduced version of the model , in which base base intrastrand interactions are limited to nearest neighbors ( nn model ) , successfully folds canonical b dna from separate complementary strands for all tested systems . the coarse - grained model of dna is built of three types of particles:neutral bead , uncharged lennard - jones sphere,charged bead , lennard - jones sphere with an electric charge located in its center,dipolar bead , lennard - jones sphere with an electric dipole located in its center . neutral bead , uncharged lennard - jones sphere , charged bead , lennard - jones sphere with an electric charge located in its center , dipolar bead , lennard - jones sphere with an electric dipole located in its center . the dna chain is built of six types of chemical units : phosphate group ( p ) , deoxyribose ( s ) , adenine ( a ) , thymine ( t ) , guanine ( g ) , and cytosine ( c ) . in the coarse - grained approximation , atoms that constitute each unit are replaced by the three types of beads defined above . the deoxyribose ring is replaced by one neutral bead , the phosphate group is replaced by one charged bead and each base is replaced by a set of dipolar beads . a schematic representation of the coarse - grained units overlapped on their atomic representation is shown in figure 1 . coarse - grained representation of fragments of the dna chain superimposed on the atomic representation . four basic building blocks a , t , g , c attached to small fragments of backbones are shown . charged beads , which replace phosphate groups , are marked red ; neutral beads , which replace deoxyriboses , are marked dark blue . dipolar beads have different colors for different bases : a , pink ; g , green ; t , cyan ; and c , yellow . the internal degrees of freedom used for the definition of the bonded part of the potential energy function ( eq 2 ) are defined in tables 14 . for clarity , symbols of beads used for definition of bonded potentials of the backbone are shown for thymine only . the longer fragment of coarse - grained strand of dna with atgc sequence overlapped on the atomic model is shown in the right panel . sequential numbers of consecutive building blocks are assigned . each chemical unit in the coarse - grained approximation constitutes a rigid body . the origin of the local coordinate frame of each rigid body is located at its center of mass , and its axes are aligned with the principal axes of the moment of inertia tensor . the position and orientation of each rigid body with respect to the global coordinate frame is described by a vector tensor pair ( r , q ) , where r is the position of the center of mass and q is the rotation matrix . in the model , spherical ( p and s ) and planar ( a , t , g , c ) . the spherical rigid bodies interact with other particles only by central forces ( see potential energy function ) , and their orientation is irrelevant ; therefore , a constant rotation matrix qsphere = const is assigned to them . the orientation of each planar rigid body is described by a 3 2 rotation matrix qplanar and varies as the rigid body changes its orientation during a simulation run . the geometry of the chain is defined in vector tensor space ( ri , qi ) where i = 1, ... ,n and n is the number of rigid bodies . the relations between global coordinates ( ri , qi ) and distances between various beads , used for definitions of nonbonded interactions , are shown in figure 2 . relations between global coordinates ( r , q ) and distances between beads for interactions of ( a ) two spherical rigid - bodies , where there is only one distance rij = |rij| ; ( b ) spherical and planar rigid body , where distances between interacting beads are functions of global coordinates given by eq 9 ; and ( c ) two planar rigid - bodies , where distances between interacting beads are given by eq 13 . rigid bodies are connected by elastic virtual bonds and form a nucleic acid chain as shown in figure 1 . three rigid bodies connected by two virtual bonds form a virtual - bond angle and four rigid bodies connected by three virtual bonds form a virtual - bond dihedral angle . all virtual bonds , virtual - bond angles , and virtual - bond dihedral angles are listed in tables 1 , 2 , and 3 , respectively . x and s b y are virtual - bond angles between the s b virtual bond and the x and y axes of the moment of inertia tensor of a rigid body . figure 1 ) p5s b x virtual - bond dihedral angle describes rotation of the base around the s the algorithm of leimkuhler and reich , which is based on the rattle constraint method applied to rotation matrices , was used for the description of rotation of rigid bodies . a slightly different version of this algorithm , based on the shake ( rather than the rattle ) constraint method , showed superior stability compared to the other commonly used quaternion - based integration scheme . however , this algorithm ( based on shake ) was not implemented in our work . the time - evolution of the system is defined in the vector - tensor phase space ( ri , pi , qi , pi ) , where pi and pi are the linear momentum and generalized angular momentum of the i - th particle , respectively . for a planar rigid body , the rotation matrix is a 3 2 tensor , subject to the orthogonality condition qq = i2 , where i2 is a 2 2 unit diagonal matrix . the leapfrog ( verlet ) algorithm was used for propagation of translations , and the r - rattle ( rotational rattle ) discretization scheme was applied to the rigid - body rotations . as in our unres model of polypeptide chains developed earlier , the effective energy function of our nucleic - acid model stems from the potential of mean force ( pmf ) of polynucleotide systems immersed in water . the pmf is then to be expanded into a cluster - cumulant series , which , in general , gives rise to the neo - classical local and two - body and nonclassical multibody terms . however , because we keep more explicit degrees of freedom than in the unres model , the multibody terms do not seem to be necessary . in particular , in contrast to the unres and nares-2p models , we do not average over any degrees of freedom of the nucleic - acid bases , the interactions between which are highly directional . in the unres model , we did average over the angles of rotation ( ) of the peptide groups about the c ... c axes ; hence , the multibody terms in the unres force field are important . in our highly reduced nares-2p model of nucleic acids , averaging is carried out over the rotation of nucleic bases about their long axes . the potential energy function of the system is a sum of the bonded and nonbonded parts:1although both the bonded and nonbonded parts depend on the cartesian - rigid - body degrees of freedom ( r , q ) , it is more convenient to define the former as a function of the internal degrees of freedom defined in tables 14 ( the internal degrees of freedom are functions of the ( r , q ) vector ) . the bonded part has a commonly used form2where:3a3b3c3dboth bond - stretching and angle - bending terms are approximated by harmonic potentials . kd and di0 are harmonic force constants and equilibrium distances for bond stretching interactions ; k and i0 are harmonic force constants and equilibrium angles for the bond - angle bending potential . the general form of the dihedral - angle torsion potentials is a fourth - order fourier series , although such a long expansion was used only for the backbone dihedral angles ( see details in the parametrization section ) . the torsional terms are the only higher ( second ) order terms that are present in the energy function ; however , they still belong to the neo - classical term set . the harmonic uimproper potential was introduced to maintain the correct position of the center of mass of the base with respect to the sugar ring and surrounding phosphate groups . k and i0 are harmonic force constant and equilibrium values of the improper dihedral angles . for a detailed geometric definition of the internal degrees of freedom related to the bonded potentials see figure 1 and tables 14 . the force constants and equilibrium positions were determined by fitting the analytical expressions to the potentials of mean force for model systems . the nonbonded energy is a sum of van der waals and electrostatic energies and is given by4where the p , b , and s symbols denote the phosphate group , base , and sugar ring , respectively . phosphate energy5a5binclude only the lennard - jones term and depend only on the distance rij between two beads . the lennard - jones potential has a commonly used form6where , alj and blj are lennard - jones coefficients . the phosphate charge repulsion term7the three remaining energy terms of eq 4 involve interactions with the planar rigid - bodies ( bases ) . the sugar base energy is a sum of excluded volume interactions between a neutral bead and the dipolar beads . this kind of all - repulsive term is also used in the unres force field to account for excluded - volume interactions between the peptide groups and side chains8where nj is the number of dipolar beads of the j - th base and rijk is the distance between the neutral bead of the i - th sugar and the k - th dipolar bead of the j - th base . it should be noted that rijk distances are functions of the distance rij between the sugar and the base and the orientation qj of the base ( see figure 2).9where rjk is the position of the k - th dipolar bead in its rigid - body local coordinate frame . the excluded volume potential has a form10where the parameters aex and bex depend on the type of interacting beads . the phosphate - base interaction additionally includes the charge - dipole terms11 the final term of eq 4 has the most complicated form and describes the interaction between the dipolar beads of two bases12where ni and nj are the number of dipolar beads located on the i - th and j - th bases , respectively . the distances rijkl between dipolar beads are functions of the relative position rij and orientations qi , qj of the rigid bodies13where ri(j)k(l ) is the position of the k - th ( l - th ) dipolar bead in the local coordinate frame of i - th ( j - th ) base . hckel model14a14b14cwhere q = eq is the negative unit charge located on phosphate group , n = r / r is a unit vector pointing from the first to the second interaction center , and pi(j ) are electric dipole vectors described in the global coordinate frame:15where pi(j ) is an electric dipole vector in the local coordinate frame of the i - th ( j - th ) base . four functions16a16b16c16dapproximate the screening of the electrostatic interactions by ions and solvent , where is the debye screening length . for charge charge interactions distance - dependent dielectric constant of the following form was applied:17where inf = 78.0 is a dielectric constant of bulk water and int is a dielectric constant of the helix the switching distances r0 and r1 , which determine boundary between unscreened and screened electrostatic interactions , were set to the values of 4 and 13 . the parameter was adjusted to keep continuity of dielectric constant . because the interactions between the nearest neighbors , that is , particles separated by less than three virtual bonds , are included in the bonded part of the potential , they were excluded from the nonbonded interactions . the nonbonded interactions and solvation effects are very important for dna folding and its conformational stability and were approximated by the excluded - volume potential ( eq 10 ) for p b and s b , the lennard - jones potential for the remaining nonbonded interactions ( eq 6 ) , and a multipole - multipole potential energy function with debye hckel screening . the optimization procedure developed previously was applied to the extended system , which now includes the phosphate group , deoxyribose , and the four bases . the parameters of the b b interactions , including the positions r of the dipolar beads in the local coordinate frame , the electric dipole vectors p in the local coordinate frame , and the lennard - jones parameters alj , blj , were retained from the 3445 model of ref ( 47 ) . for convenience , they can also be found in tables a - ii to a - v of supporting information . the missing parameters for the extended system are the charges located on neutral and charged beads , their positions in the local coordinate frame , and the lennard - jones parameters of p p , p s , s s , s b and p b interactions . the charge of a neutral bead was , by definition , set to zero , the charge of a charged bead was set to |eq| , which is equal to the charge of the phosphate group in the dna backbone . the positions of the charges in both the neutral and charged beads were fixed at the center of mass of the corresponding unit . consequently , the only parameters , which must be determined by the optimization procedure were the lennard - jones alj and blj for p the reference energy grids for missing lennard - jones interactions were computed in the same manner as the reference grids in ref ( 47 ) and the optimization procedure was applied to the extended system . although it was initially planned that the lennard - jones potential would be applied to all pairwise interactions in the system , extensive testing of dsdna dynamics showed that its stability was improved when the lennard - jones potential of p b and s b interactions was replaced by the excluded - volume one . therefore , the potential was switched to an excluded volume of the form of eq 10 , and the alj and blj parameters were retained from the fitting procedure , that is , aex = alj , bex = blj . the values of the computed lenard - jones and the excluded volume parameters , presented in the form of = ( a / b ) and = b/(4a ) , are collected in table a - i of the supporting information . the debye screening length was set to 8 , which corresponds to the physiological ionic concentration . in this work , the stability of the helix and folding efficiency was tested with several values of int and various distances r0 and r1 of the dielectric constant function ( eq 17 ) ( see conformational stability in the results and discussion section ) . the virtual bond , virtual - bond angle , and virtual - bond dihedral angle parameters were derived to reproduce the behavior of model systems in the all - atom representation . the potentials of mean force ( pmf ) of each internal degree of freedom of the coarse - grained model were calculated by using the umbrella sampling method . analytical functions of the form of eqs 3a3d were then fitted to the numerical pmf s obtained from the wham procedure . the parameters of all virtual bond , virtual - bond angle , and virtual - bond dihedral angle degrees of freedom were computed by using four model systems , which were obtained from three - nucleotide backbones with methyl end groups . the base of the middle nucleotide was either adenine , cytosine , guanine , or thymine , for a total of four different model systems . one such system with a cytosine base in the middle nucleotide is shown in figure 3 . this is a minimal size molecule for which every internal degree of freedom of our coarse - grained model can be defined . terminal bases were removed to avoid implicitly including electrostatic and van der waals interactions of bases since our model has the nonbonded parameters derived separately . model systems for parametrization were created from three - nucleotide chains by removing the bases from the nucleotides at the 5- and 3-end and replacing them with methyl groups . the middle nucleotide was left unchanged and can be cytosine ( in the figure ) , thymine , adenine , or guanine . umbrella sampling simulations , described in the supporting information , of different model systems produced results that are similar for each internal degree of freedom . figure a - i in the supporting information shows the individual and average pmfs for several virtual bonds , virtual - bond angles , and virtual - bond dihedral angles . therefore , it was decided to average the degrees of freedom for purines ( model systems with adenine or guanine ) and pyrimidines ( model systems with cytosine or thymine ) , except for the degrees of freedom such as the s p3 virtual - bond dihedral angle , which contains two deoxyriboses , and therefore , the base type can not be uniquely assigned . for these angles , the averaging was done across all four model systems ( bases ) . some degrees of freedom , which influence orientation of bases with respect to glycosidic bond , were assigned separately for each type of base ( see tables 2 and 3 ) . for each degree of freedom , the distributions of data from each window and for appropriate model systems were combined , the biasing potential was removed , and the pmf along that coordinate was calculated by using the wham procedure implemented in a program by dr . the analytical expressions of the form of eqs 3a3d were then fitted to pmf s . the fitting was done using a trust - region nonlinear least - squares fit algorithm as implemented in matlab software . figure a - ii shows the comparison of several pmfs derived from the simulations with the potential calculated using analytical expressions . although both the force constants and the equilibrium positions for the virtual bonds and virtual - bond angles were determined by fitting to the pmfs of model systems , it was decided to shift them slightly to match the equilibrium values of the ideal b - dna conformation , in the coarse - grained model . b with arbitrary force constant and an equilibrium values set to those of the ideal b - dna were incorporated . the rotation of the base around the virtual glycosidic bond ( s b ) was restricted by a cosine - shaped potential with the minimum corresponding to the anti conformation of the base and the barrier height was preliminary set to 7.0 kcal / mol . the internal degrees of freedom for which the bonded terms were determined and used in the model are listed in tables 14 . we note that a model system and bonded degrees of freedom almost identical to those presented here were used in work by morriss - andrews et al . however , the predicted values for the equilibrium values and force constants were somewhat different . there are several reasons for this , first , morriss - andrews et al . use statistics from free simulations and charm27 force field to calculate the degrees of freedom while this work used umbrella sampling along the degrees of freedom and amber s ff99bsc0 force field . most importantly though , the position of the sugar in our model is at the center of mass of sugar ring while they set it at the c1 atom . this affects not only the equilibrium values but the force constants as well , which makes the direct comparison between bonded degrees of freedom of two models difficult . the model was tested on the following tree systems , for which the structure was determined experimentally:drew dickerson dodecamer , 12 base - pair dsdna with the sequence 5-cgcgaattcgc-3 and its compliment , which has exactly the same sequence ( pdb code 1bna),narayana weiss hexadecamer , 16 base - pair dsdna with the sequence 5-actacaatgttgcaat-3 and its compliment 5-attgcaacattgtag-3 ( pdb code 3bse).21 base - pair dsdna with the sequence 5-acagcttatcatcgatcacgt-3 and its compliment 5-acgtgatcgatgataagctgt-3 ( pdb code 2jyk ) . drew dickerson dodecamer , 12 base - pair dsdna with the sequence 5-cgcgaattcgc-3 and its compliment , which has exactly the same sequence ( pdb code 1bna ) , narayana weiss hexadecamer , 16 base - pair dsdna with the sequence 5-actacaatgttgcaat-3 and its compliment 5-attgcaacattgtag-3 ( pdb code 3bse ) . 21 base - pair dsdna with the sequence 5-acagcttatcatcgatcacgt-3 and its compliment 5-acgtgatcgatgataagctgt-3 ( pdb code 2jyk ) . the following 60-nuclotide sequence of ref ( 34 ) was used for computation of persistence lengths of single and double stranded dna:hetss = d(catcctcgacaatcggaaccaggaagcgccccgcaactctgccgcgatcggtgttcgcct)hetds = hetss + complementary strand hetss = d(catcctcgacaatcggaaccaggaagcgccccgcaactctgccgcgatcggtgttcgcct ) hetds = hetss + complementary strand for canonical ensemble simulations , berendsen s thermal - bath weak - coupling algorithm was implemented . the stability of the integration algorithm was checked by simulations of the model system in the microcanonical ensemble . the energy of the system was minimized and initial velocities were assigned from the maxwell distribution ; then , the integration of the equations of motion was performed without coupling of the system to the thermal bath . the ability of the model to fold double - stranded dna from separated chains was tested with a two - stage simulated - annealing procedure . two complementary strands of each molecule were separated by a random distance and randomly rotated with respect to each other . the separation distance ensured that two strands did not overlap for any random orientation and was different for chains of various lengths . for 1bna , the separation distance between centers of mass was picked randomly from 40 to 45 interval and for 3bse and 2jyk separation intervals were 5055 and 6065 , respectively . in the first stage , the initial structure was heated up to 600 k ( 620 for 2jyk ) with velocities randomly assigned from maxwell distribution for 0.1 ns and then cooled to 300 k ( 320 for 2jyk ) with a temperature step t = 50 k. the final temperature was set approximately around 20 k below melting temperature of each molecule . the simulation at each temperature was 0.1 ns long . at the end of each simulation , a contact was assumed to be present when the centers of mass of any two bases from separate chains came within a distances of at most 6.5 . if at least one contact was present , the simulation was extended by 1.5 ns for drew dickerson ( 2.0 ns and 2.5 ns for narayana weiss and 2jyk , respectively ) at 300 k in the second stage of the canonical simulation ; otherwise , the cycle was terminated and the final structure was excluded from the analysis . the total simulation time of a two - stage simulated annealing cycle was 2.23.2 ns ( depending on the chain length ) , and each one was followed by a short energy minimization with powell s algorithm . the number of sa cycles performed for each system is shown in table 5 . dissociation of chains was prevented by application of a flat - bottom restraint on the distance between their centers of mass with a force constant kcm cm = 1 kcal/(mol ) and a minimum distance of d0 = 40 for 1bna ( 50 for 3bse and 60 for 2jyk ) , at which the attractive harmonic force was switched on . the artificial restraint force was switched off when the distance between the centers of mass dropped below 40 for 1bna ( 50 for 3bse and 60 for 2jyk ) . the first row shows the total numbers of simulated annealing cycles computed for each molecule . the second row shows the number of trajectories for which the cooling cycle ended up with at least one contact between bases . statistics of contacts for these trajectories , which were extended by 1.5 ns to 2.5 ns canonical simulation , is shown in the next 5 rows . numbers of long trajectories with various percentage of native contacts are presented in rows 37 . the final structures of the simulated annealing cycle were compared to the experimental reference by computing the all - bead rmsd . the persistence length lp is a measure of flexibility of a polymer and can be computed from decay of correlation function of unit vectors tangent to helical axis18where s is the position along the length of the chain . for ssdna the tangent vector is defined as a normalized vector between consecutive neutral beads ( centers of mass of deoxyriboses ) . for dsdna tangent vector sugar distances of complementary bases , separated by 5 base pairs . a new software package for this coarse - grained model , composed of rigid - bodies , was developed . the software is written in c / c++ programming language and currently can perform the following tasks : read the pdb - format structure and transform it into the coarse - grained representation , optimize the geometry of the molecule ( by energy minimization ) , perform microcanonical and canonical ensemble simulations with berendsen s weak thermal - bath coupling , and perform simulations of heating and simulated annealing cycles . the efficiency of the parallelization up to 12 cores is shown in figure 4 . the stability of the verlet - r - rattle algorithm was tested on the drew dickerson double - helix . microcanonical simulations of 10 ns length were run with various time - steps : dt = 10 , 12 , 14 , 16 , 18 , 20 , 22 , 24 , 26 , 28 fs . the algorithm maintains good stability up to dt = 22 fs resulting in an order of magnitude speedup compared to the commonly used all - atom time - step of 2 fs ( with shake applied on hydrogens ) , and shows superior stability compared to the quaternion - based rigid - body integration scheme , applied previously to the model of a dna chain . the drift of the energy over a 20 ns trajectory with dt = 22 fs was only 0.017 kcal / mol , compared to 10 kcal / mol over 1 ns simulation with the same time - step using a quaternion integration scheme ( see figure 5 in ref ( 19 ) ) . the drifts of the energy and the rms fluctuations for various time - steps are collected in table 6 . all canonical simulations and simulated annealing cycles utilized time - step dt = 10 fs . stability of the verlet - r - rattle integration algorithm over 0.1 s microcanonical simulation of the drew drift was calculated as a difference between the averages of the last and the first 1 ns of each trajectory . the efficiency of the model was compared to that of all - atom simulations performed with the amber 11 package . dickerson dodecamer immersed in a truncated octahedron box filled with 5099 tip3p water molecules . a 10 cutoff and the particle - mesh ewald summation method were applied for electrostatic interactions . the simulation of 1 ns took 17476 s on 8-cores using intel xeon e5645 2.4 ghz cpus of the computing cluster of the chemistry research computing facility of baker laboratory of chemistry and chemical biology , cornell university . the same simulation with our coarse - grained model and dt = 20 fs took 235 s with the same cpu , leading to almost 2 orders of magnitude speedup . it should be noted that almost 2 orders of magnitude speedup was achieved without any cutoffs applied to the nonbonded interactions . implementation of cutoffs , especially to the short - range lennard - jones ( u r ) and the dipole dipole ( u r ) interactions will further increase the speed of computation , especially for long chains . the initial crystal structure ( 3bna , 3bsa , and 2jyk ) was heated from 100 to 300 k with t = 50 k. the simulation at each temperature was 0.1 ns long , and the production run at 300 k was 0.1 s long . snapshots of trajectories were recorded every 1 ps . the initial simulations of the drew dickerson dodecamer led to a collapse of the helical structure , caused by overestimation of the strength of the lennard - jones interactions with phosphates and sugars , which were fitted to reproduce the amber potential energy in vacuum . the stability of the system was improved by scaling of lennard - jones interaction energy between neutral and charged beads ( p p , s the stability of a double - helix was also tested with varying r0 and r1 parameters of eq 17 . table 7 shows the average number of native contacts as a function of distances r0 and r1 for drew - dickerson dodecamer with int = 8.0 . the largest average number of native contacts is located in the lower right corner of table 7 , which means that sufficient electrostatic repulsion of phosphate groups of interacting chains is necessary for stability of double - helix . smaller switching distances r0 and r1 causes instability of the helix termini , which can even destabilize helix interior . on the other hand the reduced ( and less physical ) model , in which intrastrand base base interactions were limited only to nearest - neighbors , was tested . the nn model generates very stable double helices even for short switching distances r0 = 4 and r1 = 13 , as applied in the ref ( 40 ) . it seems that stronger electrostatic repulsion of phosphate groups is necessary to prevent deformations of helix termini caused by base , stronger electrostatic repulsion affects folding rate in the fast simulated annealing procedure ( see section folding of dsdna from separate chains ) . table 8 collects mean helical parameters obtained from trajectories for three model systems for full physics - based model and for the simplified nearest - neighbor model . these parameters were calculated from coarse - grained representations of experimental reference structures and the structures obtained in simulations , respectively . a precise determination of all helical parameters would require conversion of the coarse - grained structurs to an all - atom representation ; however , the values calculated by using a coarse - grained representation are sufficient for comparison of the simulated structures with the respective experimental structures . for all model systems , the average number of native contacts is larger for the nn model . for shorter molecules , 1bna and 3bse , the radius of gyration within nn approximation is close to the value obtained for experimental structure . for the pb model applied to 1bna and 3bse the radius of gyration is slightly too short , which is caused by deformation of helix termini . for the longest molecule both models overestimates the radius of gyration . both models underestimate helix radius and overestimate helical rise for all dsdnas , although these discrepancies are relatively small . larger deviations are observed for the size of minor groove , which is significantly overestimated by both models . pb and nn denotes the full physics - based and the nearest - neighbor model , respectively . the helical rise is approximated by the distance between consecutive centaral points of lines connecting centers of mass of watson crick - paired bases . persistence lengths of both hetss and hetds were computed from 0.1 s canonical simulations at 300 k with 145 mm salt concentration for both full pb and simplified nn model . pb model produces smaller persistence lengths than nn model , although the relative difference is much bigger for ssdna . this behavior is expected , because switching off some of the intrastrand base base interactions should decrease bending of the chain , especially for ssdna , in which bases are exposed to solvent and can easily form intrastrand hairpins with complementary bases . figure 6 presents typical configurations of het60ss and het60ds molecules taken from t = 300 k canonical simulation . the conformation of ssdna taken from full pb model simulation differs significantly from the one taken from nn approximation . the ssdna obtained from full pb model bends and forms hairpins , which can act as kinetic traps in the folding process of dsdna . typical configurations of het60ds ( left panel ) , het60ss within nearest - neighbor approximation ( middle panel ) and het60ss for full physics - based model ( right panel ) . persistence lengths were obtained by fitting of eq 18 the the correlation functions obtained from canonical ensemble trajectory computed at 300 k with 145 mm salt concentration . the value of persistence length of ssdna obtained with nn model agrees reasonably well with experimental values . the persistence length of dsdna appears to be around three times too small compared to experimental values , and it is close to the value computed by early version of 3spn model . although the agreement with experimental data is not perfect it appears to be quite satisfactory for bottom - up-parametrized physics - based coarse - grained model and this discrepancy will be corrected in next versions of the model . the influence of mismatches was tested on the 30-bp system of 10 g / g pairs flanked by 10 a / t pairs at both 5 and 3-end . during the 0.1 s canonical simulation at 300 k , opening of mismatched g / g pairs although interaction of complementary bases is different than that between mismatched bases , the model does not seem to be very specific with respect to base base interactions . this problem may be result of exaggeration of base base lennard - jones interactions with respect to electrostatics and will be addressed in the next version of the model . the simulated annealing ( sa ) procedure described in the methods section was applied to some model systems . only 3 out of 2100 trajectories led to the final helical structures with more than 10 native base much better folding rate was achieved with nn model in which intrastrand kinetic traps ( hairpins ) , resulting from intrastrand long - range base base interactions , were eliminated . the statistics of the folding process of three model systems within nn approximation are presented in table 5 . after the majority of simulated annealing cooling cycles ( first stage ) , the chains did not come into contact because of electrostatic repulsion of the highly charged chains and the relatively short time of the simulation . the frequency of making at least one contact after a cooling cycle is different for each simulated system and changes from 50% for the dodecamer to 6% for the hexadecamer , and to 8% for the 21-bp system . this phenomenon can be explained by increasing volume of an accessible space for longer chains , which decreases probability of making contact during fast cooling process . the effect is enhanced by increased electrostatic repulsion of the longer negatively charged backbones . after the second stage of simulated annealing , native - like structures were obtained . the percentage of the native - like structures , based on the number of native contacts , is shown in table 5 . it should be noted that the numbers summarized in table 5 were obtained from sa calculations which do not reflect the actual amount of the native - like structures that a force - field can produce . nevertheless 24% of extended trajectories led to double - helical structure with more than 10 native contacts for 1bna molecule . roughly the same number of structures broke up into separate chains or formed misfolded structures without native contacts . 41% trajectories ended up in misfolded structures with 13 native contacts , which were mostly parallel dsdna s , in which 5-end of one chain makes nonative contacts with 5-end of the other chain . figure 7 shows the potential energy of final structures as a function of all - bead rmsd computed with respect to experimental structure . the one located in the lower left corner of figure 7 is a cluster of correctly folded antiparallel dsdna s and the other one represents misfolded parallel dsdna s , in which 3-ends ( and 5-ends ) of chains are paired . the important observation that comes from this graph is that lowest energy structure is clearly located in the correctly folded group and its energy is around 10 kcal / mol lower than misfolded parallel dsdna , although the lowest energy structure is not the structure with lowest rmsd . the mean potential energy of antiparallel dsdna cluster is around 8 kcal / mol lower than the mean energy of the parallel dna cluster , which clearly favors correctly folded structures , although this difference for real systems might be larger ( experimental structures of the parallel drew dickerson dodecamer were not observed ) and further refinement of the electrostatics and/or lennard - jones balance of the model is required to increase this gap and improve specificity of base base interactions . two major clusters , corresponding to antiparallel and parallel dsdna , and the energy - minimized experimental structure are pointed by arrows . the reference structure marked by bigger dot in figure 7 is the energy - minimized experimental structure . the optimization of experimental structure was necessary to remove small steric clashes in original structure caused by imperfectness of the coarse - grained model . the potential energy of original 1bna molecule is more than 100 kcal / mol higher than that of the optimized one , but the all - bead rmsd between the optimized and original structure is only 0.4 . this effect can be expected for the coarse - grained model with force - field parameters developed in the bottom - up fashion . the potential energy of the reference structure is almost exactly the same ( < 1 kcal / mol ) as the lowest - energy structure obtained from simulated annealing procedure . the folding efficiency of longer polymers decreases as the dimensionality of phases spaces increases ( see table 5 ) . 16% of long trajectories of 3bse molecule led to final structures with more than 75% of native contacts ( 1316 contacts ) . for the 2jyk molecule , the folding rate is almost the same as that for 3bse and equals 15% . nevertheless , the energy vs rmsd graphs ( figures 8 and 9 ) are qualitatively similar to these obtained for 1bna molecule ( figure 7 ) . in both cases , two clusters of antiparallel and parallel dsdnas are clearly visible and correct antiparallel cluster has mean potential energy around 20 kcal / mol lower than this of incorrect these values should effectively discriminate misfolded structures in the real- , long - time folding process . this mean - energy difference increases to 22 kcal / mol for the longest 2jyk molecule . the energy - minimized reference structures , marked by bigger dot in figures 8 and 9 , have rmsd only 0.6 away from original structure . in both cases , the reference structures have potential energy around 10 kcal / mol lower than these obtained from simulated annealing procedure . this observation suggests that longer cooling process should lead to improvements in geometry of final structures , as the minimum of potential energy was not reached by any simulated annealing cycle for longer model systems . figure 10 presents the three lowest - energy structures of tested molecules superimposed on their experimental structure . the all - bead rmsds of 1bna , 3bse , and 2jyk molecules with respect to experimental references are 2.1 , 3.1 , and 4.2 , respectively , and as expected , they increase with the length of polymers . all structures clearly form double - helices , although some discrepancies of the geometry are visible , especially for the longest molecule , for which overestimation of the size of minor groove is clearly visible . lowest - energy structures of 1bna , 3bse , and 2jyk molecules obtained from extended simulated annealing procedure , superimposed on experimental structures . their all - bead rmsds are 2.1 , 3.1 , and 4.2 , respectively . the backbone of the computed structures is shown in blue and the bases are a ( cyan ) , t ( pink ) , g ( green ) , c ( yellow ) . analysis of 158 trajectories of 1bna , which led to structures with 1012 native contacts revealed two qualitatively different mechanisms of folding . the representative of the first most common mechanism ( 93 of 158 trajectories ) is shown in figure 11 . two separated chains ( 0 ns ) were heated to 600 k and evolved into random chains ( 0.11 ns , t = 550 k ) . at 0.56 ns ( t = 350 k ) the 5-gcgc - end established native contacts with the complementary 3-cgcg - end , and since then , more native contacts were formed , and the dsdna molecule was zipped . at 0.68 ns , all native watson snapshots of trajectory of a zippering mechanism of the folding process of the drew dickerson dodecamer . a different and less common folding process ( 65 of 158 trajectories ) it was started from two separated ssdna chains , which were heated to 600 k and , after 0.3 ns ( t = 500 k ) , evolved into a completely random conformation . first , contacts between the bases of the 5-ends of the chains were established after 0.49 ns . it should be noted that these are non - native watson crick g / c contacts . after that , the chains started to slide with respect to each other along a watson after 0.89 ns , two g / c contacts were estabilished but also a / c and a / g mismatched pairs were formed . a 1.4 ns snapshot shows a molecule with 10 contacts established and with distorted termini . snapshots of trajectory of a slithering mechanism of the folding process of the drew as can be expected , the zippering mechanism was also observed for longer molecules , 3bse and 2jyk . nevertheless , some trajectories with a slithering folding mechanism were also recorded . at first , this observation is surprising because , as opposed to the 1bna molecule , the initial mis - alignment of the two strands creates a number of mismatched base therefore , the fact that the simulated folding of 3bse and 2jyk proceeded almost as frequently by the zippering and by the slithering mechanism probably resulted from low base base pairing specificity of the model . the snapshot from helix initiation process for 3bse molecule the 3-end of a complementary strand made contacts with 3-end of the leading strand . besides three nonative watson crick contacts ( two a / t and one g / c ) and two mismatched contacts three watson crick pairs and two mismatched pairs form initial structure from which monomers start slithering with respect to each other until final proper double - helix is formed . the helix nucleation occurred between nucleotides of 3-ends but also the mechanisms where 3-end ( or 5-end ) of one chain made a contact with midsection of the other chain were observed . examples of slithering pathways of dna hybridization of ( a ) 3bse and ( b ) 2jyk molecules . for the longest chain , the slithering mechanism was observed only for two out of 22 folding trajectories . in one case , nucleation started from formation of contacts of 3-end of one chain with middle section of the other chain ( see figure 13b ) . in the second slithering trajectory , the 5-end made initial contact with middle section of the other chain . no trajectory where two 3-ends ( or 5-ends ) made initial contact was recorded . at this point , the analysis of dna duplex hybridization is reduced only to qualitative observations presented above . further development of the model is necessary , especially further reduction of computational cost via implementation of cutoffs , to obtain large set of trajectories , which is required for proper analysis of thermodynamics and kinetics of dna hybridization process . nevertheless , the mechanism of hybridization can be qualitatively compared to hybridization mechanisms proposed by de pablo and doye groups . the former group showed two mechanisms also called slithering and zippering , which follows double - helix nucleation process . the former mechanism is dominant for repetitive sequences and zippering is characteristic of random sequences . both mechanisms seem to be similar to folding mechanisms obtained with our dipolar bead model , although it should be noticed that sequences of simulated systems are not exactly the same . the drew dickerson dodecamer is symmetric and might have pathways of folding that are similar to slithering mechanism observed for 3spn model . our model shows that slithering and zippering mechanisms for 1bna are almost equally probable . the 3bse and 2jyk molecules have a sequence that can be described as random , and their dominant folding mechanism is molecular zippering . the zippering mechanism was also observed for the model of ouldridge et al . they showed that there is a bias toward initial structures with contacts between ends of the strands , an observation that can be confirmed by our model . one of the mechanisms of duplex formation observed for repetitive sequences , so call inchworm mechanism , was not observed in our simulations , although we did not test polymers with exactly the same sequence . it should also be noticed that tendency for slithering , instead of inchworm - like behavior , in our model may result from insufficient specificity of base base interactions . finally , it should be stressed that these results were obtained in unrestrained simulations started from separated chains . this mode of simulations is different from that of many other works in which simulations are started from the experimental structure . unlike many other force - fields developed for dna , our force - field , therefore , , it was shown that dihedral angle bonded potentials are not prerequisites for double - helix formation , which is rather driven by base base dipole interaction . also , the model developed by ouldridge et al . does not require backbone dihedral angle potentials to obtain dna duplex hybridization . a question can , therefore , be asked : are the backbone potentials a necessary factor for double - helix formation , or is this process solely dependent on the base stacking and pairing ? p3s3 , s5p5s p3 , s5p5s b , and s3p3s b dihedral angle potentials switched off . dickerson dodecamer showed the same efficiency of double - helix hybridization process ( 24% ) . the lowest energy structure shown in figure 14a is only 3.2 away from the experimental structure . next , improper rotamer - like potentials p5p3b s , p5s b , and p3s b were additionally switched off . the lowest energy structures have a ladder - like shape , as shown in figure 14b . it seems that backbone dihedral angle potentials are not really necessary for dna duplex hybridization . switching off rotamer - like potentials ( p5s b , p3s b , and p5p3s b ) causes formation of ladder - like structures instead of double - helices . this effect might be related to the lack of specificity of base base interactions , which might be the result of overestimation of base base van der waals interactions with respect to dipole dipole electrostatic interactions . nevertheless , the dipolar - bead model suggests that only backbone bond - stretching , angle - bending , and rotamer - like potentials are necessary for double stranded dna hybridization . lowest energy structures obtained from simulated annealing procedure with the nn model with some of bonded potentials switched off . ( b ) additionally , rotamer - like potentials p5s b , p3s b , and p5p3s b switched off . a physics - based rigid - body coarse - grained model of dna is proposed . the bonded interactions are fitted to the potentials of mean force of the model system . the equilibrium virtual - bond lengths and virtual - bond angles were set to the values of the ideal b - dna double - helix . the nonbonded interactions are approximated by lennard - jones , excluded volume and electrostatic interactions of charges and dipoles . the model does not incorporate any go-like potentials , which force the structure toward the experimental one . in the full physics - based model , nonbonded potentials do not distinguish between near - neighbors and any other base pairs . a full physics - based version of the model the nearest - neighbor approximation , in which intrastrand base base interactions are limited to nearest - neighbors only , was tested . it should be stressed that in the reduced model all interstrand base base interactions are present . the r - rattle rigid - body integration algorithm , implemented in our software package , demonstrated superior stability compared to a quaternion - based algorithm , with low drift of the total energy even for a time - step as long as 20 fs . the total speedup of computations compared to all - atom simulations is close to 2 orders of magnitude . stable double - helix trajectories were obtained for both pb and nn models after adjustment of several force - field parameters as discussed in the conformational stability section . the geometry of two experimental dna structures is preserved during 0.1 s simulations at 300 k. the persistence lengths of ssdna and dsdna are underestimated by the factor of 23 compared to experimental data , discrepancy that seems to be reasonable for physics - based model designed in the bottom - up fashion . the other drawback of the model , which seems to have the same origin as discrepancy in persistence lengths , is insufficient specificity of base base interactions , which affects simulations of dsdna with mismatches . opening of mismatched pairs was not observed during 0.1 s canonical simulations at 300 k. nevertheless , the model successfully folds short ( 1221 bp ) dnas from separated strands . the full pb model is only able to fold shortest double - stranded dna dodecamer from separated chains with relatively low efficiency caused by formation of intrastrand hairpins kinetic traps in the folding process . the reduced nn model removes kinetic traps and greatly improves the folding efficiency . all three tested molecules with lengths varying from 12 to 21 bp were folded from separate strands by nn model , although the efficiency of folding drops with increasing size of the simulated system . the possible explanation is that the cooling process is too fast to sample conformational space effectively and find the global minimum on the complicated multi - minima free energy hyper - surface . application of a replica - exchange method , which will be implemented in our software package , should help to explain the cause of the low helical populations produced by the simulated annealing procedure . the more frequent process called zippering starts from formation of native base base contacts between 3 and 5-ends of two chains and is followed by helix propagation . in the second , less frequent process called slithering , initial non - native watson crick contacts ( also mismatched contacts for 3bse and 2jyk ) are established between 5-ends ( or 3-ends ) of two strands ; then , chains slide with respect to each other along the watson this process was observed for all model systems although it is much more frequent for the dodecamer , which has specific sequence , than for other molecules . the slithering process might also be an artifact caused by insufficient specificity of base base interactions . finally , the influence of backbone dihedral angle and rotamer - like potentials on a double - helix formation process was tested . the backbone dihedral potentials do not seem to be a necessary prerequisite for dsdna hybridization , but without rotamer - like potentials , ladder - like structures instead of double helices were observed . base interactions , underestimation of persistence lengths of both single and double - stranded dna , and small influence of mismatched pairs on double - helix dynamics . the origin of mentioned problems most probably arises from overestimation of lennard - jones base base interactions , and we will address these problems in the next evaluations of the dipolar bead model .

a middle - resolution coarse - grained model of dna is proposed . the dna chain is built of spherical and planar rigid bodies connected by elastic virtual bonds . the bonded part of the potential energy function is fit to potentials of mean force of model systems . the rigid bodies are sets of neutral , charged , and dipolar beads . electrostatic and van der waals interactions are parametrized by our recently developed procedure [ maciejczyk , m. ; spasic , a. ; liwo , a. ; scheraga , h.a . j. comp . chem.2010 , 31 , 1644 ] . interactions with the solvent and an ionic cloud are approximated by a multipole multipole debye hckel model . a very efficient r - rattle algorithm , for integrating the movement of rigid bodies , is implemented . it is the first coarse - grained model , in which both bonded and nonbonded interactions were parametrized ab initio and which folds stable double helices from separated complementary strands , with the final conformation close to the geometry of experimentally determined structures .

Introduction Methods Results and Discussion Conclusions

coarse - grained models can be divided into three categories : statistical models , continuum models , and reduced interaction - center models . in the first category , the early work of zimm and rice and poland and scheraga neglected all structural and dynamical information and used only the free - energy gain per base - pair formation for computation of the partition function , which , in turn , was transformed into a thermodynamic picture of the process of helix formation . the continuum models approximate dsdna as a continuum elastic rod and by design can not be applied to the dna formation process . the last category covers all models in which groups of atoms are replaced by a reduced number of interaction centers connected by elastic and/or rigid virtual bonds . a properly designed coarse - graining procedure should reduce the number of interaction centers , leading to a significant speedup of computations , but simultaneously , the simplified potential energy function should include the most important interactions that determine the behavior of the real system . there are two common approaches to the problem of parametrization of a coarse - grained model . in the past decade , many coarse - grained models of dna have been developed , but only several of them managed to form double - stranded dna from separated complementary chains . the model was designed around the potential energy function , which effectively depends on the single parameter , which was optimized to reproduce experimental melting curves . the base base interactions were approximated by a go-like potential . the rigid - body model presented here has more interaction centers and more internal degrees of freedom than nares-2p , and consequently , it is computationally more demanding . in this paper , we present a physics - based , middle - resolution model of dna , which is capable of folding the double - helical structure from separate complementary strands . the non - bonded pairwise potentials do not depend on the sequential position of interacting bases in the polynucleotide chain because the native and non - native , nearest - neighbors , and all other pairs rely on the same potential energy function , which includes a lennard - jones and an electrostatic term . the full , physics - based ( pb model ) version of model keeps stable canonical b dna double helices in a long room - temperature simulations and folds short dna molecules . the reduced version of the model , in which base base intrastrand interactions are limited to nearest neighbors ( nn model ) , successfully folds canonical b dna from separate complementary strands for all tested systems . the coarse - grained model of dna is built of three types of particles:neutral bead , uncharged lennard - jones sphere,charged bead , lennard - jones sphere with an electric charge located in its center,dipolar bead , lennard - jones sphere with an electric dipole located in its center . the dna chain is built of six types of chemical units : phosphate group ( p ) , deoxyribose ( s ) , adenine ( a ) , thymine ( t ) , guanine ( g ) , and cytosine ( c ) . a schematic representation of the coarse - grained units overlapped on their atomic representation is shown in figure 1 . coarse - grained representation of fragments of the dna chain superimposed on the atomic representation . the internal degrees of freedom used for the definition of the bonded part of the potential energy function ( eq 2 ) are defined in tables 14 . the longer fragment of coarse - grained strand of dna with atgc sequence overlapped on the atomic model is shown in the right panel . the origin of the local coordinate frame of each rigid body is located at its center of mass , and its axes are aligned with the principal axes of the moment of inertia tensor . the position and orientation of each rigid body with respect to the global coordinate frame is described by a vector tensor pair ( r , q ) , where r is the position of the center of mass and q is the rotation matrix . in the model , spherical ( p and s ) and planar ( a , t , g , c ) . the spherical rigid bodies interact with other particles only by central forces ( see potential energy function ) , and their orientation is irrelevant ; therefore , a constant rotation matrix qsphere = const is assigned to them . the orientation of each planar rigid body is described by a 3 2 rotation matrix qplanar and varies as the rigid body changes its orientation during a simulation run . the geometry of the chain is defined in vector tensor space ( ri , qi ) where i = 1, ... ,n and n is the number of rigid bodies . relations between global coordinates ( r , q ) and distances between beads for interactions of ( a ) two spherical rigid - bodies , where there is only one distance rij = |rij| ; ( b ) spherical and planar rigid body , where distances between interacting beads are functions of global coordinates given by eq 9 ; and ( c ) two planar rigid - bodies , where distances between interacting beads are given by eq 13 . rigid bodies are connected by elastic virtual bonds and form a nucleic acid chain as shown in figure 1 . three rigid bodies connected by two virtual bonds form a virtual - bond angle and four rigid bodies connected by three virtual bonds form a virtual - bond dihedral angle . figure 1 ) p5s b x virtual - bond dihedral angle describes rotation of the base around the s the algorithm of leimkuhler and reich , which is based on the rattle constraint method applied to rotation matrices , was used for the description of rotation of rigid bodies . the leapfrog ( verlet ) algorithm was used for propagation of translations , and the r - rattle ( rotational rattle ) discretization scheme was applied to the rigid - body rotations . as in our unres model of polypeptide chains developed earlier , the effective energy function of our nucleic - acid model stems from the potential of mean force ( pmf ) of polynucleotide systems immersed in water . in particular , in contrast to the unres and nares-2p models , we do not average over any degrees of freedom of the nucleic - acid bases , the interactions between which are highly directional . the potential energy function of the system is a sum of the bonded and nonbonded parts:1although both the bonded and nonbonded parts depend on the cartesian - rigid - body degrees of freedom ( r , q ) , it is more convenient to define the former as a function of the internal degrees of freedom defined in tables 14 ( the internal degrees of freedom are functions of the ( r , q ) vector ) . the bonded part has a commonly used form2where:3a3b3c3dboth bond - stretching and angle - bending terms are approximated by harmonic potentials . for a detailed geometric definition of the internal degrees of freedom related to the bonded potentials see figure 1 and tables 14 . the force constants and equilibrium positions were determined by fitting the analytical expressions to the potentials of mean force for model systems . the nonbonded energy is a sum of van der waals and electrostatic energies and is given by4where the p , b , and s symbols denote the phosphate group , base , and sugar ring , respectively . the phosphate charge repulsion term7the three remaining energy terms of eq 4 involve interactions with the planar rigid - bodies ( bases ) . this kind of all - repulsive term is also used in the unres force field to account for excluded - volume interactions between the peptide groups and side chains8where nj is the number of dipolar beads of the j - th base and rijk is the distance between the neutral bead of the i - th sugar and the k - th dipolar bead of the j - th base . the distances rijkl between dipolar beads are functions of the relative position rij and orientations qi , qj of the rigid bodies13where ri(j)k(l ) is the position of the k - th ( l - th ) dipolar bead in the local coordinate frame of i - th ( j - th ) base . hckel model14a14b14cwhere q = eq is the negative unit charge located on phosphate group , n = r / r is a unit vector pointing from the first to the second interaction center , and pi(j ) are electric dipole vectors described in the global coordinate frame:15where pi(j ) is an electric dipole vector in the local coordinate frame of the i - th ( j - th ) base . because the interactions between the nearest neighbors , that is , particles separated by less than three virtual bonds , are included in the bonded part of the potential , they were excluded from the nonbonded interactions . the nonbonded interactions and solvation effects are very important for dna folding and its conformational stability and were approximated by the excluded - volume potential ( eq 10 ) for p b and s b , the lennard - jones potential for the remaining nonbonded interactions ( eq 6 ) , and a multipole - multipole potential energy function with debye hckel screening . the parameters of the b b interactions , including the positions r of the dipolar beads in the local coordinate frame , the electric dipole vectors p in the local coordinate frame , and the lennard - jones parameters alj , blj , were retained from the 3445 model of ref ( 47 ) . the charge of a neutral bead was , by definition , set to zero , the charge of a charged bead was set to |eq| , which is equal to the charge of the phosphate group in the dna backbone . therefore , the potential was switched to an excluded volume of the form of eq 10 , and the alj and blj parameters were retained from the fitting procedure , that is , aex = alj , bex = blj . the virtual bond , virtual - bond angle , and virtual - bond dihedral angle parameters were derived to reproduce the behavior of model systems in the all - atom representation . the potentials of mean force ( pmf ) of each internal degree of freedom of the coarse - grained model were calculated by using the umbrella sampling method . the base of the middle nucleotide was either adenine , cytosine , guanine , or thymine , for a total of four different model systems . this is a minimal size molecule for which every internal degree of freedom of our coarse - grained model can be defined . terminal bases were removed to avoid implicitly including electrostatic and van der waals interactions of bases since our model has the nonbonded parameters derived separately . for each degree of freedom , the distributions of data from each window and for appropriate model systems were combined , the biasing potential was removed , and the pmf along that coordinate was calculated by using the wham procedure implemented in a program by dr . although both the force constants and the equilibrium positions for the virtual bonds and virtual - bond angles were determined by fitting to the pmfs of model systems , it was decided to shift them slightly to match the equilibrium values of the ideal b - dna conformation , in the coarse - grained model . the rotation of the base around the virtual glycosidic bond ( s b ) was restricted by a cosine - shaped potential with the minimum corresponding to the anti conformation of the base and the barrier height was preliminary set to 7.0 kcal / mol . the final structures of the simulated annealing cycle were compared to the experimental reference by computing the all - bead rmsd . a new software package for this coarse - grained model , composed of rigid - bodies , was developed . the software is written in c / c++ programming language and currently can perform the following tasks : read the pdb - format structure and transform it into the coarse - grained representation , optimize the geometry of the molecule ( by energy minimization ) , perform microcanonical and canonical ensemble simulations with berendsen s weak thermal - bath coupling , and perform simulations of heating and simulated annealing cycles . the stability of the verlet - r - rattle algorithm was tested on the drew dickerson double - helix . the algorithm maintains good stability up to dt = 22 fs resulting in an order of magnitude speedup compared to the commonly used all - atom time - step of 2 fs ( with shake applied on hydrogens ) , and shows superior stability compared to the quaternion - based rigid - body integration scheme , applied previously to the model of a dna chain . stability of the verlet - r - rattle integration algorithm over 0.1 s microcanonical simulation of the drew drift was calculated as a difference between the averages of the last and the first 1 ns of each trajectory . the efficiency of the model was compared to that of all - atom simulations performed with the amber 11 package . the same simulation with our coarse - grained model and dt = 20 fs took 235 s with the same cpu , leading to almost 2 orders of magnitude speedup . the initial simulations of the drew dickerson dodecamer led to a collapse of the helical structure , caused by overestimation of the strength of the lennard - jones interactions with phosphates and sugars , which were fitted to reproduce the amber potential energy in vacuum . on the other hand the reduced ( and less physical ) model , in which intrastrand base base interactions were limited only to nearest - neighbors , was tested . a precise determination of all helical parameters would require conversion of the coarse - grained structurs to an all - atom representation ; however , the values calculated by using a coarse - grained representation are sufficient for comparison of the simulated structures with the respective experimental structures . for shorter molecules , 1bna and 3bse , the radius of gyration within nn approximation is close to the value obtained for experimental structure . this behavior is expected , because switching off some of the intrastrand base base interactions should decrease bending of the chain , especially for ssdna , in which bases are exposed to solvent and can easily form intrastrand hairpins with complementary bases . the persistence length of dsdna appears to be around three times too small compared to experimental values , and it is close to the value computed by early version of 3spn model . although the agreement with experimental data is not perfect it appears to be quite satisfactory for bottom - up-parametrized physics - based coarse - grained model and this discrepancy will be corrected in next versions of the model . only 3 out of 2100 trajectories led to the final helical structures with more than 10 native base much better folding rate was achieved with nn model in which intrastrand kinetic traps ( hairpins ) , resulting from intrastrand long - range base base interactions , were eliminated . 41% trajectories ended up in misfolded structures with 13 native contacts , which were mostly parallel dsdna s , in which 5-end of one chain makes nonative contacts with 5-end of the other chain . the optimization of experimental structure was necessary to remove small steric clashes in original structure caused by imperfectness of the coarse - grained model . the potential energy of original 1bna molecule is more than 100 kcal / mol higher than that of the optimized one , but the all - bead rmsd between the optimized and original structure is only 0.4 . this effect can be expected for the coarse - grained model with force - field parameters developed in the bottom - up fashion . the potential energy of the reference structure is almost exactly the same ( < 1 kcal / mol ) as the lowest - energy structure obtained from simulated annealing procedure . this observation suggests that longer cooling process should lead to improvements in geometry of final structures , as the minimum of potential energy was not reached by any simulated annealing cycle for longer model systems . all structures clearly form double - helices , although some discrepancies of the geometry are visible , especially for the longest molecule , for which overestimation of the size of minor groove is clearly visible . they showed that there is a bias toward initial structures with contacts between ends of the strands , an observation that can be confirmed by our model . this effect might be related to the lack of specificity of base base interactions , which might be the result of overestimation of base base van der waals interactions with respect to dipole dipole electrostatic interactions . a physics - based rigid - body coarse - grained model of dna is proposed . the bonded interactions are fitted to the potentials of mean force of the model system . the equilibrium virtual - bond lengths and virtual - bond angles were set to the values of the ideal b - dna double - helix . the nonbonded interactions are approximated by lennard - jones , excluded volume and electrostatic interactions of charges and dipoles . a full physics - based version of the model the nearest - neighbor approximation , in which intrastrand base base interactions are limited to nearest - neighbors only , was tested . the r - rattle rigid - body integration algorithm , implemented in our software package , demonstrated superior stability compared to a quaternion - based algorithm , with low drift of the total energy even for a time - step as long as 20 fs . the other drawback of the model , which seems to have the same origin as discrepancy in persistence lengths , is insufficient specificity of base base interactions , which affects simulations of dsdna with mismatches .

cataract surgery is one of the most commonly performed elective surgical procedures in most westernized countries . in the us medicare system , cataract is the second most expensive procedure after intravitreal injections of anti - vegf . with the growing number of older citizens , the need for eye care is expected to rise . the need for cataract surgery alone is expected to double within the next 20 years . we need to prioritize resources to be able to provide service to those most at need . immediate sequential bilateral cataract surgery ( isbcs ) , that is , surgery performed on both eyes on the same day but as separate procedures , has caused some controversy . those in favor of the procedure argue that the postoperative visual rehabilitation period is faster and that fewer visits to the clinic or hospital are needed , which saves money and time for both health professionals and patients [ 46 ] . those , who object to the procedure , argue that the risk of bilateral sight - threatening complications and the risk of postoperative refractive surprises outweigh any potential benefits that the procedure may have [ 7 , 8 ] . if the two surgeries are performed independently with strict hygienic precautions ( e.g. , rescrubbing of lids , redraping , regowning , and separate batches of surgical devices ) , the risk of bilateral endophthalmitis is small . reimbursement practices may also affect the likelihood of a surgeon considering isbcs or bilateral cataract surgery on two separate days . a study from sweden found that delayed sequential bilateral cataract surgery , that is , surgery on both eyes but on separate dates , was 14% more expensive than isbcs . a finnish study considering both the direct costs related to the surgery and transportation and time costs for the patient found that delayed sequential bilateral surgery was 849 euros more expensive than isbcs . thus , isbcs may have its economic advantages but it may be at an expense of bilateral severe complications to a few patients . how do we balance the benefits and risks ? with the increasing need for cataract surgery and a shortage of health care resources as well as the lessons learned from corneal refractive surgery where bilateral procedures are usually performed successfully on the same day , we felt that it was relevant to reconsider whether isbcs can be performed safely . the present study is a systematic review of the existing literature aimed at evaluating the safety aspects , risk , and benefits associated with isbcs . the work was undertaken after an initiation by the danish national health and medicines authority to formulate evidence - based national guidelines on surgery for age - related cataract . the aim of the present systematic review was to examine the benefits and harms associated with immediate sequential bilateral cataract surgery ( isbcs ) with specific emphasis on the rate of complications , postoperative anisometropia , and subjective visual function in order to formulate evidence - based national danish guidelines for cataract surgery . the review and resulting meta - analysis were performed based on the principles described in the grading of recommendation , assessment , development , and evaluation ( grade ) approach . the first step in the working process was to define the important questions and decide how to evaluate those questions using the pico approach . in short , pico stands for patient , intervention , comparison , and outcome . for this specific review and meta - analysis , we chose to examine the risks and advantages of isbcs for patients with bilateral age - related cataract undergoing phacoemulsification ( p ) . we extracted data from references where the patients were randomized to isbcs ( i ) or surgery on separate days ( c ) . as outcome measures ( o ) , we decided on the number of any adverse events , serious adverse events ( specifically the number of sight - threatening complications ) , and postoperative anisometropia ( > 2 diopters difference in spherical equivalent ) as well as the patient 's subjective satisfaction with the procedure . a systematic literature search was conducted in september 2014 in the embase and pubmed.gov databases and the cochrane central database using the search term : ( ( ( ( immediate sequential ) or bilateral surgery ) or same - day ) ) and ( ( ( cataract ) or cataract extraction ) or cataract surgery ) . the search was limited to references published within the last 10 years in the english or scandinavian languages . references were screened by title and abstract for eligibility . if there was any doubt as to the eligibility of the reference , the reference was obtained and read in full . study characteristics and outcome data were assessed and extracted independently by two authors ( line kessel and jesper hjortdal ) . risk of bias of the included studies was evaluated using the cochrane risk of bias tool in the review manager 5 software . in short , the cochrane risk of bias tool assesses risk of bias associated with the selection of patients ( randomization or patient allocation and concealment of allocation ) , study performance ( blinding of patients and personnel ) , detection of outcomes ( blinding of outcome assessment ) , attrition of data ( such as missing patients or drop - outs ) , reporting of study findings ( selective outcome reporting ) , or other types of bias related to the study design that could affect the internal validity . the quality of the evidence for each outcome was evaluated across the included studies and evidence profiles were prepared using the grade profiler software . the available evidence was assessed for study limitations ( risk of bias , e.g. , lack of allocation concealment or lack of blinding of patients or outcome assessors , incomplete accounting of patients and outcome , selective outcome reporting , or other limitations ) , inconsistency ( different results between studies ) , indirectness ( which was the study population and intervention comparable to the patient population and intervention , i.e. , relevant to the readers of meta - analysis and use of surrogate measures ) , imprecision ( large confidence intervals ( ci ) or the lack of statistical strength by included studies to answer the posed question ) , and risk of publication bias ( small number of studies or small number of included patients and lack of reporting of negative findings ) . continuous data were analyzed according to differences in mean treatment effects and their standard deviations . we used random - effects models to calculate pooled estimates of effects . according to danish law , no ethical committee or institutional board approval three randomized controlled clinical trials ( rcts ) examining the safety and efficacy of isbcs versus cataract surgery performed on two different days were identified by a systematic review of the literature [ 2325 ] . furthermore , 27 observational studies were identified ( including retrospective and prospective cohort studies ) . the rcts were included in the meta - analysis and all other study types were excluded . all three included rcts only included patients without competing eye diseases and with a limited range of axial lengths . we analyze the safety of isbcs compared to surgery performed on two different days with special emphasis on intra- and postoperative complications , postoperative anisometropia , and patient satisfaction . two of the included rcts provided information on the number of complications ( peri- and postoperative ) in the two groups randomized to isbcs or surgery on different days [ 24 , 25 ] . the third study provided information on the total rate of complications for the two groups combined but not for each group separately ( 6/96 = a complication rate of 6.3% , including high intraocular pressure < 30 mmhg on the first postoperative day in 2 eyes and one with a corneal edema ; at 2 months postoperatively 1 eye had iritis and at 4 months one eye had a vitreous detachment and 2 eyes ( 1 patient ) had beginnings of posterior capsule opacification ) . the reported prevalence of postoperative complications was markedly different in the remaining two studies [ 24 , 25 ] , appearing to reflect different opinions in what was considered a postoperative complication ; for example , only one of the studies included sutures in wound , first day postoperative pressure rise > 30 mmhg , or signs of posterior capsule fibrosis in the list of complications . looking at any complication ( intra- or postoperatively within the first month ) the two studies [ 24 , 25 ] reported a complication rate of 23% and 6% , respectively . the reported complications were capsule tears ( n = 17 ) , vitreous loss ( n = 5 ) , iridectomy or sphincterotomy ( n = 7 ) , sutures in wound ( n = 34 ) , intraocular pressure > 30 mmhg on the first postoperative day ( n = 67 ) , wound leak ( n = 2 ) , iol decentration or deplacement ( n = 6 ) , and corneal edema ( n = 31 ) and after one month iol decentration ( n = 2 ) , corneal edema ( n = 13 ) , anterior chamber flare ( n = 7 ) , capsular fibrosis ( n = 36 ) , and macular edema ( n = 3 ) in one study and iris prolapse ( n = 2 ) , posterior capsule tear ( n = 1 ) , corneal edema on first postoperative day ( n = 13 ) , capsule opacification ( n = 1 ) and foreign body sensation ( n = 1 ) , and dry eyes ( n = 80 ) in the other study . there was a tendency towards lower number of complications in the groups randomized to isbcs ( rr ( 95% ci ) 0.76 ( 0.55 , 1.07 ) , p = 0.12 , see figure 1 ) . due to the large inconsistency in number of reported complications and the fact that the outcome assessors in the included rcts were not blinded to patient randomization , the quality of the evidence was rated as very low ; see table 4 for a summary of the evidence and quality of the evidence assessment . most surgeons performing isbcs would recommend deferring surgery on the second eye in case of intraoperative complications . in two of the included rcts , none of the patients required deferral of second eye surgery because of intraoperative complications [ 24 , 25 ] . in the last study , three patients were excluded because of intraoperative complications . thus , three isbcs patients out of 1377 ( 0.2% ) had to have their second eye surgery deferred because of intraoperative complications . none of the studies included enough patients to be able to detect rare but serious side effects and sight - threatening complications . instead , we evaluated serious complications as the complications that could potentially be of threat to visual outcome , for example , corneal edema , macular edema , wound leakage , or iris prolapse . in total , the number of serious complications found within the three included rcts was 26 with corneal edema , three with macular edema , two with wound leakage , and 0 with iris prolapse . the rate of serious complications detected within the first postoperative month was 0.8% and 1.8% , respectively . there was no significant difference in the rate of serious postoperative complications between patients randomized to isbcs or surgery on different days ( p = 0.38 ) ; see figure 2 . one study reevaluated the hospital files one year after termination of the study and did not find any cases of retinal detachment within the first year postoperatively . due to the fact that the outcome assessors were not blinded to the patients ' randomization status and that the studies were not large enough to assess serious complications , the quality of the evidence was rated as low ; see table 4 . none of the included studies reported the number of patients who ended up with postoperative anisometropia of 2 diopters or greater , nor did they report lower grades of anisometropia . one study reported the mean difference between eyes in spherical equivalent after bilateral surgery and found that it was around 0.5 d but the range was not reported . a second study found that the postoperative refraction was within 1 day of the target at 1 month after surgery in around 90% of patients in both the isbcs and the different date groups . thus , none of the included studies could provide evidence as to the prevalence of postoperative anisometropia in patients undergoing isbcs . all three included rcts reported the subjective satisfaction with visual function postoperatively but one study did not report the standard deviation ; therefore , we could not include it in the meta - analysis . the remaining two studies [ 24 , 25 ] evaluated visual function on two different scales ( vf-7 and vf-14 ) and hence we used the standardized means method in order to include both the studies in the same meta - analysis . in the group randomized to bilateral surgery on two different days , subjective visual function was lower in the period between first and second eye surgeries . this effect disappeared when the second eye was operated on and 1 - 2 months after bilateral surgery there was no difference in subjective visual function between the groups randomized to isbcs or surgery on two different days ; see figure 3 . since none of the studies was blinded , the quality of the evidence was rated as moderate ; see table 3 . immediate sequential bilateral cataract surgery is a matter of controversy with strong arguments against and in favor of the procedure . in some countries , such as the spanish canary islands , sweden , and finland , the procedure is widely accepted and a large proportion of cataract patients are operated on both eyes on the same date . the present study was conducted to provide evidence - based recommendations on the risks associated with isbcs . since we wanted to provide evidence of the highest possible quality [ 18 , 26 ] , we chose only to include randomized trials comparing isbcs to bilateral cataract surgery performed on two different days . a review based on nonrandomized trials reporting the outcome after isbcs was published by others . however , even though we only included randomized trials , the level of evidence was low to moderate . thus , there is no strong scientific background to advice against or in favor of isbcs . after a systematic literature search , we identified three rcts including a total of 1900 patients . even though we restricted our analyses to randomized studies , the quality of evidence for each outcome across the trials ranged from low to moderate . one reason for rating down the quality of the evidence was that outcome assessment was not blinded in any of the studies . it was an inherent part of the study design that neither surgeons nor patients could be blinded as to whether both eyes were operated on the same day or on separate dates . the assessment of outcome at follow - up could , however , easily have been blinded but the studies provided no information as to whether this was the case or not . future studies could be designed with a follow - up of 3 to 6 months and the person assessing the outcome at final follow - up could be blinded to when each eye was operated on . in addition , statistical analyses can be performed with masking of data , so that the person performing the statistical analyses is blinded to which intervention each group of patients was randomized to . furthermore , the quality of evidence was rated down for inconsistency due to the large differences in reported number of complications . this probably reflects differences in opinion regarding what is considered a complication but nevertheless there is reason for concern when raised intraocular pressure on the first postoperative day is found in 7% of patients in one study but in no patients in a second study . finally , the quality of the evidence was rated down because the three rcts in combination had included too few patients to evaluate serious postoperative complications with any certainty ; for example , the rate of endophthalmitis is between 0.175% ( in the escrs study ) and 0.029% ( in sweden ) . one of the strongest arguments against isbcs is the risk of bilateral endophthalmitis . to follow this argument , the second eye should not be operated on before the first eye is safely beyond the risk of endophthalmitis . in the escrs study , four of the 29 cases ( 14% ) of endophthalmitis were diagnosed later than 2 weeks after surgery with two cases presenting at day 36 ( negative culture ) and day 132 ( propionibacterium acnes ) . this suggests that a significant amount of time should lapse between surgeries to assure that no patient has bilateral endophthalmitis . bilateral endophthalmitis [ 32 , 33 ] and bilateral early corneal decompensation requiring corneal transplantation have been both described after isbcs but the overall rate of endophthalmitis is not expected to be higher after isbcs compared to surgery on two different days . in one of the cases of bilateral endophthalmitis , surgical procedures were not optimized for isbcs with reuse of irrigating fluids and flash sterilization and in the other study instruments were autoclaved but the quality of the sterilization procedure was not checked . the risk of retinal detachment increases markedly after cataract surgery [ 37 , 38 ] but the time lapse between surgery and retinal detachment means that if it occurs , the retinal detachment will not be diagnosed in due time before second eye surgery even if bilateral surgery is performed on separate days . the evidence presented in the present systematic review does not allow for any conclusions to be drawn as to whether the risk of complications is higher or lower after isbcs than different date bilateral cataract surgery due to the low quality of evidence . if surgeries are performed on two different days , the refractive outcome of the first eye can be used to guide the refractive plan for the second eye . a study , where the biometry was based on an ultrasound application method and where the difference in axial length between eyes was very large , found that the refractive outcome of the first eye was not of value in adjusting the refractive plan of the second eye . using newer optically based methods of axial length determination , the refractive prediction of the second eye was , however , improved when information from the first eye was used [ 40 , 41 ] . none of the included rcts provided information as to the postoperative anisometropia after bilateral surgery . a retrospective study evaluating the prevalence of postoperative anisometropia in patients undergoing isbcs found that postoperative anisometropia was > 2 diopters in 1.2% of patients . to minimize the risk of postoperative anisometropia and unwanted refractive surprises , two of the included rcts excluded patients with axial lengths outside the normal range [ 23 , 25 ] . some careful consideration is required in patients with high refractive errors ; longer time between surgeries means longer periods of poor visual function due to postoperative anisometropia . most centers performing corneal refractive surgery offer surgery on both eyes on the same day to avoid postoperative anisometropia . from a surgeon 's point of view , it seems advisable to limit isbcs to the group of patients with low expected risk of peri- or postoperative complications and this may exclude some patients with extreme refractive errors . however , from a patient 's point of view , the time lapse between surgeries should be as short as possible especially if the patient has high refractive errors because of the large anisometropia when one eye has had surgery and the other has not . none of the included studies evaluated the subjective satisfaction of undergoing cataract surgery as an immediate or delayed sequential bilateral procedure . from a patient perspective a retrospective study found that a significant majority of patients ( 90% ) would recommend or recommend with pleasure isbcs to their relatives or friends whereas only 2% would not recommend the procedure . none of the studies found a significant difference in subjective visual function after bilateral surgery between patients randomized to isbcs or different date bilateral cataract surgery [ 2325 ] . in patients who were randomized to different date surgery , there was a poorer self - assessed visual function in the time period between first and second eye surgeries . based on our results , we can not say that one method provides better subjective visual outcome than the other method or that one method should be recommended over the other based on subjective visual function . the previous danish guideline for cataract surgery published by the danish ophthalmological society in 2001 stated that isbcs should not be performed because of a lack of evidence on the safety aspects of the procedure . the royal society of ophthalmologists is generally cautious about performing isbcs but advise that it may be performed in patients with a need for general anaesthesia and in whom repeated general anaesthesia is contraindicated for medical reasons . in summary , we found that there was scientific evidence of very low to moderate quality regarding the risks and benefits of isbcs . we did not find reason to suspect that complications were more or less frequent after isbcs but we can not rule out that they were . the effect on postoperative anisometropia could not be evaluated by the included randomized trials but a retrospective study indicated that this would not be a major problem . self - assessed visual function was the same after bilateral surgery no matter if patients were operated bilaterally on the same day or 2 months apart but poorer in the time interval between the surgeries for patients operated on different days . performing cataract surgery with a time interval between the two eyes allows for detection of some of the sight - threatening complications such as early endophthalmitis , but cystoid macular edema , retinal detachment , and late corneal decompensation usually present with a greater time lag than what most surgeons use between the two surgical procedures . if immediate sequential bilateral surgery is performed , it seems pertinent that the two surgeries are performed as two separate procedures including regowning of the surgeon and assistant , redraping and cleaning of the eye region , refitting the surgical equipment , and possibly also using two separate batches of viscoelastica and iols . intracameral antibiotics significantly lower the risk of endophthalmitis and it seems mandatory that intracameral antibiotics should be used if isbcs is performed . in sweden , where around 6% of cataract surgeries are performed as same - day procedure , two intracameral antibiotics ( cefuroxime and ampicillin ) are used during isbcs in order to minimize the risk of endophthalmitis and since no cases of bilateral endophthalmitis after same - day surgery have been reported in sweden ( mats lundstrm , personal communication ) , this seems to be a good advice . immediate sequential bilateral cataract surgery may offer some advantages in terms of saving of health resources and faster optical rehabilitation but it is at the risk of simultaneous , bilateral complications . the level of evidence concerning isbcs is low and hence it is not possible to formulate an evidence - based recommendation . immediate sequential bilateral cataract surgery may be a good option for patients undergoing surgery in general anaesthesia and in whom repeated general anaesthesia is associated with increased health risks . any general or ocular condition that might increase the risk for any peri- or postoperative complication conflicts with the use of isbcs . patients should be informed of and have consented to the risks associated with isbcs and it should only be performed by experienced surgeons taking meticulous care to adhere to strict hygienic standards with the two procedures being performed as independent procedures including redraping and regowning and with the use of separate batches of surgical equipment ( including viscoelastic material and iols ) [ 4 , 48 ] . furthermore , we advise that the immediate sequential approach is abandoned if complications arise during surgery on the first eye .

the aim of the present systematic review was to examine the benefits and harms associated with immediate sequential bilateral cataract surgery ( isbcs ) with specific emphasis on the rate of complications , postoperative anisometropia , and subjective visual function in order to formulate evidence - based national danish guidelines for cataract surgery . a systematic literature review in pubmed , embase , and cochrane central databases identified three randomized controlled trials that compared outcome in patients randomized to isbcs or bilateral cataract surgery on two different dates . meta - analyses were performed using the cochrane review manager software . the quality of the evidence was assessed using the grade method ( grading of recommendation , assessment , development , and evaluation ) . we did not find any difference in the risk of complications or visual outcome in patients randomized to isbcs or surgery on two different dates . the quality of evidence was rated as low to very low . none of the studies reported the prevalence of postoperative anisometropia . in conclusion , we can not provide evidence - based recommendations on the use of isbcs due to the lack of high quality evidence . therefore , the decision to perform isbcs should be taken after careful discussion between the surgeon and the patient .

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

cataract surgery is one of the most commonly performed elective surgical procedures in most westernized countries . the need for cataract surgery alone is expected to double within the next 20 years . immediate sequential bilateral cataract surgery ( isbcs ) , that is , surgery performed on both eyes on the same day but as separate procedures , has caused some controversy . those in favor of the procedure argue that the postoperative visual rehabilitation period is faster and that fewer visits to the clinic or hospital are needed , which saves money and time for both health professionals and patients [ 46 ] . those , who object to the procedure , argue that the risk of bilateral sight - threatening complications and the risk of postoperative refractive surprises outweigh any potential benefits that the procedure may have [ 7 , 8 ] . , rescrubbing of lids , redraping , regowning , and separate batches of surgical devices ) , the risk of bilateral endophthalmitis is small . reimbursement practices may also affect the likelihood of a surgeon considering isbcs or bilateral cataract surgery on two separate days . a study from sweden found that delayed sequential bilateral cataract surgery , that is , surgery on both eyes but on separate dates , was 14% more expensive than isbcs . a finnish study considering both the direct costs related to the surgery and transportation and time costs for the patient found that delayed sequential bilateral surgery was 849 euros more expensive than isbcs . how do we balance the benefits and risks ? with the increasing need for cataract surgery and a shortage of health care resources as well as the lessons learned from corneal refractive surgery where bilateral procedures are usually performed successfully on the same day , we felt that it was relevant to reconsider whether isbcs can be performed safely . the present study is a systematic review of the existing literature aimed at evaluating the safety aspects , risk , and benefits associated with isbcs . the work was undertaken after an initiation by the danish national health and medicines authority to formulate evidence - based national guidelines on surgery for age - related cataract . the aim of the present systematic review was to examine the benefits and harms associated with immediate sequential bilateral cataract surgery ( isbcs ) with specific emphasis on the rate of complications , postoperative anisometropia , and subjective visual function in order to formulate evidence - based national danish guidelines for cataract surgery . the review and resulting meta - analysis were performed based on the principles described in the grading of recommendation , assessment , development , and evaluation ( grade ) approach . the first step in the working process was to define the important questions and decide how to evaluate those questions using the pico approach . for this specific review and meta - analysis , we chose to examine the risks and advantages of isbcs for patients with bilateral age - related cataract undergoing phacoemulsification ( p ) . we extracted data from references where the patients were randomized to isbcs ( i ) or surgery on separate days ( c ) . as outcome measures ( o ) , we decided on the number of any adverse events , serious adverse events ( specifically the number of sight - threatening complications ) , and postoperative anisometropia ( > 2 diopters difference in spherical equivalent ) as well as the patient 's subjective satisfaction with the procedure . a systematic literature search was conducted in september 2014 in the embase and pubmed.gov databases and the cochrane central database using the search term : ( ( ( ( immediate sequential ) or bilateral surgery ) or same - day ) ) and ( ( ( cataract ) or cataract extraction ) or cataract surgery ) . if there was any doubt as to the eligibility of the reference , the reference was obtained and read in full . risk of bias of the included studies was evaluated using the cochrane risk of bias tool in the review manager 5 software . in short , the cochrane risk of bias tool assesses risk of bias associated with the selection of patients ( randomization or patient allocation and concealment of allocation ) , study performance ( blinding of patients and personnel ) , detection of outcomes ( blinding of outcome assessment ) , attrition of data ( such as missing patients or drop - outs ) , reporting of study findings ( selective outcome reporting ) , or other types of bias related to the study design that could affect the internal validity . the quality of the evidence for each outcome was evaluated across the included studies and evidence profiles were prepared using the grade profiler software . the available evidence was assessed for study limitations ( risk of bias , e.g. , lack of allocation concealment or lack of blinding of patients or outcome assessors , incomplete accounting of patients and outcome , selective outcome reporting , or other limitations ) , inconsistency ( different results between studies ) , indirectness ( which was the study population and intervention comparable to the patient population and intervention , i.e. , relevant to the readers of meta - analysis and use of surrogate measures ) , imprecision ( large confidence intervals ( ci ) or the lack of statistical strength by included studies to answer the posed question ) , and risk of publication bias ( small number of studies or small number of included patients and lack of reporting of negative findings ) . according to danish law , no ethical committee or institutional board approval three randomized controlled clinical trials ( rcts ) examining the safety and efficacy of isbcs versus cataract surgery performed on two different days were identified by a systematic review of the literature [ 2325 ] . the rcts were included in the meta - analysis and all other study types were excluded . we analyze the safety of isbcs compared to surgery performed on two different days with special emphasis on intra- and postoperative complications , postoperative anisometropia , and patient satisfaction . two of the included rcts provided information on the number of complications ( peri- and postoperative ) in the two groups randomized to isbcs or surgery on different days [ 24 , 25 ] . the third study provided information on the total rate of complications for the two groups combined but not for each group separately ( 6/96 = a complication rate of 6.3% , including high intraocular pressure < 30 mmhg on the first postoperative day in 2 eyes and one with a corneal edema ; at 2 months postoperatively 1 eye had iritis and at 4 months one eye had a vitreous detachment and 2 eyes ( 1 patient ) had beginnings of posterior capsule opacification ) . the reported prevalence of postoperative complications was markedly different in the remaining two studies [ 24 , 25 ] , appearing to reflect different opinions in what was considered a postoperative complication ; for example , only one of the studies included sutures in wound , first day postoperative pressure rise > 30 mmhg , or signs of posterior capsule fibrosis in the list of complications . the reported complications were capsule tears ( n = 17 ) , vitreous loss ( n = 5 ) , iridectomy or sphincterotomy ( n = 7 ) , sutures in wound ( n = 34 ) , intraocular pressure > 30 mmhg on the first postoperative day ( n = 67 ) , wound leak ( n = 2 ) , iol decentration or deplacement ( n = 6 ) , and corneal edema ( n = 31 ) and after one month iol decentration ( n = 2 ) , corneal edema ( n = 13 ) , anterior chamber flare ( n = 7 ) , capsular fibrosis ( n = 36 ) , and macular edema ( n = 3 ) in one study and iris prolapse ( n = 2 ) , posterior capsule tear ( n = 1 ) , corneal edema on first postoperative day ( n = 13 ) , capsule opacification ( n = 1 ) and foreign body sensation ( n = 1 ) , and dry eyes ( n = 80 ) in the other study . there was a tendency towards lower number of complications in the groups randomized to isbcs ( rr ( 95% ci ) 0.76 ( 0.55 , 1.07 ) , p = 0.12 , see figure 1 ) . due to the large inconsistency in number of reported complications and the fact that the outcome assessors in the included rcts were not blinded to patient randomization , the quality of the evidence was rated as very low ; see table 4 for a summary of the evidence and quality of the evidence assessment . in two of the included rcts , none of the patients required deferral of second eye surgery because of intraoperative complications [ 24 , 25 ] . none of the studies included enough patients to be able to detect rare but serious side effects and sight - threatening complications . instead , we evaluated serious complications as the complications that could potentially be of threat to visual outcome , for example , corneal edema , macular edema , wound leakage , or iris prolapse . in total , the number of serious complications found within the three included rcts was 26 with corneal edema , three with macular edema , two with wound leakage , and 0 with iris prolapse . the rate of serious complications detected within the first postoperative month was 0.8% and 1.8% , respectively . there was no significant difference in the rate of serious postoperative complications between patients randomized to isbcs or surgery on different days ( p = 0.38 ) ; see figure 2 . one study reevaluated the hospital files one year after termination of the study and did not find any cases of retinal detachment within the first year postoperatively . due to the fact that the outcome assessors were not blinded to the patients ' randomization status and that the studies were not large enough to assess serious complications , the quality of the evidence was rated as low ; see table 4 . none of the included studies reported the number of patients who ended up with postoperative anisometropia of 2 diopters or greater , nor did they report lower grades of anisometropia . thus , none of the included studies could provide evidence as to the prevalence of postoperative anisometropia in patients undergoing isbcs . all three included rcts reported the subjective satisfaction with visual function postoperatively but one study did not report the standard deviation ; therefore , we could not include it in the meta - analysis . the remaining two studies [ 24 , 25 ] evaluated visual function on two different scales ( vf-7 and vf-14 ) and hence we used the standardized means method in order to include both the studies in the same meta - analysis . in the group randomized to bilateral surgery on two different days , subjective visual function was lower in the period between first and second eye surgeries . this effect disappeared when the second eye was operated on and 1 - 2 months after bilateral surgery there was no difference in subjective visual function between the groups randomized to isbcs or surgery on two different days ; see figure 3 . since none of the studies was blinded , the quality of the evidence was rated as moderate ; see table 3 . immediate sequential bilateral cataract surgery is a matter of controversy with strong arguments against and in favor of the procedure . in some countries , such as the spanish canary islands , sweden , and finland , the procedure is widely accepted and a large proportion of cataract patients are operated on both eyes on the same date . the present study was conducted to provide evidence - based recommendations on the risks associated with isbcs . since we wanted to provide evidence of the highest possible quality [ 18 , 26 ] , we chose only to include randomized trials comparing isbcs to bilateral cataract surgery performed on two different days . however , even though we only included randomized trials , the level of evidence was low to moderate . after a systematic literature search , we identified three rcts including a total of 1900 patients . even though we restricted our analyses to randomized studies , the quality of evidence for each outcome across the trials ranged from low to moderate . one reason for rating down the quality of the evidence was that outcome assessment was not blinded in any of the studies . it was an inherent part of the study design that neither surgeons nor patients could be blinded as to whether both eyes were operated on the same day or on separate dates . furthermore , the quality of evidence was rated down for inconsistency due to the large differences in reported number of complications . finally , the quality of the evidence was rated down because the three rcts in combination had included too few patients to evaluate serious postoperative complications with any certainty ; for example , the rate of endophthalmitis is between 0.175% ( in the escrs study ) and 0.029% ( in sweden ) . one of the strongest arguments against isbcs is the risk of bilateral endophthalmitis . to follow this argument , the second eye should not be operated on before the first eye is safely beyond the risk of endophthalmitis . in the escrs study , four of the 29 cases ( 14% ) of endophthalmitis were diagnosed later than 2 weeks after surgery with two cases presenting at day 36 ( negative culture ) and day 132 ( propionibacterium acnes ) . bilateral endophthalmitis [ 32 , 33 ] and bilateral early corneal decompensation requiring corneal transplantation have been both described after isbcs but the overall rate of endophthalmitis is not expected to be higher after isbcs compared to surgery on two different days . in one of the cases of bilateral endophthalmitis , surgical procedures were not optimized for isbcs with reuse of irrigating fluids and flash sterilization and in the other study instruments were autoclaved but the quality of the sterilization procedure was not checked . the risk of retinal detachment increases markedly after cataract surgery [ 37 , 38 ] but the time lapse between surgery and retinal detachment means that if it occurs , the retinal detachment will not be diagnosed in due time before second eye surgery even if bilateral surgery is performed on separate days . the evidence presented in the present systematic review does not allow for any conclusions to be drawn as to whether the risk of complications is higher or lower after isbcs than different date bilateral cataract surgery due to the low quality of evidence . if surgeries are performed on two different days , the refractive outcome of the first eye can be used to guide the refractive plan for the second eye . a study , where the biometry was based on an ultrasound application method and where the difference in axial length between eyes was very large , found that the refractive outcome of the first eye was not of value in adjusting the refractive plan of the second eye . using newer optically based methods of axial length determination , the refractive prediction of the second eye was , however , improved when information from the first eye was used [ 40 , 41 ] . none of the included rcts provided information as to the postoperative anisometropia after bilateral surgery . a retrospective study evaluating the prevalence of postoperative anisometropia in patients undergoing isbcs found that postoperative anisometropia was > 2 diopters in 1.2% of patients . to minimize the risk of postoperative anisometropia and unwanted refractive surprises , two of the included rcts excluded patients with axial lengths outside the normal range [ 23 , 25 ] . some careful consideration is required in patients with high refractive errors ; longer time between surgeries means longer periods of poor visual function due to postoperative anisometropia . most centers performing corneal refractive surgery offer surgery on both eyes on the same day to avoid postoperative anisometropia . from a surgeon 's point of view , it seems advisable to limit isbcs to the group of patients with low expected risk of peri- or postoperative complications and this may exclude some patients with extreme refractive errors . however , from a patient 's point of view , the time lapse between surgeries should be as short as possible especially if the patient has high refractive errors because of the large anisometropia when one eye has had surgery and the other has not . none of the included studies evaluated the subjective satisfaction of undergoing cataract surgery as an immediate or delayed sequential bilateral procedure . none of the studies found a significant difference in subjective visual function after bilateral surgery between patients randomized to isbcs or different date bilateral cataract surgery [ 2325 ] . in patients who were randomized to different date surgery , there was a poorer self - assessed visual function in the time period between first and second eye surgeries . based on our results , we can not say that one method provides better subjective visual outcome than the other method or that one method should be recommended over the other based on subjective visual function . the previous danish guideline for cataract surgery published by the danish ophthalmological society in 2001 stated that isbcs should not be performed because of a lack of evidence on the safety aspects of the procedure . in summary , we found that there was scientific evidence of very low to moderate quality regarding the risks and benefits of isbcs . we did not find reason to suspect that complications were more or less frequent after isbcs but we can not rule out that they were . self - assessed visual function was the same after bilateral surgery no matter if patients were operated bilaterally on the same day or 2 months apart but poorer in the time interval between the surgeries for patients operated on different days . performing cataract surgery with a time interval between the two eyes allows for detection of some of the sight - threatening complications such as early endophthalmitis , but cystoid macular edema , retinal detachment , and late corneal decompensation usually present with a greater time lag than what most surgeons use between the two surgical procedures . if immediate sequential bilateral surgery is performed , it seems pertinent that the two surgeries are performed as two separate procedures including regowning of the surgeon and assistant , redraping and cleaning of the eye region , refitting the surgical equipment , and possibly also using two separate batches of viscoelastica and iols . intracameral antibiotics significantly lower the risk of endophthalmitis and it seems mandatory that intracameral antibiotics should be used if isbcs is performed . in sweden , where around 6% of cataract surgeries are performed as same - day procedure , two intracameral antibiotics ( cefuroxime and ampicillin ) are used during isbcs in order to minimize the risk of endophthalmitis and since no cases of bilateral endophthalmitis after same - day surgery have been reported in sweden ( mats lundstrm , personal communication ) , this seems to be a good advice . immediate sequential bilateral cataract surgery may offer some advantages in terms of saving of health resources and faster optical rehabilitation but it is at the risk of simultaneous , bilateral complications . the level of evidence concerning isbcs is low and hence it is not possible to formulate an evidence - based recommendation . immediate sequential bilateral cataract surgery may be a good option for patients undergoing surgery in general anaesthesia and in whom repeated general anaesthesia is associated with increased health risks . any general or ocular condition that might increase the risk for any peri- or postoperative complication conflicts with the use of isbcs . patients should be informed of and have consented to the risks associated with isbcs and it should only be performed by experienced surgeons taking meticulous care to adhere to strict hygienic standards with the two procedures being performed as independent procedures including redraping and regowning and with the use of separate batches of surgical equipment ( including viscoelastic material and iols ) [ 4 , 48 ] . furthermore , we advise that the immediate sequential approach is abandoned if complications arise during surgery on the first eye .

several types of monochromators have been developed for electron microscopy in this decade and are now available as a tool for material characterization . they can reduce the energy spread of incident electrons to milli - electron - volt level . monochromators can improve a diverse range of electron microscopy techniques , including not only electron energy - loss spectroscopy ( eels ) but also other imaging techniques , such as transmission electron microscopy ( tem ) and scanning transmission electron microscopy ( stem ) . a monochromator is an electron - optical system including energy - dispersive deflectors and an energy - selecting slit , and there are many types of deflectors and their combinations , which decide the inherent performance of the instrument . for instance , most monochromators function at a high tension , but a few function at the earth ground , yielding different performance properties . monochromators change the illumination conditions , such as the convergence angle ; therefore , the tem imaging properties are affected in terms of both chromatic and spatial partial coherence . although monochromators disperse the electron source image on the energy - selecting slit , a probe - forming lens focusses the source image on the specimen in stem imaging . thus , the monochromator properties are also related to the stem imaging performance from the viewpoint of not only chromatic aberration but also spatial incoherence . in short , the energy - dispersed source image on the specimen might degrade the stem spatial resolution under an inappropriate alignment condition . in this review , then , a few critical properties of these monochromators are pointed out through a discussion of their differences . the advantages and side effects of using these monochromators in eels , tem and stem imaging are also discussed . several monochromators developed for electron microscopy have already been studied by pioneering researchers [ 24 ] , and manufacturers have recently provided several types of monochromators . table 1 outlines the four monochromators described in this review : ( a ) a single wien filter monochromator developed by fei [ 57 ] , ( b ) a double wien filter monochromator by jeol , ( c ) an omega - shaped electrostatic monochromator by ceos and ( d ) an alpha - type magnetic monochromator by nion . here a few properties of these monochromators are outlined through a discussion of their differences . table 1.basic structures of currently available monochromators(a ) single wien filter(fei)(b ) double wien filter(jeol)(c ) omega - shaped electrostatic(ceos)(d ) alpha - type magnetic(nion)basic structurewien filter(acc . tube)m - sector x2slitm - sector x2electron energy in energy dispersion partlowlowlowhighelectron energy in energy - selecting slithighlowlowhighspatial chromaticityresidualcorrectedcorrectedcorrectedangular chromaticityresidualresidualcorrectedcorrectedacc . tube , acceleration tube ; e - sector , electrostatic toroidal sector deflector ; m - sector , magnetic sector . basic structures of currently available monochromators acc . tube , acceleration tube ; e - sector , electrostatic toroidal sector deflector ; m - sector , magnetic sector . the single wien filter monochromator ( a ) consists of a wien filter and an energy - selecting slit ; a wien filter analyses the speed of electrons using the crossing magnetic and electrostatic fields , and the energy - selecting slit mechanically chooses a portion of the dispersed electrons . the double wien filter monochromator ( b ) includes two wien filters with an energy - selecting slit inserted between them . the second wien filter eliminates the energy dispersion at the conjugate plane of the electron source . the omega - shaped electrostatic monochromator ( c ) constructs an omega - shaped electron path using four electrostatic toroidal sector deflectors . the alpha - type magnetic monochromator ( d ) is attached after the acceleration tube , and four energy dispersions , which are actually formed by one uniform - field sector ( functioning twice ) and two gradient - field sectors , are used to produce an alpha - shaped electron path . the omega- or alpha - shaped electron paths minimize the aberrations of each monochromator by acting as in - column energy filters , because the energy - selecting slit and energy dispersion plane of the monochromators lie on the midplane of the electron path . the electron energy in the energy dispersion part depends on the arrangement of the monochromator and the acceleration tube and yields a critical difference in the monochromator properties . in many cases ( i.e. ( a)(c ) ) , the electron energy in the energy dispersion part is relatively low ( e.g. a few kev ) to obtain sufficient energy dispersion at the energy - selecting slit and , as a result , many monochromators are mounted between the electron gun and the acceleration tube . there are a few technical challenges in mounting a monochromator at a high tension in terms of its electronics , mechanics and the requirement of ultrahigh vacuum in the electron gun chamber . note that a monochromator at a high tension can not filter out the instability of the acceleration voltage ; however , it can be easily used at various acceleration voltages under the same monochromator condition . the performance of the energy dispersion part depends not only on the electron optics but also on the boersch effect ; therefore , the length of the electron path , the probe current and the number of crossovers should be minimized . in contrast , monochromator ( d ) attached after the acceleration tube works at the earth ground , and the electron energy in the energy dispersion part is high . therefore , monochromator ( d ) requires additional electron optics to magnify the energy dispersion , and nion adopted a multipole lens system as well as a parallel eel spectrometer or a post - column energy filter . the high - tension instability directly affects the projected position on the energy - selecting slit , allowing us to stabilize the high tension . monochromator ( d ) has been combined with a cold field - emission gun ( cfeg ) , which requires ultrahigh vacuum , because monochromator ( d ) can be fitted on the microscope column below the acceleration tube . the energy - selecting slit is an indispensable part of a monochromator , and the required slit width is generally of the order of a micrometre . because monochromators ( b ) and ( c ) select electrons at a relatively low energy , the energy - selecting slit should be clean and free from charge - up . in addition , the mechanical movable components of the energy - selecting slit should not degrade the vacuum in the adjacent electron gun chamber . in contrast , the energy - selecting slits of monochromators ( a ) and ( d ) select high - energy electrons in the microscope columns , and a variety of slits and a complicated mechanical / electrical system can be implemented . in the case of the single wien filter monochromator , the acceleration tube between the wien filter and the energy - selecting slit magnifies the source image and the energy dispersion , which is advantageous for energy selection . in monochromator ( d ) , a current detection system is implemented on the energy - selecting slit for the additional stabilization of high tension . figure 1 shows schematics of the four monochromators , in which the electron path is drawn as a straight line for simplicity . dotted curves represent the axial paths of electrons with a different energy , and open circles correspond to the source images . small closed circles are astigmatically focussed ( i.e. line - focussed ) source images . the multiple energy - dispersive deflectors in monochromators ( b)(d ) finally yield an achromatic source image , which is schematically represented as the coincidence of an open circle and a dotted line in fig . 1b and d. the symmetric optical arrangement of monochromators ( c ) and ( d ) yields a symmetric electron path to the midplane at the energy - selecting slit , resulting in the minimized aberration of each system . 1.schematics of four monochromators : ( a ) single wien filter monochromator , ( b ) double wien filter monochromator , ( c ) omega - shaped electrostatic monochromator and ( d ) alpha - type magnetic monochromator . the electron path with the standard energy is schematically drawn as a straight line , and dotted curves represent the axial paths of electrons with a different energy . open circles correspond to the conjugate planes to the electron source and small closed circles are the positions of astigmatically focussed ( i.e. line - focussed ) source images . rectangles , triangles and rounded rectangles represent monochromator units , energy dispersion parts and acceleration tubes , respectively . the arrows at the bottom represent the residual energy dispersion at the final source image . schematics of four monochromators : ( a ) single wien filter monochromator , ( b ) double wien filter monochromator , ( c ) omega - shaped electrostatic monochromator and ( d ) alpha - type magnetic monochromator . the electron path with the standard energy is schematically drawn as a straight line , and dotted curves represent the axial paths of electrons with a different energy . open circles correspond to the conjugate planes to the electron source and small closed circles are the positions of astigmatically focussed ( i.e. line - focussed ) source images . rectangles , triangles and rounded rectangles represent monochromator units , energy dispersion parts and acceleration tubes , respectively . the arrows at the bottom represent the residual energy dispersion at the final source image . spatial chromaticity means that there is energy dispersion at the final source image , which is eliminated in monochromators ( b)(d ) . angular chromaticity means that electrons propagating in different directions have different energies , which is eliminated in monochromators ( c ) and ( d ) owing to their symmetric electron paths . it is worth mentioning that the angular chromaticity in the double wien filter monochromator ( b ) can be controlled by changing the length of the wien filters . for instance , the deflection induced by the lorentz force becomes zero ( i.e. the rotation angle equals 2 ) at a specific filter length , and the angular chromaticity is eliminated under this condition . figure 1b depicts an example setting , in which the total angle of rotation induced by the lorentz force equals , which was reported in a previous paper . stabilities at a high tension and power supply of the monochromator are critical technical requirements to realize high energy resolution . the high - tension instability can not be corrected by a monochromator at a high tension , and a highly stabilized high tension is required to realize high energy resolution . note that a stable energy - dispersed source image on an energy - selecting slit only ensures the stability of the voltage ( e.g. a few kv ) of the electrons in the monochromator . in contrast , monochromator ( d ) at the earth ground level can eliminate high - tension instability . because high - tension instability causes instability in a probe current of monochromator ( d ) , in addition , a monochromator at the earth ground level requires ultrahigh resolving power in comparison with that of a monochromator at a high tension , and its power supply should also be highly stabilized . although the aberration figure on the selecting slit can be easily observed in the single wien filter monochromator , those of the other monochromators are not observable because of the energy dispersion deflector(s ) after the energy - selecting slit , making their alignment indirect . the eel spectrometer should have high energy resolution ( small non - isochromaticity ) and be highly stabilized . a few pioneering studies on high - resolution eels have been performed with the combination of a monochromator and a retarding analyzer , in which the potentials of both energy dispersion deflectors were connected . for this combination , the potential instability does not result in spectroscopic instability , even if the actual energy of incident electrons at the specimen fluctuates . a similar concept is embraced in monochromator ( d ) , in which the power supplies of the monochromator and analyzer are synchronized . the electron gun brightness is generally defined as the current j per unit solid angle and unit area [ a sr m ] . from the viewpoint of monochromated electron microscopy , the brightness of each electron gun should be re - examined on the basis of each energy spread . namely , the brightness should be normalized by the energy spread ( e.g. [ a sr m ev ] ) , in which the current j is replaced by the maximum of the energy distribution , dj(e)/de , where e is the energy of the incident electrons . the electron gun brightness related to the energy spread has already been investigated as the differential brightness in the theoretical study of shimoyama and maruse and as the reduced brightness in the experimental study reported by schwind et al . . the latter reported an improvement of the reduced brightness in the sharpened tip of a schottky emission gun ( seg ) . because a cfeg has the highest reduced brightness among existing electron guns , the use of a cfeg or seg with a sharpened tip is preferable for a monochromator . an example of the energy distributions obtained using a cfeg and the single wien filter monochromator is shown in fig . 2 . the energy distribution of a cfeg is limited by the fermi tail and the tunnelling tail on the low- and high - energy - loss sides , respectively . the fermi tail has a steep onset comparable to that in our previous report on a monochromator ; however , recent monochromators have sharp onsets as shown in fig . this is important because the energy spread of incident electrons should be narrower than the natural fermi edge of a specimen to reveal the energy - loss near - edge structure of the specimen . because the tunnelling tail is problematic in the observation of the fine structure in low - energy - loss spectra , monochromators are effective for low - loss spectroscopy . the energy resolution is usually specified by the full width at half maximum ( fwhm ) of a zero - loss peak ; however , the full width at 100th maximum is often appropriate in low - loss spectroscopy . in the practical use of a monochromator , the energy spread , for instance , the energy resolution and probe size for atomic - column stem eels elemental mapping should be optimized on the basis of the lifetime energy broadening and the delocalization in inelastic scattering , respectively . otherwise , the low probe current becomes the practical limiting factor preventing the visualization of atomic arrangements in terms of the minimum detectable atomic fraction . numbers in parentheses are fwhms . a narrow energy spread of the monochromator can be realized with a short exposure time of 1 ms . numbers in parentheses are fwhms . a narrow energy spread of the monochromator can be realized with a short exposure time of 1 ms . in addition to the energy spread of a zero - loss peak , several technical factors should be optimized to realize high energy resolution , such as the stabilities of the high - tension and monochromator power supplies , low - noise circumstances ( e.g. stray magnetic field ) , a narrow point spread function ( psf ) of a spectrometer detector and a small chromatic aberration of post - specimen lenses . conventional techniques for multiple fast acquisition and drift correction are still effective even for advanced monochromated microscopy . for instance , the narrow energy spread of the monochromator in fig . 2 is degraded when a longer exposure time is used . we performed quasi - simultaneous acquisition ( e.g. low - loss and core - loss spectra ) to accurately evaluate a chemical shift , and a similar concept has been commercialized in a more sophisticated product ( gatan , dualeels ) . because the psf can degrade fine spectral features , a high energy dispersion ( e.g. < 10 mev ch ) should be applied , resulting in a narrow energy range ( e.g. 200 ev ) of an acquired spectrum . a more advanced detector that has a narrow psf or many channels is preferable . we have recently recognized the last factor , i.e. the effect of the chromatic aberration of the post - specimen lenses , as being particularly important for low - acceleration - voltage electron microscopy . we usually use the drift tube in a spectrometer to switch the energy range , such as from low - loss to high - loss spectroscopy , and we found a substantial difference in each spectroscopic focus , which is due to the vertical shift of the final crossover in the microscope column . the spherical aberration corrector is a breakthrough in tem , yielding the new category of low - voltage high - resolution tem . because the third - order spherical aberration coefficient c3 can be eliminated , the information limit has become more important than the conventional scherzer limit . there are two envelope functions that restrict the information limit : the spatial envelope ks and the chromatic envelope kc . because c3 and the optimum defocus become close to zero in c3-corrected microscopy , the effect of the spatial envelope becomes small even under a convergent illumination condition . accordingly , the chromatic envelope , which is quantified as the defocus spread , is critical in high - resolution tem . the defocus spread d depends on ( i ) the energy spread of the incident electrons e , ( ii ) the high - tension instability v and ( iii ) the instability of the objective lens current i , and is given asd = cc(ee0)2+(vv0)2+(2ii0)2 . monochromators can reduce only the effect of factor ( i ) . however , we demonstrated the advantages of a monochromator in low - voltage high - resolution tem , in which the information limit of 80 kv tem was improved from about 0.2 nm to 90 pm by reducing the energy spread from 0.9 to 0.1 ev . this means that the energy spread of the incident electrons ( i ) is a major limiting factor compared with other instabilities ( ii ) and ( iii ) . note that the chromatic aberration cc corrector produces negative chromatic aberration ; however , the defocus spread due to the current instability ( iii ) can not be eliminated . thus , the energy - dependent defocus spread ( e.g. ( i ) and ( ii ) ) and the current - dependent defocus spread ( iii ) should be separately discussed . the current - dependent defocus spread of the cc corrector system consists of many factors including the current instabilities in the objective lens , the spherical aberration corrector and the cc corrector itself . in addition to reducing the energy spread , a monochromator changes the illumination conditions , which affect high - resolution tem imaging . as mentioned above , some monochromators have a residual energy dispersion , and here the effects of spatial and angular chromaticity on high - resolution tem are elaborated . spatial chromaticity is not critical for high - resolution tem because each illuminated local area retains a high energy resolution and high - resolution tem images are acquired with small defocus . the single wien filter monochromator provides spatial - chromatic illumination , and one of its illumination modes is energy - dispersive critical illumination ( so - called rainbow illumination ) . a drawback of rainbow illumination is that the current density on the specimen can not be changed without degradation of the energy spread . however , this can be easily avoided by using a narrow energy - selecting slit and a defocussed source image , and such a defocussed source with a narrow energy - selecting slit can reduce the convergence angle . 3.3d fourier transforms of through - focus tem images at ( a ) 80 kv and ( b ) 40 kv . the energy spreads of the tem imaging were 100 and 60 mev at 80 and 40 kv , respectively . the two arrows show the observable limits of the ewald spheres , i.e. the information limit . 3d fourier transforms of through - focus tem images at ( a ) 80 kv and ( b ) 40 kv . the energy spreads of the tem imaging were 100 and 60 mev at 80 and 40 kv , respectively . the two arrows show the observable limits of the ewald spheres , i.e. the information limit . angular chromaticity exists in monochromators ( a ) and ( b ) owing to asymmetric electron paths . because electrons with different angles have different energies , their tem images theoretically show different amounts of defocus . if the variation in defocus of each electron is negligible , the spatial envelope function under such angular chromaticity is equal to the conventional spatial envelope function . note that electrons of different directions are considered to be incoherent to each other in the partial coherent imaging theory . in the case of monochromators ( a ) and ( b ) , the acceleration tube can minimize the angular dispersion ; as a result , the angular chromaticity can be minimized . consequently , spatial / angular chromaticity does not lead to problems in c3-corrected high - resolution tem imaging . the tem imaging performance can be assessed using the three - dimensional ( 3d ) fourier transform of through - focus images . because the experimental through - focus series is considered to be the convolution between a defocus spread function and an ideal ( i.e. no defocus spread ) through - focus series , the 3d fourier transform can deduce the defocus spread function as an envelope function . in the 3d fourier transform data obtained using an amorphous specimen , there are twin ewald spheres attached at the origin . the information limit can be estimated as the observable lateral frequency in the ewald spheres . figure 3a and b shows cross sections of 3d fourier transforms , which were obtained using a monochromated tem instrument ( fei , titan ) with an image corrector ( ceos , cetcor ) and an amorphous carbon film . high lateral spatial frequencies of about 11 ( 90 pm ) and 9 nm ( 111 pm ) can be observed in the 3d fourier transforms at 80 and 40 kv , respectively , showing a high information limit . the monochromator is an indispensable instrument for realizing high spatial resolution in low - voltage tem imaging . electrons with different energies are defocussed owing to the chromatic aberration of probe - forming lenses , producing a weak and wide tail of the incident probe and resulting in the elongation of the depth of focus and a contrast reduction of annular dark - field ( adf ) images . because the defocus spread does not change the fwhm of the incident probe , the energy spread is not critical to the achievable spatial resolution of stem adf imaging . the demagnified source image projected on the specimen is considered to be spatial incoherent , and the geometric size and shape of the projected source limit the achievable spatial resolution . monochromators ( b)(d ) , which consist of multiple energy dispersion parts , can construct an achromatic source image ; however , the aberration of the monochromator is critical for constructing a circular source image . in the case of the single wien filter monochromator , various slits or apertures can be inserted on the energy - dispersed source image , because the energy - selecting slit units are located in the microscope column at the earth ground . the aperture is thus a source for the following stem optics . using a circular aperture ( e.g. an aperture of 1 m diameter ) , the source image projected on the specimen always becomes circular even when the monochromator is misaligned . an example of monochromated stem imaging is shown in fig . 4 , where the images were obtained using the single wien filter monochromator at an acceleration voltage of 300 kv ( fei , titan ) . a srtio3 ( 001 ) specimen was prepared by mechanical thinning and ar ion milling ( gatan , pips and technoorg - linda , gentlemill ) . an adf image with a high signal - to - noise ratio 4a ) shows no anisotropy , and a spatial frequency of about 70 pm is observed . the illumination conditions , such as the probe current , probe size and energy spread , can be changed via a variety of instrument parameters including the spot size ( i.e. demagnification in the condenser lens system ) , convergence angle , energy dispersion and energy slit width of the monochromator . 4a and d realize a high probe current ( 56 pa ) and high spatial resolution ( 70 pm ) with a moderate energy resolution ( 0.15 ev ) , which are suitable conditions for element - selective atomic - column imaging using stem eels . ( a ) adf image , ( b ) zero - loss peak , ( c ) abf image and ( d ) bf image . a high probe current ( 56 pa ) and high spatial resolution ( 70 pm ) with a moderate energy resolution ( 0.15 ev ) are simultaneously realized . ( a ) adf image , ( b ) zero - loss peak , ( c ) abf image and ( d ) bf image . a high probe current ( 56 pa ) and high spatial resolution ( 70 pm ) with a moderate energy resolution ( 0.15 ev ) are simultaneously realized . under a high - demagnification condition of the condenser lens system , small - angle electrons emitted from the electron source in other words , the objective aperture of the probe - forming lens is coherently illuminated even in the case of angular chromaticity . note that a coherently illuminated objective aperture is necessary for reciprocity , which is fundamental for the interpretation of bright - field ( bf ) stem image contrast . under such high - resolution stem imaging conditions , bf stem images can be acquired as well as conventional stem images . figure 4c and d shows annular bright - field ( abf ) and bf images , respectively , where a low - pass filter is applied four monochromators , the single wien filter monochromator , the double wien filter monochromator , the omega - shaped electrostatic monochromator and the alpha - shaped magnetic monochromator , were compared . a few critical properties of the monochromators , such as spatial and angular chromaticity , were discussed . the advantages and side effects of the monochromators in eel spectroscopy and tem and stem imaging were pointed out . the monochromators are effective not only for eel spectroscopy but also for high - resolution tem imaging , and spatial / angular chromaticity is not problematic in tem and stem imaging . the monochromator is one of the frontiers of advanced electron microscopy , and other techniques such as the use of a spin - polarized electron gun and a pulsed electron gun are promising techniques . a monochromator for surface analysis has already achieved 1 mev resolution in a unified system consisting of a monochromator and a spectrometer , and there is still room for an improvement of monochromators for electron microscopy . this study was partly supported by the jst research acceleration program and the nano platform program of mext , japan . funding to pay the open access publication charges for this article was provided by national institute for materials science .

a few practical aspects of monochromators recently developed for transmission electron microscopy are briefly reviewed . the basic structures and properties of four monochromators , a single wien filter monochromator , a double wien filter monochromator , an omega - shaped electrostatic monochromator and an alpha - shaped magnetic monochromator , are outlined . the advantages and side effects of these monochromators in spectroscopy and imaging are pointed out . a few properties of the monochromators in imaging , such as spatial or angular chromaticity , are also discussed .

Introduction Basic features of four monochromators Monochromators used for EELS Monochromators used for TEM imaging Monochromators used for STEM imaging Concluding remarks Funding

several types of monochromators have been developed for electron microscopy in this decade and are now available as a tool for material characterization . monochromators can improve a diverse range of electron microscopy techniques , including not only electron energy - loss spectroscopy ( eels ) but also other imaging techniques , such as transmission electron microscopy ( tem ) and scanning transmission electron microscopy ( stem ) . a monochromator is an electron - optical system including energy - dispersive deflectors and an energy - selecting slit , and there are many types of deflectors and their combinations , which decide the inherent performance of the instrument . for instance , most monochromators function at a high tension , but a few function at the earth ground , yielding different performance properties . monochromators change the illumination conditions , such as the convergence angle ; therefore , the tem imaging properties are affected in terms of both chromatic and spatial partial coherence . although monochromators disperse the electron source image on the energy - selecting slit , a probe - forming lens focusses the source image on the specimen in stem imaging . thus , the monochromator properties are also related to the stem imaging performance from the viewpoint of not only chromatic aberration but also spatial incoherence . in this review , then , a few critical properties of these monochromators are pointed out through a discussion of their differences . the advantages and side effects of using these monochromators in eels , tem and stem imaging are also discussed . several monochromators developed for electron microscopy have already been studied by pioneering researchers [ 24 ] , and manufacturers have recently provided several types of monochromators . table 1 outlines the four monochromators described in this review : ( a ) a single wien filter monochromator developed by fei [ 57 ] , ( b ) a double wien filter monochromator by jeol , ( c ) an omega - shaped electrostatic monochromator by ceos and ( d ) an alpha - type magnetic monochromator by nion . here a few properties of these monochromators are outlined through a discussion of their differences . table 1.basic structures of currently available monochromators(a ) single wien filter(fei)(b ) double wien filter(jeol)(c ) omega - shaped electrostatic(ceos)(d ) alpha - type magnetic(nion)basic structurewien filter(acc . basic structures of currently available monochromators acc . tube , acceleration tube ; e - sector , electrostatic toroidal sector deflector ; m - sector , magnetic sector . the single wien filter monochromator ( a ) consists of a wien filter and an energy - selecting slit ; a wien filter analyses the speed of electrons using the crossing magnetic and electrostatic fields , and the energy - selecting slit mechanically chooses a portion of the dispersed electrons . the double wien filter monochromator ( b ) includes two wien filters with an energy - selecting slit inserted between them . the second wien filter eliminates the energy dispersion at the conjugate plane of the electron source . the omega - shaped electrostatic monochromator ( c ) constructs an omega - shaped electron path using four electrostatic toroidal sector deflectors . the alpha - type magnetic monochromator ( d ) is attached after the acceleration tube , and four energy dispersions , which are actually formed by one uniform - field sector ( functioning twice ) and two gradient - field sectors , are used to produce an alpha - shaped electron path . the omega- or alpha - shaped electron paths minimize the aberrations of each monochromator by acting as in - column energy filters , because the energy - selecting slit and energy dispersion plane of the monochromators lie on the midplane of the electron path . the electron energy in the energy dispersion part depends on the arrangement of the monochromator and the acceleration tube and yields a critical difference in the monochromator properties . in many cases ( i.e. a few kev ) to obtain sufficient energy dispersion at the energy - selecting slit and , as a result , many monochromators are mounted between the electron gun and the acceleration tube . there are a few technical challenges in mounting a monochromator at a high tension in terms of its electronics , mechanics and the requirement of ultrahigh vacuum in the electron gun chamber . note that a monochromator at a high tension can not filter out the instability of the acceleration voltage ; however , it can be easily used at various acceleration voltages under the same monochromator condition . the performance of the energy dispersion part depends not only on the electron optics but also on the boersch effect ; therefore , the length of the electron path , the probe current and the number of crossovers should be minimized . the energy - selecting slit is an indispensable part of a monochromator , and the required slit width is generally of the order of a micrometre . in addition , the mechanical movable components of the energy - selecting slit should not degrade the vacuum in the adjacent electron gun chamber . in contrast , the energy - selecting slits of monochromators ( a ) and ( d ) select high - energy electrons in the microscope columns , and a variety of slits and a complicated mechanical / electrical system can be implemented . in the case of the single wien filter monochromator , the acceleration tube between the wien filter and the energy - selecting slit magnifies the source image and the energy dispersion , which is advantageous for energy selection . in monochromator ( d ) , a current detection system is implemented on the energy - selecting slit for the additional stabilization of high tension . figure 1 shows schematics of the four monochromators , in which the electron path is drawn as a straight line for simplicity . small closed circles are astigmatically focussed ( i.e. 1b and d. the symmetric optical arrangement of monochromators ( c ) and ( d ) yields a symmetric electron path to the midplane at the energy - selecting slit , resulting in the minimized aberration of each system . 1.schematics of four monochromators : ( a ) single wien filter monochromator , ( b ) double wien filter monochromator , ( c ) omega - shaped electrostatic monochromator and ( d ) alpha - type magnetic monochromator . the electron path with the standard energy is schematically drawn as a straight line , and dotted curves represent the axial paths of electrons with a different energy . open circles correspond to the conjugate planes to the electron source and small closed circles are the positions of astigmatically focussed ( i.e. line - focussed ) source images . rectangles , triangles and rounded rectangles represent monochromator units , energy dispersion parts and acceleration tubes , respectively . the arrows at the bottom represent the residual energy dispersion at the final source image . schematics of four monochromators : ( a ) single wien filter monochromator , ( b ) double wien filter monochromator , ( c ) omega - shaped electrostatic monochromator and ( d ) alpha - type magnetic monochromator . line - focussed ) source images . the arrows at the bottom represent the residual energy dispersion at the final source image . angular chromaticity means that electrons propagating in different directions have different energies , which is eliminated in monochromators ( c ) and ( d ) owing to their symmetric electron paths . it is worth mentioning that the angular chromaticity in the double wien filter monochromator ( b ) can be controlled by changing the length of the wien filters . for instance , the deflection induced by the lorentz force becomes zero ( i.e. the rotation angle equals 2 ) at a specific filter length , and the angular chromaticity is eliminated under this condition . figure 1b depicts an example setting , in which the total angle of rotation induced by the lorentz force equals , which was reported in a previous paper . stabilities at a high tension and power supply of the monochromator are critical technical requirements to realize high energy resolution . note that a stable energy - dispersed source image on an energy - selecting slit only ensures the stability of the voltage ( e.g. a few kv ) of the electrons in the monochromator . because high - tension instability causes instability in a probe current of monochromator ( d ) , in addition , a monochromator at the earth ground level requires ultrahigh resolving power in comparison with that of a monochromator at a high tension , and its power supply should also be highly stabilized . although the aberration figure on the selecting slit can be easily observed in the single wien filter monochromator , those of the other monochromators are not observable because of the energy dispersion deflector(s ) after the energy - selecting slit , making their alignment indirect . a few pioneering studies on high - resolution eels have been performed with the combination of a monochromator and a retarding analyzer , in which the potentials of both energy dispersion deflectors were connected . a similar concept is embraced in monochromator ( d ) , in which the power supplies of the monochromator and analyzer are synchronized . from the viewpoint of monochromated electron microscopy , the brightness of each electron gun should be re - examined on the basis of each energy spread . [ a sr m ev ] ) , in which the current j is replaced by the maximum of the energy distribution , dj(e)/de , where e is the energy of the incident electrons . the latter reported an improvement of the reduced brightness in the sharpened tip of a schottky emission gun ( seg ) . because a cfeg has the highest reduced brightness among existing electron guns , the use of a cfeg or seg with a sharpened tip is preferable for a monochromator . an example of the energy distributions obtained using a cfeg and the single wien filter monochromator is shown in fig . 2 . this is important because the energy spread of incident electrons should be narrower than the natural fermi edge of a specimen to reveal the energy - loss near - edge structure of the specimen . because the tunnelling tail is problematic in the observation of the fine structure in low - energy - loss spectra , monochromators are effective for low - loss spectroscopy . in the practical use of a monochromator , the energy spread , for instance , the energy resolution and probe size for atomic - column stem eels elemental mapping should be optimized on the basis of the lifetime energy broadening and the delocalization in inelastic scattering , respectively . otherwise , the low probe current becomes the practical limiting factor preventing the visualization of atomic arrangements in terms of the minimum detectable atomic fraction . a narrow energy spread of the monochromator can be realized with a short exposure time of 1 ms . numbers in parentheses are fwhms . a narrow energy spread of the monochromator can be realized with a short exposure time of 1 ms . in addition to the energy spread of a zero - loss peak , several technical factors should be optimized to realize high energy resolution , such as the stabilities of the high - tension and monochromator power supplies , low - noise circumstances ( e.g. stray magnetic field ) , a narrow point spread function ( psf ) of a spectrometer detector and a small chromatic aberration of post - specimen lenses . for instance , the narrow energy spread of the monochromator in fig . because the psf can degrade fine spectral features , a high energy dispersion ( e.g. the effect of the chromatic aberration of the post - specimen lenses , as being particularly important for low - acceleration - voltage electron microscopy . we usually use the drift tube in a spectrometer to switch the energy range , such as from low - loss to high - loss spectroscopy , and we found a substantial difference in each spectroscopic focus , which is due to the vertical shift of the final crossover in the microscope column . because c3 and the optimum defocus become close to zero in c3-corrected microscopy , the effect of the spatial envelope becomes small even under a convergent illumination condition . accordingly , the chromatic envelope , which is quantified as the defocus spread , is critical in high - resolution tem . the defocus spread d depends on ( i ) the energy spread of the incident electrons e , ( ii ) the high - tension instability v and ( iii ) the instability of the objective lens current i , and is given asd = cc(ee0)2+(vv0)2+(2ii0)2 . monochromators can reduce only the effect of factor ( i ) . however , we demonstrated the advantages of a monochromator in low - voltage high - resolution tem , in which the information limit of 80 kv tem was improved from about 0.2 nm to 90 pm by reducing the energy spread from 0.9 to 0.1 ev . this means that the energy spread of the incident electrons ( i ) is a major limiting factor compared with other instabilities ( ii ) and ( iii ) . ( i ) and ( ii ) ) and the current - dependent defocus spread ( iii ) should be separately discussed . the current - dependent defocus spread of the cc corrector system consists of many factors including the current instabilities in the objective lens , the spherical aberration corrector and the cc corrector itself . in addition to reducing the energy spread , a monochromator changes the illumination conditions , which affect high - resolution tem imaging . as mentioned above , some monochromators have a residual energy dispersion , and here the effects of spatial and angular chromaticity on high - resolution tem are elaborated . spatial chromaticity is not critical for high - resolution tem because each illuminated local area retains a high energy resolution and high - resolution tem images are acquired with small defocus . the single wien filter monochromator provides spatial - chromatic illumination , and one of its illumination modes is energy - dispersive critical illumination ( so - called rainbow illumination ) . a drawback of rainbow illumination is that the current density on the specimen can not be changed without degradation of the energy spread . the energy spreads of the tem imaging were 100 and 60 mev at 80 and 40 kv , respectively . the two arrows show the observable limits of the ewald spheres , i.e. the information limit . the energy spreads of the tem imaging were 100 and 60 mev at 80 and 40 kv , respectively . the two arrows show the observable limits of the ewald spheres , i.e. angular chromaticity exists in monochromators ( a ) and ( b ) owing to asymmetric electron paths . if the variation in defocus of each electron is negligible , the spatial envelope function under such angular chromaticity is equal to the conventional spatial envelope function . in the case of monochromators ( a ) and ( b ) , the acceleration tube can minimize the angular dispersion ; as a result , the angular chromaticity can be minimized . consequently , spatial / angular chromaticity does not lead to problems in c3-corrected high - resolution tem imaging . because the experimental through - focus series is considered to be the convolution between a defocus spread function and an ideal ( i.e. in the 3d fourier transform data obtained using an amorphous specimen , there are twin ewald spheres attached at the origin . the information limit can be estimated as the observable lateral frequency in the ewald spheres . figure 3a and b shows cross sections of 3d fourier transforms , which were obtained using a monochromated tem instrument ( fei , titan ) with an image corrector ( ceos , cetcor ) and an amorphous carbon film . high lateral spatial frequencies of about 11 ( 90 pm ) and 9 nm ( 111 pm ) can be observed in the 3d fourier transforms at 80 and 40 kv , respectively , showing a high information limit . electrons with different energies are defocussed owing to the chromatic aberration of probe - forming lenses , producing a weak and wide tail of the incident probe and resulting in the elongation of the depth of focus and a contrast reduction of annular dark - field ( adf ) images . because the defocus spread does not change the fwhm of the incident probe , the energy spread is not critical to the achievable spatial resolution of stem adf imaging . the demagnified source image projected on the specimen is considered to be spatial incoherent , and the geometric size and shape of the projected source limit the achievable spatial resolution . monochromators ( b)(d ) , which consist of multiple energy dispersion parts , can construct an achromatic source image ; however , the aberration of the monochromator is critical for constructing a circular source image . in the case of the single wien filter monochromator , various slits or apertures can be inserted on the energy - dispersed source image , because the energy - selecting slit units are located in the microscope column at the earth ground . the aperture is thus a source for the following stem optics . using a circular aperture ( e.g. an example of monochromated stem imaging is shown in fig . 4 , where the images were obtained using the single wien filter monochromator at an acceleration voltage of 300 kv ( fei , titan ) . the illumination conditions , such as the probe current , probe size and energy spread , can be changed via a variety of instrument parameters including the spot size ( i.e. demagnification in the condenser lens system ) , convergence angle , energy dispersion and energy slit width of the monochromator . a high probe current ( 56 pa ) and high spatial resolution ( 70 pm ) with a moderate energy resolution ( 0.15 ev ) are simultaneously realized . under a high - demagnification condition of the condenser lens system , small - angle electrons emitted from the electron source in other words , the objective aperture of the probe - forming lens is coherently illuminated even in the case of angular chromaticity . under such high - resolution stem imaging conditions , bf stem images can be acquired as well as conventional stem images . figure 4c and d shows annular bright - field ( abf ) and bf images , respectively , where a low - pass filter is applied four monochromators , the single wien filter monochromator , the double wien filter monochromator , the omega - shaped electrostatic monochromator and the alpha - shaped magnetic monochromator , were compared . a few critical properties of the monochromators , such as spatial and angular chromaticity , were discussed . the advantages and side effects of the monochromators in eel spectroscopy and tem and stem imaging were pointed out . the monochromators are effective not only for eel spectroscopy but also for high - resolution tem imaging , and spatial / angular chromaticity is not problematic in tem and stem imaging . the monochromator is one of the frontiers of advanced electron microscopy , and other techniques such as the use of a spin - polarized electron gun and a pulsed electron gun are promising techniques . a monochromator for surface analysis has already achieved 1 mev resolution in a unified system consisting of a monochromator and a spectrometer , and there is still room for an improvement of monochromators for electron microscopy .

respiratory syncytial virus ( rsv ) is an enveloped negative sense single - strand rna virus of the paramyxoviridae family and pneumovirus genus . rsv infection is most common in infants and young children : nearly all children are infected by 2 years of age . rsv infection of infants can lead to serious respiratory disease , sometimes requiring hospitalization . in a screen for viral and atypical bacterial respiratory pathogens , rsv was the most prevalent pathogen ( 43.3 % ) infecting children less than 5 years old with acute respiratory infection ( ari ) , as well as extremely common agent ( 44.1 % ) in co - infection cases of 2 or more pathogens in the same cohort . in conjunction , it has long been speculated that acute rsv infection during infancy correlates with a greater risk of allergic asthma later in life [ 3 , 4 ] . this correlation is evident in a longitudinal study by sigurs and colleagues of 47 children who were hospitalized with rsv lower respiratory tract infection ( lrti ) when they were less than 1 year old [ 5 ] . in a recent follow - up with the patients at age 18 , the rsv lrti cohort had increased incidence of asthma with recurrent wheeze , asthma without recurrent wheeze , and allergic rhinoconjunctivitis ( arc ) compared with 92 age - matched control patients ( table 1 ) [ 5 ] . in addition , the lrti cohort had increased sensitization to animal dander and perennial allergens as well as decreased spirometric function [ 5].table 1allergic symptoms in 18-year - old cohortrsv group ( n = 46)control group ( n = 92)current asthma / rw**39 % 9 % current asthma only**33 % 7 % allergic rhinoconjunctivitis ( arc)*43 % 17 % atopic dermatitis11 % 9 % animal dander sensitization * ( positive skin prick test)33 % 11 % perennial allergen sensitization * * ( positive skin prick test)41 % 14 % positive phadiatop response * ( allergic sensitization as defined by phadiatop)56 % 28 % * p 0.005 ; * * p 0.001 allergic symptoms in 18-year - old cohort * p 0.005 ; * * p 0.001 these and other epidemiological data support 2 notions . rsv could have a long - lasting effect via an alteration of the immune response ; it is also possible that early rsv infection serves as an indication of inherent differences in an individual s immune system , which leaves it not only vulnerable to serious rsv infection , but also to other , chronic respiratory conditions ( eg , recurrent wheeze and asthma ) . many of the rsv studies have explored these broad hypotheses by examining the effects of rsv on different aspects of the immune response in both human patients and mouse models . recent work has done a great deal to expound the mechanisms underlying the immune response to rsv infection through investigating the formative signaling pathways and genetic underpinnings to the subsequent differential cellular responses . rsv infection causes a wide array of immunologic responses ; clarifying the ways in which rsv triggers these changes may lead to the development of effective therapeutics that can ameliorate or abolish acute and chronic disease that result from ineffective immune system response . rsv predominantly infects primary airway epithelial cells , but can also infect other structural airway and immune cells . in addition , new research shows tlr7 activation during rsv infection [ 10 ] . these receptors in turn promote expression and secretion of inflammatory cytokines ( table 2 ) , which mount the earlier innate immune response and subsequent adaptive immune response . to elicit these responses , several different signaling pathways and their respective molecules these include pathways that include protein kinase c ( pkc ) , mitogen - activated protein kinase ( mapk ) , and nuclear factor-b ( nf-b).table 2summary of cytokines and chemokines produced during rsv infectioncytokinesourceeffectsifn-nk cells , cd4 t cells , cd8 t cellsincreases nk activation , th1 differentiationil-1macrophagesincreases expression of cytokines mediated by nf-bil-4cd4 + t cellsincreases th2 differentiation , decreases th1 differentiationil-8epithelial cells , macrophagesneutrophil recruitmentil-10cd4 + t cellsinhibits expression of inflammatory cytokines , suppresses inflammatory responseil-17acd4 + t cellsincreases th17 differentiation , increases inflammatory cytokine production summary of cytokines and chemokines produced during rsv infection p38 mapk and extracellular signal - regulated kinase ( erk ) mapk are both involved in rsv replication in human airway epithelial cells . pharmacologic inhibitors for p38 and erk ( sb203580 and u0126 , respectively ) both decreased viral replication when cells were treated 30 minutes prior to infection . in primary fibroblasts from myeloid differentiation factor 88 ( myd88 ) knockout ( ko ) mice , rsv - induced p38 activation was myd88-dependent , whereas erk activation was myd88-independent . in the presence of anti - tlr4 antibodies , in addition , immunofluorescence confocal microscopy revealed that tlr4 clustering at the point of contact between the virus and cell was related to p38 activation , helping to elucidate the mechanism of viral entry . upon viral entry and activation of signaling complexes , inflammatory cytokines and chemokines one important chemokine during this response is il-8 , which , as a chemoattractant , recruits neutrophils to the location of infection . rsv induces il-8 expression , which is mediated by nf-b and ap-1 . in rsv - infected a549 cells , co - transfection of a reporter luciferase gene coupled to the il-8 promoter , and a dominant negative ( dn ) mutation revealed that either of the 2 ikk dns used significantly decreased il-8 promoter activation . an ikk inhibitor , nemo binding domain ( nbd ) fusion peptide , significantly decreased il-8 secretion . therefore , nf-b was activated through the canonical pathway . inhibition of jnk significantly reduced transfected ap-1 reporter activity , while leaving nf-b reporter activity unaffected [ 14 ] . mekk1 and tak1 are serine / threonine kinases that activate both jnk and ikks [ 15 , 16 ] . when a549 cells were infected with rsv , tak1 was essential to both ap-1 and nf-b activation [ 14 ] . thus , rsv infection caused il-8 expression through nf-b and ap-1 induction via the canonical and the jnk pathway , respectively , and tak1 was an upstream regulator able to mediate both responses . thus , there are likely other kinases that regulate ikk and nf-b activation during rsv infection [ 14 ] . the canonical pathway is not the only recently reported means of induction of nf-b during rsv infection in human airway epithelial cells . in addition , the importance of a non - canonical pathway mediated by the virus - activated kinase ( vak ) complex ( composed of tbk-1 and ikk ) is also evident . in rsv - infected 293 cells transfected with ikk dn , il-8 promoter activity was significantly down - regulated in comparison to control cells at 12 hours post - infection . using sirna in a549 cells and mouse embryonic fibroblasts from ikk ko mice , ikk was determined to regulate nf-b during rsv infection through p65 ser536 phosphorylation . thus , there is evidence for 2 separate pathways of nf-b activation during rsv infection . in a similar vein , rsv infection leads to il-1 expression , which is an inflammatory cytokine critical to antiviral immune response . il-1 works in an autocrine and paracrine fashion , and ultimately causes the expression of a number of nf-b - dependent cytokines and chemokines . mature il-1 is produced and secreted by activated macrophages following assembly and activation of inflammasomes in the cytoplasm [ 19 , 20 ] . this is achieved through 2 signals : first , stimulation of a pattern recognition receptor ensures expression of pro - il-1 ( the il-1 precursor ) and inflammasome components ; second , the inflammasome complex assembly and caspase-1 activation cleave pro - il-1 into il-1. this second signal is either initiated by reactive oxygen species ( ros ) , cellular potassium efflux , or cathepsin leakage into the cytosol after lysomal disintegration . nf-b activation through a tlr2/myd88-dependent pathway is necessary for the first signal of il-1 production in macrophages during rsv infection . the second signal is a combination of ros production and potassium efflux via an atp - sensitive potassium channel [ 20 ] . these new findings elucidate the basic mechanism of il-1 production in rsv - infected macrophages . likewise , rsv induces il-15 expression in both epithelial cell lines and primary bronchial epithelial cells . il-15 is an inflammatory cytokine that is suggested to augment antiviral defense through up - regulation of ifn - related transcription factors , nf-b , synthesis of type 1 effector molecules , and survival and stimulation of nk cells and cd8 t cells [ 2224 ] . thus , effective expression of il-15 could be beneficial to effective rsv clearance ; further research is required to determine the importance of il-15 in shaping the immune response to rsv infection . in addition to regulating inflammatory cytokine production , nf-b activation is associated with tight junction formation in rsv - infected human nasal epithelial cells . tight junctions are important for epithelial cell polarity and maintenance of tissue integrity . during rsv infection , genes for tight junction molecules , claudin-2 , -4 , -7 , -9 , -14 , -19 ; occludin ; zo-2 ; cingulin and mag-1 interestingly , inhibition of nf-b and pkc led to decreased viral replication and decreased formation of virus filaments . this , along with immunocytochemistry of tight junction proteins and viral proteins ( rsv proteins f and g ) seem to suggest that virally - induced tight junction formation could augment cell polarity during rsv infection allowing for characteristic viral budding at the apical surface . rsv infection also leads to a specific adaptive immune response . the characteristics of the adaptive immune response ( ie , which t helper cell subset and associated cytokines dominate the response [ table 2 ] ) can help to understand acute rsv disease and the implication of rsv infection for potential long - term respiratory disease . therefore , it is important to realize any effects rsv infection has on predisposition to a certain adaptive immune response and the mechanism by which it occurs . rsv infection has been associated with skewing the immune system away from an anti - viral th1 response and towards a th2 response . new data shed light on how this occurs and introduced a role of il-17a ( the principle cytokine of th17 response ) in rsv infection as well , as antibody blockade of il-17a led to significant reduction of viral protein mrna expression in the lungs and th2 cytokine protein expression in the lymph nodes in rsv - infected mice compared with mice treated with control antibody . this is interesting because il-17a has recently been implicated in the development of severe forms of asthma . epithelial cells , in addition to providing a link to the initial innate immune response , aid in adaptive immune system activation after rsv infection . when human bronchial epithelial cells ( hbecs ) were co - cultured with peripheral blood mononuclear cells ( pbmcs ) , there was an induction of ifn- , il-4 , and il-17 cytokine production , which suggested activation of lymphocytes as these are the characteristic cytokines of t helper cell subsets th1 , th2 , and th17 , respectively . when isolated lymphocytes were exposed to the supernatant of rsv - infected hbecs , flow cytometry revealed that compared with controls exposed to complete cell culture medium , th2 and th17 differentiation was significantly induced , while treg differentiation was clearly suppressed . in addition to inherent major histocompatibility complex ( mhc ) i , epithelial cells may be able to express mhc ii in times of stress or disease , as well as costimulatory molecules cd80 and cd86 during infection [ 3032 ] . therefore , it is conceivable that hbecs directly activate nave cd4 lymphocytes during rsv infection . rsv infection of primary rat airway epithelial cells ( praecs ) caused upregulated thymic stromal lymphopoietin ( tslp ) 6 to 18 hours post - infection . when rsv - infected praecs were cocultured with myeloid dendritic cells ( mdcs ) , the mdcs had increased major histocompatibility complex ii ( mhc ii ) and cd86 , but not cd80 expression . in the same coculture system , sirna for tslp effectively decreased tslp expression and also the expression of mhc ii and cd86 , without changing cd80 expression . in this mdc- and rsv - infected praec coculture system , mdcs also up - regulated mrna expression of molecules important for th2 polarization : thymus- and activation - regulation chemokine ( tarc ) and ox40l , while tnf- mrna was down - regulated . functional mdc maturation was verified , as mdcs exposed to rsv - infected praecs significantly increased t cell proliferation in comparison to uninfected praecs . thus , it is possible that rsv infection of epithelial cells leads to a differential t helper cell response through the polarization of mdcs , and potentially directly through epithelial cell antigen presentation . however , this study did not assess the ability of affected mdcs to alter nave t cell differentiation . further study is necessary to further elucidate the mechanisms of t cell activation and differentiation during rsv infection . during rsv infection , natural killer ( nk ) cells interestingly , past clinical studies show that increased nk deficiency is linked to increased respiratory disease symptoms . specifically , nk cell numbers were reduced in hospitalized bronchiolitis cases , and ventilated infants exhibited an even greater ( 3-fold ) reduction in nk cells . in fatal rsv bronchiolitis cases , patients were nearly devoid of nk cells [ 3537 ] . in balb / c mice , antibody - mediated nk cell reduction during rsv infection resulted in lower ifn- production and an increase in th2 cytokines . this result was dependent on il-25 produced by airway epithelial cells : il-25 up - regulated jagged1 on dendritic cells and led to increased th2 differentiation . just as the immune system needs to effectively activate and clear a pathogen , it also needs an effective system of self - regulation and inhibition to inhibit an unbalanced immune response and collateral damage of host tissue . studies examining humans and mouse models reveal that inefficient immune system regulation escalates respiratory disease pathology following rsv infection , and can also lead to less optimal viral clearance . it mediates suppression of the activation and effector functions of many immune system cells . as can be expected , it is important in rsv infection and resultant disease pathogenesis . in hospitalized infants with rsv bronchiolitis , il-10 concentration in nasopharyngeal aspirates were significantly higher in infants who later developed post - bronchiolitis wheeze ( pbw ) than in those who did not . however , this difference in il-10 concentration was not correlated to the rs1800872 single - nucleotide polymorphism ( snp ) in the il10 promoter region . this snp was considered because heterozygous expression was previously correlated with protection from rsv bronchiolitis [ 39 , 40 ] . so , although there is some support for a protective role from this il10 genetic variation , the potential functional role during rsv infection remains unclear . that said , it is also possible that during rsv infection the il-10 response is modulated via indirect genetic factors . for instance , il-19 and il-20 are both il-10 family cytokines and their genes are clustered with il10 ( as well as il24 ) [ 4143 ] . in 1 recent study of 166 hospitalized infants with rsv lrti , 1 snps of the il19 gene ( rs2243191 and rs2243188 ) and 1 snp of il20 gene ( rs2981573 ) were associated with protection from recurrent wheeze ( or 0.4 , 0.5 , and 0.4 , respectively ) . from these snps , 3 haplotypes comprised 99 % of all the il19/il20 haplotypes in the patients . only 1 of these haplotypes , tgg ( the nucleotides of rs2243191 , rs2981572 , and rs2981573 , respectively ) , was significantly related to recurrent wheeze after ltri . perhaps changes in il-10 family cytokine expression through snps play a role in direct immune system regulation during rsv infection . it is also possible that their expression influences il-10 expression during infection because il-10 family members cross - regulate each other in other models [ 44 , 45 ] . these cohort studies show that il-10 has some influence in disease pathogenesis , prompting studies to focus on elucidating the mechanism of il-10 and other factors of immunosuppression during rsv infection in mouse models . cd4 and cd8 t cells both produced il-10 in the lung during rsv infection in mice , with production peaking at 8 days post infection [ 46 ] . the majority of il-10 producing t cells were cd4 during rsv infection . the vast majority of cd8 t cells that produce il-10 co - produced ifn- , while this was less true for cd4 t cells however , cd8 cells also may have modulated il-10 production , as depletion of cd8 cells led to a significant increase in il-10 . thus , these data support that cd8 cells play a suppressive role in cd4 il-10 production [ 46 ] . although cd4 t cells are the major producers of il-10 in mouse rsv infection , the subset that contributes most to this production is not as clear . one group has shown t regulatory cells ( cd4foxp3 ) was the most common il-10 expressing cells in the lung , mediastinal lymph node ( mln ) and bal fluid of infected mice , while another has shown that cd4foxp3 are the majority of il-10 expressers [ 46 , 47 ] . thus , it needs to be determined under which circumstances , if any , each subset expresses the most il-10 . in il-10 ko mice and wt mice treated with anti - il-10r antibody , rsv infection increased disease and delayed recovery when compared to il-10 producing and control antibody treated mice , respectively [ 46 , 47 ] . il-10 depletion was associated with increased weight loss , airway obstruction , edema , tissue inflammation , and mucus production . these macroscopic changes were marked by a congruous increase in inflammatory cytokines ( il-6 , il-17a , il-17f , ifn - y , tnf - a ) and chemokines ( cxcl1 , cxcl10 , cxcl9 , ccl1 , ccl2 , ccl3 ) [ 46 , 4749 ] . thus , a lack of il-10 resulted in an altered adaptive immune response , providing evidence that il-10 is an important regulator of inflammation during rsv infection . this is reflected at the cellular level : with il-10 depletion there was a decrease in total cell recruitment to the bal at day 4 , whereas there was increased airway inflammation 6 and 8 days post - infection [ 47 , 49 ] . this trend could be responsible for the lag in viral clearance in comparison to control mice . in addition , there was a decrease in natural t regulatory cells ( cd4foxp3helios ) when mice were given anti - il-10r antibody [ 46 ] . as il-10 is an important regulatory cytokine , t regulatory cells ( tregs ; cd4foxp3 ) are responsible for suppressing the adaptive immune response . after rsv infection in mice , there was a rapid accumulation of tregs in the airways and secondary lymphoid tissue . by 4 days post - infection there is a 50-fold increase in tregs in the bal fluid of infected mice compared to nave mice . this drastic increase was amplified 8 days post - infection to an 86-fold increase over nave mice . there was also significant treg accumulation observed in the lung parenchyma and mlns and a slight increase in the spleen . local proliferation of tregs occurred with 78 % and 69 % of tregs expressing the proliferation marker ki-67 in the bal and lung parenchyma , respectively , 6 days post - infection . in addition , the majority of tregs expressed activation markers , which were up - regulated post - infection . cd43 , icos , ctla-4 , cd69 , ox40 , and pdl-1 were all significantly increased 6 days post - infection . cd25 tregs present in the airways increased significantly as well . with the clearance of rsv treg efflux took longer in the bal and mlns than in the lung parenchyma and spleen . a 60 % decrease in cd4foxp3cd25 tregs using cd25-neutralizing antibodies was extremely detrimental to immune system efficiency , leading to a significant increase in time of viral clearance . this delay could be traced to a delay in the recruitment in rsv - specific cd8 t cells to the lung . this finding argues that tregs are more than just immunosuppressors ; they also act as conductors of an effective immune response for rsv clearance . as can be surmised in understanding the vast and intricate effects of rsv infection on the immune system response , it is important to acknowledge that there is variability of immune response to rsv infection from case to case . there are many different strains of rsv that each contains significant genetic variability , leading to altered virulence and subsequent disease pathogenesis . also , the characteristics of the infected host cell ( ie , species and type of cell ) can lead to differential cellular and systematic responses . this is especially important to keep in mind in examining in vitro studies that focus on a small number of cell types . obviously , it is necessary to use basic systems to garner mechanistic understanding , but we must be mindful of the plasticity inherent in these mechanisms and their phenotypic manifestations . inherent differences lead to differences in the innate and adaptive immune responses triggered by rsv infection . several recently published studies examine the effects of intrinsic differences on rsv pathogenesis and immune response . in examination of the pathogenesis of several rsv isolates in a balb / cj mouse model , both laboratory strains ( a2 , long , and line19 ) and clinical strains ( a2001/2 - 20 , a2001/3 - 12 , a1997/12 - 35 , a2000/3 - 4 , a1998/3 - 2 , and a1998/12 - 21 ) were studied [ 51 ] . of the laboratory strains , only a2 caused host weight loss , while 3 of 6 clinical isolates ( 312 , 220 , 1235 ) caused weight loss post - infection . while those 4 isolates caused weight loss , they all caused different patterns of weight loss : early , late , and bimodal patterns were all represented . similarly , strains caused differing characteristics and disease phenotypes including : varying expression of lung il-13 and gob-5 protein , amount of in vitro replication , in vivo viral load , and other characteristics . indeed , in a comparison of a2 , 220 , 312 , and line 19 at 8 days post - infection , a2 infection had a significantly greater amount of cd8 t cells in the lung that produced ifn- than 220 and line 19 . interestingly , this matched pas staining data that showed a2 as nonmucogenic , whereas 220 and line 19 were mucogenic . this finding links the role of t cell differentiation and disease pathogenesis [ 51 ] . a similar study examined a variety of rsv strains ( a2 and 3 clinical isolates : bt2a , bt3a , bt4a ) in primary human bronchial epithelial cells ( pbecs ) from healthy children . as in the aforementioned mouse model , the strains were characterized by different patterns of infection . the strains differed drastically in their ability to infect pbecs , as revealed by host cell expression of viral f protein . a2 was much more infectious ; at 72 hours post - infection , most host cells were infected . in contrast , the clinical isolates were not nearly as infectious , and displayed differential infectivity ( bt2a was most infectious , bt4a was least ) . this difference carried over to viral growth kinetics : a2 grew more quickly and to higher titers than the clinical isolates . a2 titers reached 5.25 log10 tcid50/ml at an moi 0.1 , whereas the clinical isolates bt2a , bt3a , and bt4a reached 4.20 , 3.80 , 3.25 log10 tcid50/ml , respectively . interestingly , all strains initiated a post - infection increase in the concentration of the chemokines and cytokines rantes , ip-10 , il-6 , and il-8 in supernatants . however , by 72 hours post - infection , a2 had significantly higher concentrations of ip-10 and rantes than the mock infections and clinical isolates . similarly , il-8 was significantly up - regulated by only a2 and bt4a and il-6 only by bt4a . so , inflammatory chemokine and cytokine release varied by rsv strain , which would likely elicit a variable immune system response in an in vivo model . from the opposite perspective , the types of cells used for experimental infection are also important . for instance , a group examined rsv infection in airway epithelial cells ( aecs ) from lungs of healthy children and cells of the beas-2b cell line [ 53 ] . they determined that the peak of rsv replication ( as determined by real - time pcr for rsv n - gene expression ) was 2 to 3 log - fold greater in aec cultures than in beas-2b cultures . although both cell culture supernatants contained high concentrations ( > 500 pg / ml ) of il-8 and il-6 from 3 to 48 hours post - infection , aecs had significantly greater concentrations of il-8 than beas-2b from 6 to 24 hours post - infection [ 53 ] . in short , the response to rsv infection differed between epithelial cells derived from epithelial cell lines and from the lungs of children . recent advancements in rsv research have illuminated the signal transduction pathways that are activated to initiate transcription of inflammatory cytokines and chemokines once rsv is recognized by a tlr on the cell surface . in addition , th2 and th17 differentiation and role in disease progression during rsv infection are better understood . that said , it is necessary to better characterize the link between the innate and adaptive immune systems post - infection , and how variations in this link can lead to variations in acute and chronic airway disease . understanding the influence of regulatory agents ( eg , il-10 , tregs ) host genetic predisposition , viral strain , and other factors on this combined immune response may help glean new therapeutic avenues .

respiratory syncytial virus ( rsv ) most often causes severe respiratory disease in the very young and the elderly . acute disease can also cause exacerbations of asthma in any age group . recent findings provide insight into how the innate and adaptive immune systems respond to rsv infection and provide preliminary evidence that these effects vary significantly by rsv strain and host . components of cell signaling pathways that induce inflammatory cytokine expression during the innate immune response and alter epithelial cell polarity through activating transcription factors , namely nf-b , are now more clearly understood . new studies also reveal how rsv infection skews t helper ( th ) cell differentiation away from the cell - mediated th1 subset and towards the th2 subset . there are also new data supporting preferential th17 differentiation during rsv infection . in addition , effective immune system regulation of il-10 expression and t regulatory cell ( treg ) airway accumulation are essential for effective rsv clearance .

Introduction Initial RSV Infection, Cell Signaling, and the Innate Immune System Response Adaptive Immune System Response Immune System Regulation During RSV Infection Different Strains, Different Cells: Different Response Conclusions

respiratory syncytial virus ( rsv ) is an enveloped negative sense single - strand rna virus of the paramyxoviridae family and pneumovirus genus . rsv infection is most common in infants and young children : nearly all children are infected by 2 years of age . rsv infection of infants can lead to serious respiratory disease , sometimes requiring hospitalization . in a screen for viral and atypical bacterial respiratory pathogens , rsv was the most prevalent pathogen ( 43.3 % ) infecting children less than 5 years old with acute respiratory infection ( ari ) , as well as extremely common agent ( 44.1 % ) in co - infection cases of 2 or more pathogens in the same cohort . in conjunction , it has long been speculated that acute rsv infection during infancy correlates with a greater risk of allergic asthma later in life [ 3 , 4 ] . in a recent follow - up with the patients at age 18 , the rsv lrti cohort had increased incidence of asthma with recurrent wheeze , asthma without recurrent wheeze , and allergic rhinoconjunctivitis ( arc ) compared with 92 age - matched control patients ( table 1 ) [ 5 ] . in addition , the lrti cohort had increased sensitization to animal dander and perennial allergens as well as decreased spirometric function [ 5].table 1allergic symptoms in 18-year - old cohortrsv group ( n = 46)control group ( n = 92)current asthma / rw**39 % 9 % current asthma only**33 % 7 % allergic rhinoconjunctivitis ( arc)*43 % 17 % atopic dermatitis11 % 9 % animal dander sensitization * ( positive skin prick test)33 % 11 % perennial allergen sensitization * * ( positive skin prick test)41 % 14 % positive phadiatop response * ( allergic sensitization as defined by phadiatop)56 % 28 % * p 0.005 ; * * p 0.001 allergic symptoms in 18-year - old cohort * p 0.005 ; * * p 0.001 these and other epidemiological data support 2 notions . rsv could have a long - lasting effect via an alteration of the immune response ; it is also possible that early rsv infection serves as an indication of inherent differences in an individual s immune system , which leaves it not only vulnerable to serious rsv infection , but also to other , chronic respiratory conditions ( eg , recurrent wheeze and asthma ) . many of the rsv studies have explored these broad hypotheses by examining the effects of rsv on different aspects of the immune response in both human patients and mouse models . recent work has done a great deal to expound the mechanisms underlying the immune response to rsv infection through investigating the formative signaling pathways and genetic underpinnings to the subsequent differential cellular responses . rsv infection causes a wide array of immunologic responses ; clarifying the ways in which rsv triggers these changes may lead to the development of effective therapeutics that can ameliorate or abolish acute and chronic disease that result from ineffective immune system response . rsv predominantly infects primary airway epithelial cells , but can also infect other structural airway and immune cells . in addition , new research shows tlr7 activation during rsv infection [ 10 ] . these receptors in turn promote expression and secretion of inflammatory cytokines ( table 2 ) , which mount the earlier innate immune response and subsequent adaptive immune response . to elicit these responses , several different signaling pathways and their respective molecules these include pathways that include protein kinase c ( pkc ) , mitogen - activated protein kinase ( mapk ) , and nuclear factor-b ( nf-b).table 2summary of cytokines and chemokines produced during rsv infectioncytokinesourceeffectsifn-nk cells , cd4 t cells , cd8 t cellsincreases nk activation , th1 differentiationil-1macrophagesincreases expression of cytokines mediated by nf-bil-4cd4 + t cellsincreases th2 differentiation , decreases th1 differentiationil-8epithelial cells , macrophagesneutrophil recruitmentil-10cd4 + t cellsinhibits expression of inflammatory cytokines , suppresses inflammatory responseil-17acd4 + t cellsincreases th17 differentiation , increases inflammatory cytokine production summary of cytokines and chemokines produced during rsv infection p38 mapk and extracellular signal - regulated kinase ( erk ) mapk are both involved in rsv replication in human airway epithelial cells . in the presence of anti - tlr4 antibodies , in addition , immunofluorescence confocal microscopy revealed that tlr4 clustering at the point of contact between the virus and cell was related to p38 activation , helping to elucidate the mechanism of viral entry . thus , rsv infection caused il-8 expression through nf-b and ap-1 induction via the canonical and the jnk pathway , respectively , and tak1 was an upstream regulator able to mediate both responses . thus , there are likely other kinases that regulate ikk and nf-b activation during rsv infection [ 14 ] . the canonical pathway is not the only recently reported means of induction of nf-b during rsv infection in human airway epithelial cells . in addition , the importance of a non - canonical pathway mediated by the virus - activated kinase ( vak ) complex ( composed of tbk-1 and ikk ) is also evident . using sirna in a549 cells and mouse embryonic fibroblasts from ikk ko mice , ikk was determined to regulate nf-b during rsv infection through p65 ser536 phosphorylation . thus , there is evidence for 2 separate pathways of nf-b activation during rsv infection . in a similar vein , rsv infection leads to il-1 expression , which is an inflammatory cytokine critical to antiviral immune response . mature il-1 is produced and secreted by activated macrophages following assembly and activation of inflammasomes in the cytoplasm [ 19 , 20 ] . this is achieved through 2 signals : first , stimulation of a pattern recognition receptor ensures expression of pro - il-1 ( the il-1 precursor ) and inflammasome components ; second , the inflammasome complex assembly and caspase-1 activation cleave pro - il-1 into il-1. nf-b activation through a tlr2/myd88-dependent pathway is necessary for the first signal of il-1 production in macrophages during rsv infection . likewise , rsv induces il-15 expression in both epithelial cell lines and primary bronchial epithelial cells . il-15 is an inflammatory cytokine that is suggested to augment antiviral defense through up - regulation of ifn - related transcription factors , nf-b , synthesis of type 1 effector molecules , and survival and stimulation of nk cells and cd8 t cells [ 2224 ] . thus , effective expression of il-15 could be beneficial to effective rsv clearance ; further research is required to determine the importance of il-15 in shaping the immune response to rsv infection . in addition to regulating inflammatory cytokine production , nf-b activation is associated with tight junction formation in rsv - infected human nasal epithelial cells . tight junctions are important for epithelial cell polarity and maintenance of tissue integrity . during rsv infection , genes for tight junction molecules , claudin-2 , -4 , -7 , -9 , -14 , -19 ; occludin ; zo-2 ; cingulin and mag-1 interestingly , inhibition of nf-b and pkc led to decreased viral replication and decreased formation of virus filaments . this , along with immunocytochemistry of tight junction proteins and viral proteins ( rsv proteins f and g ) seem to suggest that virally - induced tight junction formation could augment cell polarity during rsv infection allowing for characteristic viral budding at the apical surface . rsv infection also leads to a specific adaptive immune response . the characteristics of the adaptive immune response ( ie , which t helper cell subset and associated cytokines dominate the response [ table 2 ] ) can help to understand acute rsv disease and the implication of rsv infection for potential long - term respiratory disease . therefore , it is important to realize any effects rsv infection has on predisposition to a certain adaptive immune response and the mechanism by which it occurs . rsv infection has been associated with skewing the immune system away from an anti - viral th1 response and towards a th2 response . new data shed light on how this occurs and introduced a role of il-17a ( the principle cytokine of th17 response ) in rsv infection as well , as antibody blockade of il-17a led to significant reduction of viral protein mrna expression in the lungs and th2 cytokine protein expression in the lymph nodes in rsv - infected mice compared with mice treated with control antibody . this is interesting because il-17a has recently been implicated in the development of severe forms of asthma . epithelial cells , in addition to providing a link to the initial innate immune response , aid in adaptive immune system activation after rsv infection . when human bronchial epithelial cells ( hbecs ) were co - cultured with peripheral blood mononuclear cells ( pbmcs ) , there was an induction of ifn- , il-4 , and il-17 cytokine production , which suggested activation of lymphocytes as these are the characteristic cytokines of t helper cell subsets th1 , th2 , and th17 , respectively . when isolated lymphocytes were exposed to the supernatant of rsv - infected hbecs , flow cytometry revealed that compared with controls exposed to complete cell culture medium , th2 and th17 differentiation was significantly induced , while treg differentiation was clearly suppressed . in addition to inherent major histocompatibility complex ( mhc ) i , epithelial cells may be able to express mhc ii in times of stress or disease , as well as costimulatory molecules cd80 and cd86 during infection [ 3032 ] . therefore , it is conceivable that hbecs directly activate nave cd4 lymphocytes during rsv infection . rsv infection of primary rat airway epithelial cells ( praecs ) caused upregulated thymic stromal lymphopoietin ( tslp ) 6 to 18 hours post - infection . in the same coculture system , sirna for tslp effectively decreased tslp expression and also the expression of mhc ii and cd86 , without changing cd80 expression . functional mdc maturation was verified , as mdcs exposed to rsv - infected praecs significantly increased t cell proliferation in comparison to uninfected praecs . thus , it is possible that rsv infection of epithelial cells leads to a differential t helper cell response through the polarization of mdcs , and potentially directly through epithelial cell antigen presentation . however , this study did not assess the ability of affected mdcs to alter nave t cell differentiation . further study is necessary to further elucidate the mechanisms of t cell activation and differentiation during rsv infection . during rsv infection , natural killer ( nk ) cells interestingly , past clinical studies show that increased nk deficiency is linked to increased respiratory disease symptoms . in balb / c mice , antibody - mediated nk cell reduction during rsv infection resulted in lower ifn- production and an increase in th2 cytokines . just as the immune system needs to effectively activate and clear a pathogen , it also needs an effective system of self - regulation and inhibition to inhibit an unbalanced immune response and collateral damage of host tissue . studies examining humans and mouse models reveal that inefficient immune system regulation escalates respiratory disease pathology following rsv infection , and can also lead to less optimal viral clearance . it mediates suppression of the activation and effector functions of many immune system cells . as can be expected , it is important in rsv infection and resultant disease pathogenesis . however , this difference in il-10 concentration was not correlated to the rs1800872 single - nucleotide polymorphism ( snp ) in the il10 promoter region . so , although there is some support for a protective role from this il10 genetic variation , the potential functional role during rsv infection remains unclear . that said , it is also possible that during rsv infection the il-10 response is modulated via indirect genetic factors . in 1 recent study of 166 hospitalized infants with rsv lrti , 1 snps of the il19 gene ( rs2243191 and rs2243188 ) and 1 snp of il20 gene ( rs2981573 ) were associated with protection from recurrent wheeze ( or 0.4 , 0.5 , and 0.4 , respectively ) . from these snps , 3 haplotypes comprised 99 % of all the il19/il20 haplotypes in the patients . perhaps changes in il-10 family cytokine expression through snps play a role in direct immune system regulation during rsv infection . it is also possible that their expression influences il-10 expression during infection because il-10 family members cross - regulate each other in other models [ 44 , 45 ] . these cohort studies show that il-10 has some influence in disease pathogenesis , prompting studies to focus on elucidating the mechanism of il-10 and other factors of immunosuppression during rsv infection in mouse models . cd4 and cd8 t cells both produced il-10 in the lung during rsv infection in mice , with production peaking at 8 days post infection [ 46 ] . the majority of il-10 producing t cells were cd4 during rsv infection . although cd4 t cells are the major producers of il-10 in mouse rsv infection , the subset that contributes most to this production is not as clear . one group has shown t regulatory cells ( cd4foxp3 ) was the most common il-10 expressing cells in the lung , mediastinal lymph node ( mln ) and bal fluid of infected mice , while another has shown that cd4foxp3 are the majority of il-10 expressers [ 46 , 47 ] . in il-10 ko mice and wt mice treated with anti - il-10r antibody , rsv infection increased disease and delayed recovery when compared to il-10 producing and control antibody treated mice , respectively [ 46 , 47 ] . thus , a lack of il-10 resulted in an altered adaptive immune response , providing evidence that il-10 is an important regulator of inflammation during rsv infection . in addition , there was a decrease in natural t regulatory cells ( cd4foxp3helios ) when mice were given anti - il-10r antibody [ 46 ] . as il-10 is an important regulatory cytokine , t regulatory cells ( tregs ; cd4foxp3 ) are responsible for suppressing the adaptive immune response . after rsv infection in mice , there was a rapid accumulation of tregs in the airways and secondary lymphoid tissue . by 4 days post - infection there is a 50-fold increase in tregs in the bal fluid of infected mice compared to nave mice . this drastic increase was amplified 8 days post - infection to an 86-fold increase over nave mice . there was also significant treg accumulation observed in the lung parenchyma and mlns and a slight increase in the spleen . local proliferation of tregs occurred with 78 % and 69 % of tregs expressing the proliferation marker ki-67 in the bal and lung parenchyma , respectively , 6 days post - infection . in addition , the majority of tregs expressed activation markers , which were up - regulated post - infection . cd25 tregs present in the airways increased significantly as well . with the clearance of rsv treg efflux took longer in the bal and mlns than in the lung parenchyma and spleen . a 60 % decrease in cd4foxp3cd25 tregs using cd25-neutralizing antibodies was extremely detrimental to immune system efficiency , leading to a significant increase in time of viral clearance . this delay could be traced to a delay in the recruitment in rsv - specific cd8 t cells to the lung . this finding argues that tregs are more than just immunosuppressors ; they also act as conductors of an effective immune response for rsv clearance . as can be surmised in understanding the vast and intricate effects of rsv infection on the immune system response , it is important to acknowledge that there is variability of immune response to rsv infection from case to case . there are many different strains of rsv that each contains significant genetic variability , leading to altered virulence and subsequent disease pathogenesis . also , the characteristics of the infected host cell ( ie , species and type of cell ) can lead to differential cellular and systematic responses . this is especially important to keep in mind in examining in vitro studies that focus on a small number of cell types . inherent differences lead to differences in the innate and adaptive immune responses triggered by rsv infection . several recently published studies examine the effects of intrinsic differences on rsv pathogenesis and immune response . indeed , in a comparison of a2 , 220 , 312 , and line 19 at 8 days post - infection , a2 infection had a significantly greater amount of cd8 t cells in the lung that produced ifn- than 220 and line 19 . interestingly , this matched pas staining data that showed a2 as nonmucogenic , whereas 220 and line 19 were mucogenic . this finding links the role of t cell differentiation and disease pathogenesis [ 51 ] . as in the aforementioned mouse model , the strains were characterized by different patterns of infection . a2 was much more infectious ; at 72 hours post - infection , most host cells were infected . in contrast , the clinical isolates were not nearly as infectious , and displayed differential infectivity ( bt2a was most infectious , bt4a was least ) . this difference carried over to viral growth kinetics : a2 grew more quickly and to higher titers than the clinical isolates . interestingly , all strains initiated a post - infection increase in the concentration of the chemokines and cytokines rantes , ip-10 , il-6 , and il-8 in supernatants . so , inflammatory chemokine and cytokine release varied by rsv strain , which would likely elicit a variable immune system response in an in vivo model . from the opposite perspective , the types of cells used for experimental infection are also important . for instance , a group examined rsv infection in airway epithelial cells ( aecs ) from lungs of healthy children and cells of the beas-2b cell line [ 53 ] . in short , the response to rsv infection differed between epithelial cells derived from epithelial cell lines and from the lungs of children . recent advancements in rsv research have illuminated the signal transduction pathways that are activated to initiate transcription of inflammatory cytokines and chemokines once rsv is recognized by a tlr on the cell surface . in addition , th2 and th17 differentiation and role in disease progression during rsv infection are better understood . that said , it is necessary to better characterize the link between the innate and adaptive immune systems post - infection , and how variations in this link can lead to variations in acute and chronic airway disease . understanding the influence of regulatory agents ( eg , il-10 , tregs ) host genetic predisposition , viral strain , and other factors on this combined immune response may help glean new therapeutic avenues .

the ecology of tularemia is represented by a literature that reflects the diversity of this complex zoonosis . like the literature of many other infections , the sheer volume of observations makes it difficult to organize and synthesize sets of working hypotheses for how the causative agent exists in nature . an organized understanding of tularemia ecology serves as the basis for developing public health interventions and to predict or explain changes in incidence or distribution . in addition , knowing how the agent is currently maintained in nature provides information that helps us to reconstruct its evolutionary history . we review herein features of tularemia ecology that are particularly critical and suggest lacunae that hinder us from distinguishing major themes from variations on themes . the reader is referred to excellent reviews of the subject by hopla ( 1974 ) , friend ( 2006 ) , and petersen et al . ( 2009 ) for significantly more detail on the breadth and diversity of the ecological literature . we use some basic terms and concepts in the population biology of infectious agents that help to organize our interpretation of the existing literature . maintenance refers to the life cycle of the agent : how one infection gives rise to at least one other infection . perpetuation of zoonotic infections may involve a vector , an intermediary in the life cycle that imparts directionality to the agent . hematophagous arthropods are vectors because they require blood and thus directly transport an infectious agent to a relevant host . in contrast , scalars may also maintain an agent , but there is no directionality ; copepods containing dracunculus medinensis nematodes , for example , are passively imbibed with water . vectors may support biological transmission of an agent , a process that involves replication or developmental changes . vectorial capacity ( spielman and rossignol , 1984 ) refers to the sum of vector traits that ensures that the basic reproduction number ( brn ) of an infection exceeds unity and comprises competence ( ability to support replication and effectively deliver the agent ) as well as factors such as abundance , longevity , and narrowness of host range . a mosquito that requires a large dose of pathogen and rarely passes it during feeding has poor vector competence and thus might not contribute much to brn , but even a highly competent vector ( agent replicates well and is readily ejected during feeding ) can have poor vectorial capacity if it feeds only on an animal that is a the central question in the ecology of infectious agents is to describe how an agent ensures that brn > 1 ( anderson and may , 1981 ) ; brn < 1 implies unstable transmission and extinction . quantitative modeling of brn helps to synthesize diverse field observations and rank the contributions of factors and influences . basic reproduction number models may be developed from simple flow charts representing the presumed life cycle of the infectious agent ; boxes can be outcomes ( e.g. , number of non - immune hosts ) and arrows are processes ( e.g. , infection ) . such compartmental models ( figure 1 ) can then be quantified by expressing the transitions between boxes as differential equations . we could adopt for the purposes of constructing a general tularemia model conditions similar to that for pasteurellosis in mice ( anderson and may , 1979 ) . the agents of tularemia and pasteurellosis are categorized as microparasites : they are physically small with short generation times ; they have a high replicative rate within a host ; they tend to induce immunity to reinfection in survivors ; and , the duration of infection is short relative to host lifespan . in addition , these infections may modify the demography of their host . if for the purposes of constructing a model , we assume that the only mode of tularemia transmission is direct ( no vectors ; transmission by contact with , inhalation of or ingestion of saliva , excreta , or blood ) , then model development may proceed as outlined in anderson and may ( 1979 ) . the model then distils down to critical variables such as absolute number of susceptibles ( non - infected ) ; number that are infected ; number immune ; the natural mortality rate of the host population ; the rate of introduction of susceptibles ( immigration , birth ) ; and a measure of the acquisition of infection ( contact rate of susceptibles with infected ) . even at this rudimentary level of discussion , we can see how difficult it would be to have an empirical basis for the model : we still debate the identity of the reservoir host for francisella tularensis or whether the reservoir might be environmental and even if we assumed a specific animal , would we have data on its demography , behavior ( contact between individuals ) , or prevalence of infection ? however , by constructing such models , we can prioritize the field observations required for us to refine and validate the models , which summarize our current understanding of the life cycle . resources should be expended in describing the circumstances of acquiring infection over determining the number of infected hosts , for example , because in the model the process drives the outcome . flow chart ( compartmental model ) for general model of f. tularensis perpetuation . tularemia is a specific infectious disease due to bacterium tularense and is transmitted from rodents to man by the bite of an infected bloodsucking insect or by the handling and dissecting of infected rodents by market men or laboratory workers . the first written account of tularemia in the us was in 1907 noting signs and symptoms compatible with tularemia in native americans who had handled jackrabbits ( barnes , 1928 ) . plague - like disease in ground squirrels was identified in 1909 during animal surveillance in california but microscopic observation of tissue sections demonstrated organisms inconsistent with the characteristic safety pin the bacterium was quickly cultivated and it was apparent that a new entity had been discovered ( mccoy and chapin , 1912 ) . although pearse ( 1911 ) first described deer fly fever in utah residents bitten by tabanid flies , francis ( 1922 ) demonstrated its identity with the ground squirrel disease , and proposed the name tularemia . he provided definitive evidence by isolating the agent from fly bitten humans , from jackrabbits , and ground squirrels . francis also provided experimental evidence for transmission of tularemia by the bites of deerflies , lice , and bugs ( francis , 1922 ) . investigations of the bitterroot valley rocky mountain spotted fever epidemic in the 1920s isolated f. tularensis from the main rmsf vector , dermacentor andersoni ( parker et al . , 1924 ) . other human biting ticks ( dermacentor variabilis , amblyomma americanum ) were soon documented as vectors ( philip and jellison , 1934 ; parker , 1934 ) . therefore , within 20 years of its discovery as an infection of rodents in california , the most important aspects of the epidemiology ( factors relating to human risk ) of tularemia in the us had been described , as summarized by francis pithy statement , but taken together , the sum of knowledge would not allow quantitative modeling of brn ( ecology ) for f. tularensis in any site . it is not clear that 80 years later that we have sufficient information to do so . francis ( 1937 ) noted that > 90% of the > 6000 tularemia case reports that he had compiled from 1924 to 1935 were associated with exposure to cottontail rabbits or hares , and this analysis surely helped to formally develop tularemia 's reputation as rabbit fever . it is possible that the strong rabbit association was due to an active market for rabbit meat in the north central states where there was a tradition of rabbit hunting ( yeatter and thompson , 1952 ) . this rabbit association , interestingly , obscured the fact that in the south central us , tick exposure accounted for 70% of all cases ( brooks and buchanan , 1970 ) during the 1960s . tularemia incidence in the us started to diminish in the 1960s ( boyce , 1975 ) , perhaps as a result of a loss in popularity of rabbits as food and of hunting in general . it seems unlikely that the force of transmission of the agent diminished in nature during this time . the tick vectors ( d. andersoni , d. variabilis , and a. americanum ) for tick - transmitted tularemia in the us are the same as those for rmsf , which increased in incidence during the 1960s and 1970s ( childs and paddock , 2002 ) . russia and japan had concurrently discovered tularemia ( francis , 1934 ; pollitzer , 1967 ) . episodes of morbidity and mortality in hares were associated with an increase in the number of human cases of yato - byo and the disease could be acquired by skin contact with hare tissues ( ohara , 1926 ) . apparently , 90% of all japanese tularemia cases were associated with exposure to the hare lepus brachyurus ( toyoshima and ohara , 1967 ) . thus , in north america and japan , during the very first decades of epidemiologic investigations of the disease , lagomorphs ( rabbits and hares ) were the main focus of attention . researchers in the former soviet union were extremely active in investigations of tularemia , producing 1300 publications from 1928 to 1960 ( pavlovsky , 1966 ) , and were the first to describe at least six perpetuation scenarios centered around habitat types ( floodplain swamp ; meadow field ; forest ; steppe ; piedmont river ; and desert floodplain ) ; lagomorphs were requisites for none . paradoxically , a hypothesis for the evolution of f. tularensis by russia 's most prominent tularemia researcher ( olsufiev , 1963 ) focused on associations with lagomorphs , mainly based on their great degree of susceptibility and sensitivity to infection as well as a scenario for the zoogeography of the steppes , which were thought by the former ussr workers to have been a pivotal habitat for tularemia . differences in distribution , ecology , biochemistry , and virulence led to the seminal classification of tularemia into distinct types ( olsufiev et al . , 1959 ) . type a organisms ( now known as f. tularensis tularensis ) are prevalent in north america but not in eurasia , are frequently transmitted by ticks , and may cause severe disease . type b ( f. tularensis holarctica ) causes episodic outbreaks ( epizootics ) in beavers , muskrats , and arvicoline rodents in either north america or eurasia , may be isolated from water or soil , and may cause a milder disease ( jellison et al . these eco - epidemiological paradigms retain tremendous utility and argue for modeling brn separately . the perception that tularemia was due to lagomorphs was largely the influence of francis himself and also of william jellison of the rocky mountain laboratories , who compiled and interpreted the existing literature on tularemia biology in a seminal monograph ( jellison , 1974 ) . jellison argued that human risk and geographic distribution of north american tularemia was strongly associated with cottontail rabbits ( jellison and parker , 1945 ; jellison et al . , 1961 ) . cottontail rabbits are very susceptible to infection by type a , dying within 7 days , and are large enough animals to attract attention when there is an epizootic , making them good sentinels for transmission activity . furthermore , because of their value as food , their populations were a focus of attention by local residents and by state game management divisions : 25,000,000 rabbits were killed annually with a value of $ 5,000,000 during the 1920s ( henderson and craig , 1932 ) . whether cottontail rabbits are required for type a brn > 1 remains unproven and requires further study . it may be that the question has been considered resolved due to conflating the requirements for maintenance with the proximal determinants for human exposure . of course , human exposure ( the subject of epidemiology ) may provide clues to the mode of perpetuation ( ecology ) but this is not axiomatic . zoonotic infections may exist in sites with no implied human risk in the absence of an effective epidemiological bridge . the classical theory of natural nidality ( pavlovsky , 1966 ) posits that most zoonotic agents exist in longstanding foci that comprise optimal physical ( weather , geology ) and biological ( fauna , flora ) associations and that humans only become aware of their existence when they intrude . accordingly , rabbits and hares may only be the epidemiological bridge and are not necessarily an element of natural focality . the concept of rabbits as central to type a ecology was bolstered by the identification of the rabbit tick , haemaphysalis leporispalustris , as an effective enzootic vector ( parker 1934 ) . these ticks are widely distributed across north america and their feeding is focused primarily on lagomorphs . narrowness of host range ( bites focused on relevant hosts ) greatly contributes to vectorial capacity and brn ( spielman and rossignol , 1984 ) . these ticks transmit f. tularensis and pass the agent by inheritance ( transovarial transmission ; parker 1934 ) ; presumed type a isolates were made from field collected h. leporispalustris ( philip and parker , 1938 ) . rabbits may be infested by hundreds of these ticks and all stages may infest a rabbit simultaneously , thereby providing an opportunity for non - systemic ( co - feeding ) transmission ( randolph et al . , 1996 ) . thus , rabbits and rabbit ticks could serve to powerfully maintain type a. in addition , because subadults ( larvae and nymphs ) of this tick will infest ground - inhabiting birds , including those that migrate , the agent could be readily transported . this fact begs the question : why has f. tularensis not been detected in latin america south of mexico , particularly given the presence of rabbits throughout south america ? the central importance of rabbits and their ticks in type a ecology is not supported by recent studies on martha 's vineyard ( mv ) . this is the only site in the us which has endemic primary ( inhalational ) pneumonic tularemia ( matyas et al . , 2007 ) ; of more than 100 tularemia cases reported from mv from 2000 to 2010 , case control studies demonstrate that landscapers are the major risk group and use of lawnmowers or leaf blowers are the main risk factors , suggesting environmental contamination . landscapers there insist that they rarely mow over animal carcasses because they visually inspect properties prior to their activities to reduce hazard due to rocks and other debris . the nature of the fomites that served to infect these case - patients remains undescribed but unseen remnants of animal carcasses , animal feces , urine - soaked soil , ticks , tick feces , fleas , and contaminated water are possible sources . why mv alone reports numerous pneumonic tularemia cases when tularemia is more prevalent in the south central us where lawnmowers are certainly used remains enigmatic . it is possible that the heavily salt - spray influenced landscape of the ocean facing southern edge of mv is more conducive to the agent remaining viable for a longer duration than elsewhere in the us ( berrada and telford , in press ) . ecological studies , extended from lyme disease surveillance starting in 1994 , quickly suggested that dog ticks ( d. variabilis ) were important to type a perpetuation ( goethert et al . , 2004 ; matyas et al . , 2007 ; goethert and telford , 2009 ) . infection has been found in mv dog ticks every year to date , comprising a large degree of genetic heterogeneity ( goethert et al . , 2009 ) . rabbits were indeed infected but virtually disappeared on mv , probably due to tularemia mortality ; their disappearance did not influence the force of transmission of type a , which continued to remain prevalent in dog ticks . intensive studies of cottontail rabbits had been undertaken on nantucket island , which is visible from the eastern portion of mv . rabbits attained densities of 1520 per hectare and were heavily infested by h. leporispalustris ( telford and spielman , 1989 ; goethert and telford , 2003 ) . evidence of tularemia had never been detected in more than 200 rabbits sampled on nantucket , even though five human cases had been identified from 1990 to 2005 . one of these cases was definitively associated with rabbits : a worker who had helped his colleague move a rabbit that had been mutilated by a lawnmower developed pneumonic tularemia ( goethert and telford , 2005 ) . this event demonstrated that even though type a had been introduced at least once to nantucket , despite the presence of dense rabbits and heavy h. leporispalustris infestations , the agent did not perpetuate . no mass die - offs of rabbits were noted during the year when the lawn mowing incident occurred , nor did active surveillance for rabbit carcasses by landscapers yield any evidence of mortality due to tularemia . there is one important difference in the ecology of nantucket relative to mv : nantucket lacks appreciable numbers of dog ticks ( indeed , the tick may now be extirpated from that island due to the recent widespread use of topical anti - ectoparasiticides on dogs ) because it does not have their reproductive hosts , skunks , raccoons , foxes , or coyotes . although one exception to the rule of rabbits maintain tularemia does not necessarily invalidate the rule , we note that mv is one of few sites where longitudinal ecological studies have been undertaken and thus where incidental findings can be distinguished from general findings . we suspect that rabbits are not necessarily critical to the brn of type a , or if they are , it is a function of local conditions . this dependence on local conditions , in fact , is the challenge of developing a quantitative model for the brn of tularemia : should there be a general model , or should we approach the subject as did the researchers of the former ussr , focusing on independent natural foci ? at one extreme , the 4 genotypes / subclades of type a and 11 of type b ( kugeler et al . , 2009 ; vogler et al . , 2009 ) might each require a specific brn model . but , a general model would have to assume that all elements for the ecology of type a would apply to type b and vice versa , not necessarily a good assumption . although our hypothesis is that dog ticks are critical to brn on mv , experimentally infected as well as naturally infected ticks die more quickly than do those that are not ( reese et al . these findings stand in contrast to our empirical observation of infected ticks each year , and suggest that there may be factors that mitigate the negative fitness of infection in nature . tularemia in eurasia and non - rabbit or tick associated infection in north america seem to have a strong environmental basis , acquired from agricultural activities such as hay threshing ; from water contaminated by muskrats or water voles ; or during the trapping of furbearers ( pavlovsky , 1966 ; syrjala et al . , 1985 ; reintjes et al . , 2002 ; allue et al . , 2008 ) . in addition , transmission of the pathogen could occur via contamination of foodstuffs by urine or fecal material from infectious rodents ( karpoff and antonoff , 1936 ; parker et al . , 1951 ) . experimental studies with voles suggested the possibility that some f. tularensis - infected animals developed a chronic nephritis and bacteriuria that could serve as a protracted source of environmental contamination ( bell and stewart , 1975 ) . of particular interest was the suggestion that voles became partially immune due to low level infection resulting from cannibalism of tularemic carcasses and that this immunity allowed survival of the vole during subsequent infection , increasing the probability for shedding in the excreta . ( cannibalism of moribund cagemates is well known as a mode of transmission for type a in the laboratory , owen and buker , 1956 , and could be a complementary factor in perpetuation . ) this suggestion of orally induced immunity has not been explored further but if confirmed could be a critical factor for the brn of type b , particularly in the context of environmental persistence . ticks may be infected by type b and are said to be the reservoir ( pavlovsky , 1966 ) . in the former soviet union , 17 species of ixodid ticks have been found to be naturally infected ( balashov , 1972 ) , presumably by type b inasmuch as type a is virtually restricted to north america . in addition , type b is well known to be tick - transmitted in north america and both types may be present in ticks in the same site ( markowitz et al . , 1985 ) . human cases certainly result from tick exposure but this mode of transmission is less common than exposure to furbearers or contaminated water ( pavlovsky , 1966 ) . dermacentor marginatus and dermacentor reticulatus appear to be the main vectors there as well as into central europe . as with type a , type b - infected d. marginatus or d. variabilis die more rapidly than do uninfected ticks ( petrov , 1960 ; reese et al . , 2010 ) , which again raises the question of whether a non - adaptive trait may be associated with stable brn . mosquitoes are strongly implicated as vectors in sweden , given that tularemic ulcers are most frequently found on the upper back , neck , and ears of case - patients ( eliasson and back , 2007 ) , where mosquitoes are more likely to feed . in addition , the agent has been isolated from mosquitoes there ( olin , 1942 ) and recent studies provide evidence for mosquito larvae acquiring infection from water , perhaps by the ingestion of predatory protozoa ( mathisen et al . , gis studies of the orebro endemic area in sweden demonstrate that there is temporal spatial association of incidence with mosquito breeding ( svensson et al . , 2009 ) . one recent study reported a third of mosquito pools to be infected in alaska ( triebenbach et al . , 2010 ) when tested by pcr , but this finding was at odds with the epidemiological evidence as well as with the difficulty of finding infection in animals . older studies in alaska failed to isolate f. tularensis from mosquitoes ( hopla , 1974 ) and thus it is not clear what the pcr findings represent ; it should be noted that the assay that was used might also detect francisella novicida . future mosquito surveys should always attempt to confirm pcr findings with a complementary assay such as culture or even indirect immunofluorescence using monoclonal antibodies . at the very least definitive evidence for biological transmission by naturally infected mosquitoes might be provided by the use of deliberately placed sentinel mice but given the difficulties of animal experimentation in sweden , not a likely approach . the recent suggestion that infectious agents may be detected by assaying sugar sources probed by mosquitoes ( hall - mendelin et al . , 2010 ) may be an effective alternative approach to demonstrating that naturally infected mosquitoes can transmit . given that mechanical transmission causes infection and thus brn > 1 , it might be considered academic to determine whether biological transmission occurs . but , the duration of mosquito infectivity would differ depending on whether it was mechanical or biological transmission , thereby impacting the magnitude of brn . in addition , the possibility that mosquito larvae may acquire infection from water might greatly enhance brn if those larvae became adult females that transmitted . whether there is a main mode of perpetuation ( e.g. , ticks and rodents ) with ancillary cycles ( spillover into a water cycle , mechanical transmission by mosquitoes ) , whether it is the other way around ( perpetuation within water and spillover into rodents and their ectoparasites ) or whether there may be multiple parallel cycles in sites where there are ticks would be difficult to answer with field studies but might be facilitated by mathematical modeling of brn . such questions have more than just academic interest : if ticks and rodents drive the ecology of type b , then intervention could be considered to reduce risk , for example , by rodent or tick control . if water drives the ecology , then risk reduction would have to focus on personal protection ( e.g. , with vaccination ) given the difficulty of manipulating water ecosystems . both type a and type b are highly infectious and may be transmitted mechanically on the mouthparts of various hematophagous arthropods , or by contact with body or tissue fluids through abraded and even intact skin , and by ingestion , in addition to true biological ( vector ) transmission . such a wide spectrum of modes of exposure and great infectivity helps to explain the wide range of kinds of animals known to be exposed or infected ( burroughs et al . , 1945 ; friend , 2006 ) . it is likely that most of these animals do not serve as amplifying hosts ( reservoirs ) that increase the brn of the agent of tularemia , but are incidental many of the reports of an animal serving as host simply document exposure ( seroreactivity ) in a limited sample which does not allow inference about whether the exposure might be common over many sampling periods or among many sites . if an animal contributes significantly to brn , it should do so for successive generations and in more than one site . this is the rationale for undertaking longitudinal ecologic studies : to determine whether an observation is incidental or is a generality . the reason it is important to determine whether there is a main theme for perpetuation ( one important reservoir host such as a lagomorph ) as opposed to many themes ( almost anything can serve as a reservoir ) is that an ecological paradox otherwise exists : if virtually any hematophagous arthropod and vertebrate could maintain infection , then tularemia should be readily perpetuated and extremely common across the holarctic . thorough search of any site should document the presence of f. tularensis . from an epidemiological standpoint , tularemia is only moderately common , with global incidence in the range of 1001000 cases annually ( paddock and telford , in press ) . tick surveys in known endemic sites generally record prevalence of f. tularensis infection in the range of 0.15% ( e.g. , green , 1931 ; hopla , 1960 ; hubalek and halouzka , 1997 ; goethert et al . , 2004 ) which is similar to that for tick borne encephalitis ( tbe ) virus in i. persulcatus complex ticks ( gresikova and nosek , 1967 ; korenberg , 1994 ; schafer et al . , 1999 ) . tbe is considered to be a common tick borne infection , with 100010,000 new cases each year ( paddock and telford , in press ) . f. tularensis has been detected in i. ricinus , the main european vector of tbe . with the potential for transmission by mosquitoes , in addition to tick transmission and environmental exposure , tularemia risk ( human incidence ) over the palearctic should approach or exceed that of tbe . becomes even more vexing given the possibility of environmental persistence , that is , perpetuation that may be independent of vertebrate hosts . surface water and sediment yielded indisputable dna sequence evidence of contamination with type b in swedish endemic sites , even in years with little epidemiological activity ( broman et al . , infected carcasses contaminated water for 10 days and contaminated water stored in the cold infected animals after 2 weeks . naturally contaminated mud remained infectious as long as 10 weeks ( parker et al . , 1951 ) . experimental microcosm experiments demonstrated that f. tularensis in silt could infect animals for 2 months . about half of rodents immersed in contaminated water became infected with exposure to as few as 1001000 cfu / ml ( pavlovsky , 1966 ) , which appears to be large dose but the spleen alone of a mouse dying of tularemia may have 10 cfu ( molins et al . , 2010 ) and it would not take many carcasses to contaminate a contained body of water . indeed , many have speculated that environmental persistence depends on continual contamination by dead animals . however , water invertebrates such as shrimp or snails could retain viable organisms for 20 days ( mironchuk and mazepa , 2002 ) , and indeed , invertebrates were first described as contributing to f. tularensis survival within water by former ussr research ( pavlovsky , 1966 ) . more recent reports ( anda et al . , 2001 ) of crayfish infection suggest that additional surveys using modern methods are warranted . even more interesting is the hypothesis that free - living amebae serve as hosts ( berdal et al . , 1996 ; abd et al . , 2003 ; el - etr et al . , 2009 ) , which would allow for even greater duration of persistence and even the possibility of amplification in the absence of vertebrates . in terms of brn , environmental persistence within aquatic invertebrates or protozoa would have implications for ensuring that brn > 1 when vertebrates are scarce but water contamination could serve to greatly increase brn and trigger an epizootic or outbreak . the ecology of tularemia is sufficiently complex that limited resources for research should be targeted to the most relevant questions . developing quantitative models of the brn for tularemia , probably separately for type a and type b , would appear to be a priority because such models provide structure for observations . critical life cycle breakpoints and influential variables can be hypothesized a priori and tested by field observations . surveys should be undertaken to identify useful study sites where the agent is reliably detected and longitudinal observations ( on a scale of years ) may be undertaken . cooperative agreements should be developed among researchers and long term funding jointly sought to support such studies . we have outlined some of the more interesting lacunae in our knowledge of tularemia ecology , and additional information for all of these would greatly enhance iterative refinements of brn quantitative models in the future . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

the epidemiology of tularemia has influenced , perhaps incorrectly skewed , our views on the ecology of the agent of tularemia . in particular , the central role of lagomorphs needs to be reexamined . diverse observations , some incidental , and some that are more generally reproducible , have not been synthesized so that the critical elements of the perpetuation of francisella tularensis can be identified . developing a quantitative model of the basic reproduction number of f. tularensis may require separate treatments for type a and type b given the fundamental differences in their ecology .

Introduction General Comments on the Ecology of Infectious Agents The Role of Rabbits in Type a Ecology: Conflation with Epidemiologic Risk Type B ecology: Driven by Water or by Rodents? Why is Tularemia Relatively Rare? Priorities for Tularemia Ecology Research Conflict of Interest Statement

the ecology of tularemia is represented by a literature that reflects the diversity of this complex zoonosis . like the literature of many other infections , the sheer volume of observations makes it difficult to organize and synthesize sets of working hypotheses for how the causative agent exists in nature . an organized understanding of tularemia ecology serves as the basis for developing public health interventions and to predict or explain changes in incidence or distribution . in addition , knowing how the agent is currently maintained in nature provides information that helps us to reconstruct its evolutionary history . we review herein features of tularemia ecology that are particularly critical and suggest lacunae that hinder us from distinguishing major themes from variations on themes . the reader is referred to excellent reviews of the subject by hopla ( 1974 ) , friend ( 2006 ) , and petersen et al . ( 2009 ) for significantly more detail on the breadth and diversity of the ecological literature . we use some basic terms and concepts in the population biology of infectious agents that help to organize our interpretation of the existing literature . maintenance refers to the life cycle of the agent : how one infection gives rise to at least one other infection . perpetuation of zoonotic infections may involve a vector , an intermediary in the life cycle that imparts directionality to the agent . vectorial capacity ( spielman and rossignol , 1984 ) refers to the sum of vector traits that ensures that the basic reproduction number ( brn ) of an infection exceeds unity and comprises competence ( ability to support replication and effectively deliver the agent ) as well as factors such as abundance , longevity , and narrowness of host range . a mosquito that requires a large dose of pathogen and rarely passes it during feeding has poor vector competence and thus might not contribute much to brn , but even a highly competent vector ( agent replicates well and is readily ejected during feeding ) can have poor vectorial capacity if it feeds only on an animal that is a the central question in the ecology of infectious agents is to describe how an agent ensures that brn > 1 ( anderson and may , 1981 ) ; brn < 1 implies unstable transmission and extinction . basic reproduction number models may be developed from simple flow charts representing the presumed life cycle of the infectious agent ; boxes can be outcomes ( e.g. , number of non - immune hosts ) and arrows are processes ( e.g. the agents of tularemia and pasteurellosis are categorized as microparasites : they are physically small with short generation times ; they have a high replicative rate within a host ; they tend to induce immunity to reinfection in survivors ; and , the duration of infection is short relative to host lifespan . if for the purposes of constructing a model , we assume that the only mode of tularemia transmission is direct ( no vectors ; transmission by contact with , inhalation of or ingestion of saliva , excreta , or blood ) , then model development may proceed as outlined in anderson and may ( 1979 ) . the model then distils down to critical variables such as absolute number of susceptibles ( non - infected ) ; number that are infected ; number immune ; the natural mortality rate of the host population ; the rate of introduction of susceptibles ( immigration , birth ) ; and a measure of the acquisition of infection ( contact rate of susceptibles with infected ) . even at this rudimentary level of discussion , we can see how difficult it would be to have an empirical basis for the model : we still debate the identity of the reservoir host for francisella tularensis or whether the reservoir might be environmental and even if we assumed a specific animal , would we have data on its demography , behavior ( contact between individuals ) , or prevalence of infection ? however , by constructing such models , we can prioritize the field observations required for us to refine and validate the models , which summarize our current understanding of the life cycle . flow chart ( compartmental model ) for general model of f. tularensis perpetuation . the first written account of tularemia in the us was in 1907 noting signs and symptoms compatible with tularemia in native americans who had handled jackrabbits ( barnes , 1928 ) . although pearse ( 1911 ) first described deer fly fever in utah residents bitten by tabanid flies , francis ( 1922 ) demonstrated its identity with the ground squirrel disease , and proposed the name tularemia . he provided definitive evidence by isolating the agent from fly bitten humans , from jackrabbits , and ground squirrels . francis also provided experimental evidence for transmission of tularemia by the bites of deerflies , lice , and bugs ( francis , 1922 ) . investigations of the bitterroot valley rocky mountain spotted fever epidemic in the 1920s isolated f. tularensis from the main rmsf vector , dermacentor andersoni ( parker et al . therefore , within 20 years of its discovery as an infection of rodents in california , the most important aspects of the epidemiology ( factors relating to human risk ) of tularemia in the us had been described , as summarized by francis pithy statement , but taken together , the sum of knowledge would not allow quantitative modeling of brn ( ecology ) for f. tularensis in any site . francis ( 1937 ) noted that > 90% of the > 6000 tularemia case reports that he had compiled from 1924 to 1935 were associated with exposure to cottontail rabbits or hares , and this analysis surely helped to formally develop tularemia 's reputation as rabbit fever . it is possible that the strong rabbit association was due to an active market for rabbit meat in the north central states where there was a tradition of rabbit hunting ( yeatter and thompson , 1952 ) . tularemia incidence in the us started to diminish in the 1960s ( boyce , 1975 ) , perhaps as a result of a loss in popularity of rabbits as food and of hunting in general . it seems unlikely that the force of transmission of the agent diminished in nature during this time . the tick vectors ( d. andersoni , d. variabilis , and a. americanum ) for tick - transmitted tularemia in the us are the same as those for rmsf , which increased in incidence during the 1960s and 1970s ( childs and paddock , 2002 ) . episodes of morbidity and mortality in hares were associated with an increase in the number of human cases of yato - byo and the disease could be acquired by skin contact with hare tissues ( ohara , 1926 ) . thus , in north america and japan , during the very first decades of epidemiologic investigations of the disease , lagomorphs ( rabbits and hares ) were the main focus of attention . researchers in the former soviet union were extremely active in investigations of tularemia , producing 1300 publications from 1928 to 1960 ( pavlovsky , 1966 ) , and were the first to describe at least six perpetuation scenarios centered around habitat types ( floodplain swamp ; meadow field ; forest ; steppe ; piedmont river ; and desert floodplain ) ; lagomorphs were requisites for none . paradoxically , a hypothesis for the evolution of f. tularensis by russia 's most prominent tularemia researcher ( olsufiev , 1963 ) focused on associations with lagomorphs , mainly based on their great degree of susceptibility and sensitivity to infection as well as a scenario for the zoogeography of the steppes , which were thought by the former ussr workers to have been a pivotal habitat for tularemia . differences in distribution , ecology , biochemistry , and virulence led to the seminal classification of tularemia into distinct types ( olsufiev et al . type a organisms ( now known as f. tularensis tularensis ) are prevalent in north america but not in eurasia , are frequently transmitted by ticks , and may cause severe disease . type b ( f. tularensis holarctica ) causes episodic outbreaks ( epizootics ) in beavers , muskrats , and arvicoline rodents in either north america or eurasia , may be isolated from water or soil , and may cause a milder disease ( jellison et al . the perception that tularemia was due to lagomorphs was largely the influence of francis himself and also of william jellison of the rocky mountain laboratories , who compiled and interpreted the existing literature on tularemia biology in a seminal monograph ( jellison , 1974 ) . cottontail rabbits are very susceptible to infection by type a , dying within 7 days , and are large enough animals to attract attention when there is an epizootic , making them good sentinels for transmission activity . whether cottontail rabbits are required for type a brn > 1 remains unproven and requires further study . it may be that the question has been considered resolved due to conflating the requirements for maintenance with the proximal determinants for human exposure . the concept of rabbits as central to type a ecology was bolstered by the identification of the rabbit tick , haemaphysalis leporispalustris , as an effective enzootic vector ( parker 1934 ) . these ticks transmit f. tularensis and pass the agent by inheritance ( transovarial transmission ; parker 1934 ) ; presumed type a isolates were made from field collected h. leporispalustris ( philip and parker , 1938 ) . thus , rabbits and rabbit ticks could serve to powerfully maintain type a. in addition , because subadults ( larvae and nymphs ) of this tick will infest ground - inhabiting birds , including those that migrate , the agent could be readily transported . this fact begs the question : why has f. tularensis not been detected in latin america south of mexico , particularly given the presence of rabbits throughout south america ? the central importance of rabbits and their ticks in type a ecology is not supported by recent studies on martha 's vineyard ( mv ) . the nature of the fomites that served to infect these case - patients remains undescribed but unseen remnants of animal carcasses , animal feces , urine - soaked soil , ticks , tick feces , fleas , and contaminated water are possible sources . it is possible that the heavily salt - spray influenced landscape of the ocean facing southern edge of mv is more conducive to the agent remaining viable for a longer duration than elsewhere in the us ( berrada and telford , in press ) . ecological studies , extended from lyme disease surveillance starting in 1994 , quickly suggested that dog ticks ( d. variabilis ) were important to type a perpetuation ( goethert et al . rabbits were indeed infected but virtually disappeared on mv , probably due to tularemia mortality ; their disappearance did not influence the force of transmission of type a , which continued to remain prevalent in dog ticks . evidence of tularemia had never been detected in more than 200 rabbits sampled on nantucket , even though five human cases had been identified from 1990 to 2005 . this event demonstrated that even though type a had been introduced at least once to nantucket , despite the presence of dense rabbits and heavy h. leporispalustris infestations , the agent did not perpetuate . there is one important difference in the ecology of nantucket relative to mv : nantucket lacks appreciable numbers of dog ticks ( indeed , the tick may now be extirpated from that island due to the recent widespread use of topical anti - ectoparasiticides on dogs ) because it does not have their reproductive hosts , skunks , raccoons , foxes , or coyotes . we suspect that rabbits are not necessarily critical to the brn of type a , or if they are , it is a function of local conditions . this dependence on local conditions , in fact , is the challenge of developing a quantitative model for the brn of tularemia : should there be a general model , or should we approach the subject as did the researchers of the former ussr , focusing on independent natural foci ? at one extreme , the 4 genotypes / subclades of type a and 11 of type b ( kugeler et al . but , a general model would have to assume that all elements for the ecology of type a would apply to type b and vice versa , not necessarily a good assumption . although our hypothesis is that dog ticks are critical to brn on mv , experimentally infected as well as naturally infected ticks die more quickly than do those that are not ( reese et al . these findings stand in contrast to our empirical observation of infected ticks each year , and suggest that there may be factors that mitigate the negative fitness of infection in nature . tularemia in eurasia and non - rabbit or tick associated infection in north america seem to have a strong environmental basis , acquired from agricultural activities such as hay threshing ; from water contaminated by muskrats or water voles ; or during the trapping of furbearers ( pavlovsky , 1966 ; syrjala et al . in addition , transmission of the pathogen could occur via contamination of foodstuffs by urine or fecal material from infectious rodents ( karpoff and antonoff , 1936 ; parker et al . experimental studies with voles suggested the possibility that some f. tularensis - infected animals developed a chronic nephritis and bacteriuria that could serve as a protracted source of environmental contamination ( bell and stewart , 1975 ) . of particular interest was the suggestion that voles became partially immune due to low level infection resulting from cannibalism of tularemic carcasses and that this immunity allowed survival of the vole during subsequent infection , increasing the probability for shedding in the excreta . ( cannibalism of moribund cagemates is well known as a mode of transmission for type a in the laboratory , owen and buker , 1956 , and could be a complementary factor in perpetuation . ) this suggestion of orally induced immunity has not been explored further but if confirmed could be a critical factor for the brn of type b , particularly in the context of environmental persistence . ticks may be infected by type b and are said to be the reservoir ( pavlovsky , 1966 ) . in the former soviet union , 17 species of ixodid ticks have been found to be naturally infected ( balashov , 1972 ) , presumably by type b inasmuch as type a is virtually restricted to north america . in addition , type b is well known to be tick - transmitted in north america and both types may be present in ticks in the same site ( markowitz et al . dermacentor marginatus and dermacentor reticulatus appear to be the main vectors there as well as into central europe . as with type a , type b - infected d. marginatus or d. variabilis die more rapidly than do uninfected ticks ( petrov , 1960 ; reese et al . mosquitoes are strongly implicated as vectors in sweden , given that tularemic ulcers are most frequently found on the upper back , neck , and ears of case - patients ( eliasson and back , 2007 ) , where mosquitoes are more likely to feed . in addition , the agent has been isolated from mosquitoes there ( olin , 1942 ) and recent studies provide evidence for mosquito larvae acquiring infection from water , perhaps by the ingestion of predatory protozoa ( mathisen et al . , gis studies of the orebro endemic area in sweden demonstrate that there is temporal spatial association of incidence with mosquito breeding ( svensson et al . one recent study reported a third of mosquito pools to be infected in alaska ( triebenbach et al . older studies in alaska failed to isolate f. tularensis from mosquitoes ( hopla , 1974 ) and thus it is not clear what the pcr findings represent ; it should be noted that the assay that was used might also detect francisella novicida . but , the duration of mosquito infectivity would differ depending on whether it was mechanical or biological transmission , thereby impacting the magnitude of brn . in addition , the possibility that mosquito larvae may acquire infection from water might greatly enhance brn if those larvae became adult females that transmitted . such questions have more than just academic interest : if ticks and rodents drive the ecology of type b , then intervention could be considered to reduce risk , for example , by rodent or tick control . , with vaccination ) given the difficulty of manipulating water ecosystems . both type a and type b are highly infectious and may be transmitted mechanically on the mouthparts of various hematophagous arthropods , or by contact with body or tissue fluids through abraded and even intact skin , and by ingestion , in addition to true biological ( vector ) transmission . such a wide spectrum of modes of exposure and great infectivity helps to explain the wide range of kinds of animals known to be exposed or infected ( burroughs et al . it is likely that most of these animals do not serve as amplifying hosts ( reservoirs ) that increase the brn of the agent of tularemia , but are incidental many of the reports of an animal serving as host simply document exposure ( seroreactivity ) in a limited sample which does not allow inference about whether the exposure might be common over many sampling periods or among many sites . thorough search of any site should document the presence of f. tularensis . tick surveys in known endemic sites generally record prevalence of f. tularensis infection in the range of 0.15% ( e.g. tbe is considered to be a common tick borne infection , with 100010,000 new cases each year ( paddock and telford , in press ) . f. tularensis has been detected in i. ricinus , the main european vector of tbe . becomes even more vexing given the possibility of environmental persistence , that is , perpetuation that may be independent of vertebrate hosts . surface water and sediment yielded indisputable dna sequence evidence of contamination with type b in swedish endemic sites , even in years with little epidemiological activity ( broman et al . experimental microcosm experiments demonstrated that f. tularensis in silt could infect animals for 2 months . about half of rodents immersed in contaminated water became infected with exposure to as few as 1001000 cfu / ml ( pavlovsky , 1966 ) , which appears to be large dose but the spleen alone of a mouse dying of tularemia may have 10 cfu ( molins et al . however , water invertebrates such as shrimp or snails could retain viable organisms for 20 days ( mironchuk and mazepa , 2002 ) , and indeed , invertebrates were first described as contributing to f. tularensis survival within water by former ussr research ( pavlovsky , 1966 ) . the ecology of tularemia is sufficiently complex that limited resources for research should be targeted to the most relevant questions . developing quantitative models of the brn for tularemia , probably separately for type a and type b , would appear to be a priority because such models provide structure for observations . critical life cycle breakpoints and influential variables can be hypothesized a priori and tested by field observations . surveys should be undertaken to identify useful study sites where the agent is reliably detected and longitudinal observations ( on a scale of years ) may be undertaken . we have outlined some of the more interesting lacunae in our knowledge of tularemia ecology , and additional information for all of these would greatly enhance iterative refinements of brn quantitative models in the future .

paramphistomosis has been an ignored trematode infectious disease in ruminants however , it is distributed cosmopolitan and has appeared as an important cause of productivity loss ( 1 ) . its economic loss may be greater than those caused by many other parasites ( 2 ) . death due to immature paramphistomes is very high , and may be as high as 8090% in domesticated ruminants ( 3 , 4 ) . paramphistomosisis caused by specific species of the parasite depending on the regions ( 1 ) . in egypt , paramphistomum microbothrium is a standout amongst the vast majority species of paramphistomes ( 5 ) . adult paramphistomes are the main parasites in the rumen and reticulum of sheep , goats , cattle and water buffaloes . although treatment for adult fluke has no direct benefit to the animal , it may reduce the source of infection for the snail intermediate host . this then reduces the size of the next generation of infective fluke larvae on pasture . desert date , a member of the family zygophyllaceae , is one of the most common but neglected wild plant species of the dry land areas of africa and south asia ( 6 ) . b. aegyptiaca fruits are commonly used to purge intestinal parasites , and have been found to be effective against fasciola gigantica , schistosoma japonicum ( 7 ) , trichinella spiralis ( 8) and toxocara vitulorum ( 9 ) . the fruit mesocarps are used for control of fresh water snails that act as intermediate host of bilharzia ( 10 ) and of water flea that acts as an alternate host of the guinea worm ( 6 ) . the methanolic extract of b. aegyptiaca revealed potent larvicidal effects against aedes aegypti mosquito larvae ( 11 ) . these findings motivated this study in order to assess the in vitro effects of methanolic extract of b. aegyptiaca fruits on attachment organs and tegument of adult p. microbothrium ; as the integrity of the trematode tegument is essential for nutritive , immunoprotective and osmoregulatory functions ( 12 ) . the methanolic extract of b. aegyptiaca fruits was obtained as described previously ( 8) . it was concentrated under reduced pressure on a rotatory evaporator , and then dissolved in a vehicle mixture of liquid paraffin and tween 80 ( v / v ) to obtain a 10% liquid extract . adult worms of p. microbothrium were collected from the rumen of cattle slaughtered in a cairo abattoir , and were identified according to the method of sey and abdel - rahman ( 13 ) . under sterile conditions in a laminar flow cabinet , worms were washed in several changes of warm ( 37c ) , sterile complete rpmi 1640 culture medium containing antibiotics ( penicillin , 50 iu / ml ; streptomycin , 50 mg / ml ) . they were subsequently transferred to fresh culture medium containing 50% ( v / v ) heat denatured rabbit serum ; 2% ( v / v ) rabbit red blood cells , as recommended by ibarra and jenkins ( 14 ) ; and bae at four different concentrations ; 10 , 50 , 100 and 200 g / ml . then the whole worms were incubated for 24 h at 37c in an atmosphere of 5% co 2 . following incubation , adult worms of p. microbothrium were cut into small , 5-mm pieces before being fixed in 10% buffered formol saline . after dehydration , samples were embedded in paraffin and sectioned at 46 m . sections were stained with hematoxylin and eosin according to the method of bancroft et al . the tegument of adult flukes was studied and photographed using an olympus cx41 microscope . following incubation , adult worms of p. microbothrium were fixed intact for 12 h in a 3:1 mixture of 4% ( w / v ) glutaraldehyde in 0.12 m millonig s buffer , ph 7.4 and 1% aqueous osmium tetroxide . after this , the specimens were processed for sem following a method previously reported ( 16 ) . the methanolic extract of b. aegyptiaca fruits was obtained as described previously ( 8) . it was concentrated under reduced pressure on a rotatory evaporator , and then dissolved in a vehicle mixture of liquid paraffin and tween 80 ( v / v ) to obtain a 10% liquid extract . adult worms of p. microbothrium were collected from the rumen of cattle slaughtered in a cairo abattoir , and were identified according to the method of sey and abdel - rahman ( 13 ) . under sterile conditions in a laminar flow cabinet , worms were washed in several changes of warm ( 37c ) , sterile complete rpmi 1640 culture medium containing antibiotics ( penicillin , 50 iu / ml ; streptomycin , 50 mg / ml ) . they were subsequently transferred to fresh culture medium containing 50% ( v / v ) heat denatured rabbit serum ; 2% ( v / v ) rabbit red blood cells , as recommended by ibarra and jenkins ( 14 ) ; and bae at four different concentrations ; 10 , 50 , 100 and 200 g / ml . then the whole worms were incubated for 24 h at 37c in an atmosphere of 5% co 2 . solvent control worms were incubated for 24 h in rpmi1640 culture medium containing 0.2 % ( v / v ) mixture of liquid paraffin and tween 80 . following incubation , adult worms of p. microbothrium were cut into small , 5-mm pieces before being fixed in 10% buffered formol saline . after dehydration sections were stained with hematoxylin and eosin according to the method of bancroft et al . following incubation , adult worms of p. microbothrium were fixed intact for 12 h in a 3:1 mixture of 4% ( w / v ) glutaraldehyde in 0.12 m millonig s buffer , ph 7.4 and 1% aqueous osmium tetroxide . after this , the specimens were processed for sem following a method previously reported ( 16 ) . it rested on layer of connective tissue called basal lamina , which connected the former to the underlying and deeply stained two muscular layers . the interior of the parasite was filled with the parenchyma composed of loose connective tissues and parenchymal cells ( fig.1a , b ) . no significant differences were observed between fresh and control flukes incubated for 24 h in solvent ( 0.2 % ( v / v ) mixture of liquid paraffin and tween 80 ) . ( c ) following 24 h incubation with 10 g / ml b. aegyptiaca extract ( bae ) . t tegument , bl basal lamina , m muscular layer , tc tegumental cell , p parenchyma flukes incubated in 10 g / ml bae for 24 h revealed only relatively slight swelling of tegument accompanied with corrugated appearance of its surface ( fig.1c ) . this swelling became pronounced , on increasing the concentration to 50 g / ml ( fig.1d ) , and appeared so severe , with folded tegumental surface and blebbing on increasing the concentration to 100 g / ml ( fig.1e ) . the strongest bae effects were reached with concentration of 200 g / ml ( fig.1f ) , where the treated flukes showed severe tegumental disruption , patches of tegument had been completely removed , exposing the basal lamina and severe damage was observed in muscle bundles and parenchymal tissues . adult p. microbothrium is pear - shaped with a broadly rounded posterior end and narrower anterior end . the oral aperture is terminally positioned surrounded by the oral sucker , and the acetabulum is subterminally positioned ( fig.2a ) . the anterior third of the body has clearly visible concentric aggregations of dome - shaped papillae ( fig.2b , inset ) . these papillae are very densely arranged towards the oral end , but decrease gradually toward the middle of the body before disappearance . around the acetabular aperture , the tegument has folds irregularly directed with prominent domed papillae arranged singly or in groups of a few between the folds . the tegument covering the body is transversely folded or ridged ( fig.2a , inset ) , the folds being closer and more numerous near the anterior end , decreasing in number posteriorly . no significant differences were observed between fresh and control flukes incubated for 24 h in solvent ( 0.2 % ( v / v ) mixture of liquid paraffin and tween 80 ) . ( a ) sem of entire fluke showing broadly rounded posterior end and narrower anterior end . the tegument shows folds or ridges , closer to each other near the anterior end of the fluke ( inset ) . ( b ) sem of anterior third of the body showing concentric aggregations of dome - shaped papillae ( inset ) arranged towards the oral end . ( c f ) following 24 h incubation with 10 g / ml bae . the anterior third and the mid - body regions of the fluke s body show slightly swollen tegument and papillae , with a few blebs covering their surfaces . os oral sucker , a acetabulum , p papilla after 24 h of incubation with 10 g / ml bae , the changes in adult flukes concerned the anterior end other than the posterior one , which retained its normal morphology ( fig.2c ) . at the anterior third of the fluke s body , the tegument appeared to be slightly more swollen than normal with contractions to sucker s opening ( fig.2d ) . the papillae surrounding the oral aperture were slightly swollen with a few blebs covering their surfaces ( fig.2e ) . swelling was also present towards the mid - body region of the fluke ( fig.2f ) . after 24 h of incubation with 50 g / ml bae , the tegumental alterations were similar to that described for 10 g / ml concentration , except that the blebbing became more pronounced ( fig.3a d ) . the papillae surrounding the oral aperture exhibited severe blebbing in the form of small bulbous structures at their surfaces ; some of them were disrupted causing a number of pits on the tegumental surface ( fig.3c ) . besides , a few large blebs extended towards the mid - body region of the fluke ( fig.3 d ) . the degrees of tegumental changes became more severe , on increasing the concentration of bae to 100 g / ml . both oral sucker and acetabulum were distorted in most of examined specimens ( fig.4a , b ) . the tegumental surface around the oral sucker exhibited severe blebbing on top of papillae ( fig.4c ) . there were wide and deep furrows between the transverse folds ; because of tegumental swelling ( fig.4 d ) . sems of adult p. microbothrium following 24 h incubation with 50 g / ml bae . ( a c ) sems of anterior and posterior ends reveal severe blebbing surrounding the oral aperture ( white arrow ) , and a number of pits resulted from disruption of some blebs ( black arrow ) . ( d ) sem of the mid - body region showing a few large blebs ( white arrow ) . os oral sucker , a acetabulum , p papilla sems of adult p. microbothrium following 24 h incubation with 100 g / ml bae . ( c ) sem of the tegumental surface showing severe blebbing on top of papillae ( arrow ) . ( d ) sem of the tegument showing wide and deep furrows between the transverse folds . os oral sucker , a acetabulum , p papilla with 200 g / ml bae , severe tegumental disruption was evident in all regions of the fluke ( fig.5a ) in the majority of examined specimens . the oral sucker was retracted and deformed , and the concentric tegumental rings encircling the oral aperture were no longer seen , but were substituted by tegumental ridges ( fig.5b ) . the tegument at the anterior third of the fluke s body was more swollen so that the papillae appeared to be submerged by the tegument , which gave the surface a smooth appearance . some areas of the tegument were characterized by a number of pits caused by rupture of papillae ( fig.5b , inset ) . in the mid - body region , severe damage was apparent in the form of focal erosions of the surface ; resulted from rupture of blebs ( fig.5 c ) . this disruption was more severe in the acetabular region , with large areas of tegument were completely removed ; exposing the basal lamina ( fig.5d ) . in this region , several flukes displayed a large swelling that was clearly visible to the naked eye . one fluke , however , displayed extreme disruption with a large hole that had penetrated completely through the tegument ( fig.5 a ) . sems of adult p. microbothrium following 24 h incubation with 200 g / ml bae . ( a ) entire worm revealing tegumental disruption in all regions of the fluke , with a large hole penetrated completely through the tegument ( arrow ) . ( b ) tegumental swelling at the anterior third of the fluke s body so that the papillae appeared to be submerged by the tegument ( arrow ) . inset shows a number of pits caused by rupture of papillae ( head arrow ) . ( c ) focal erosions of the surface resulted from rupture of blebs ( arrow ) . ( d ) large area of the tegument , at the acetabular region , is completely removed exposing the basal lamina ( arrow ) . it rested on layer of connective tissue called basal lamina , which connected the former to the underlying and deeply stained two muscular layers . the interior of the parasite was filled with the parenchyma composed of loose connective tissues and parenchymal cells ( fig.1a , b ) . no significant differences were observed between fresh and control flukes incubated for 24 h in solvent ( 0.2 % ( v / v ) mixture of liquid paraffin and tween 80 ) . ( c ) following 24 h incubation with 10 g / ml b. aegyptiaca extract ( bae ) . t tegument , bl basal lamina , m muscular layer , tc tegumental cell , p parenchyma flukes incubated in 10 g / ml bae for 24 h revealed only relatively slight swelling of tegument accompanied with corrugated appearance of its surface ( fig.1c ) . this swelling became pronounced , on increasing the concentration to 50 g / ml ( fig.1d ) , and appeared so severe , with folded tegumental surface and blebbing on increasing the concentration to 100 g / ml ( fig.1e ) . the strongest bae effects were reached with concentration of 200 g / ml ( fig.1f ) , where the treated flukes showed severe tegumental disruption , patches of tegument had been completely removed , exposing the basal lamina and severe damage was observed in muscle bundles and parenchymal tissues . it rested on layer of connective tissue called basal lamina , which connected the former to the underlying and deeply stained two muscular layers . the interior of the parasite was filled with the parenchyma composed of loose connective tissues and parenchymal cells ( fig.1a , b ) . no significant differences were observed between fresh and control flukes incubated for 24 h in solvent ( 0.2 % ( v / v ) mixture of liquid paraffin and tween 80 ) . ( c ) following 24 h incubation with 10 g / ml b. aegyptiaca extract ( bae ) . t tegument , bl basal lamina , m muscular layer , tc tegumental cell , p parenchyma flukes incubated in 10 g / ml bae for 24 h revealed only relatively slight swelling of tegument accompanied with corrugated appearance of its surface ( fig.1c ) . this swelling became pronounced , on increasing the concentration to 50 g / ml ( fig.1d ) , and appeared so severe , with folded tegumental surface and blebbing on increasing the concentration to 100 g / ml ( fig.1e ) . the strongest bae effects were reached with concentration of 200 g / ml ( fig.1f ) , where the treated flukes showed severe tegumental disruption , patches of tegument had been completely removed , exposing the basal lamina and severe damage was observed in muscle bundles and parenchymal tissues . adult p. microbothrium is pear - shaped with a broadly rounded posterior end and narrower anterior end . the oral aperture is terminally positioned surrounded by the oral sucker , and the acetabulum is subterminally positioned ( fig.2a ) . the anterior third of the body has clearly visible concentric aggregations of dome - shaped papillae ( fig.2b , inset ) . these papillae are very densely arranged towards the oral end , but decrease gradually toward the middle of the body before disappearance . around the acetabular aperture , the tegument has folds irregularly directed with prominent domed papillae arranged singly or in groups of a few between the folds . the tegument covering the body is transversely folded or ridged ( fig.2a , inset ) , the folds being closer and more numerous near the anterior end , decreasing in number posteriorly . no significant differences were observed between fresh and control flukes incubated for 24 h in solvent ( 0.2 % ( v / v ) mixture of liquid paraffin and tween 80 ) . scanning electron micrographs ( sems ) of adult p. microbothrium . ( a ) sem of entire fluke showing broadly rounded posterior end and narrower anterior end . the tegument shows folds or ridges , closer to each other near the anterior end of the fluke ( inset ) . ( b ) sem of anterior third of the body showing concentric aggregations of dome - shaped papillae ( inset ) arranged towards the oral end . ( c f ) following 24 h incubation with 10 g / ml bae . the anterior third and the mid - body regions of the fluke s body show slightly swollen tegument and papillae , with a few blebs covering their surfaces . os oral sucker , a acetabulum , p papilla after 24 h of incubation with 10 g / ml bae , the changes in adult flukes concerned the anterior end other than the posterior one , which retained its normal morphology ( fig.2c ) . at the anterior third of the fluke s body , the tegument appeared to be slightly more swollen than normal with contractions to sucker s opening ( fig.2d ) . the papillae surrounding the oral aperture were slightly swollen with a few blebs covering their surfaces ( fig.2e ) . swelling was also present towards the mid - body region of the fluke ( fig.2f ) . after 24 h of incubation with 50 g / ml bae , the tegumental alterations were similar to that described for 10 g / ml concentration , except that the blebbing became more pronounced ( fig.3a d ) . the papillae surrounding the oral aperture exhibited severe blebbing in the form of small bulbous structures at their surfaces ; some of them were disrupted causing a number of pits on the tegumental surface ( fig.3c ) . besides , a few large blebs extended towards the mid - body region of the fluke ( fig.3 d ) . the degrees of tegumental changes became more severe , on increasing the concentration of bae to 100 g / ml . both oral sucker and acetabulum were distorted in most of examined specimens ( fig.4a , b ) . the tegumental surface around the oral sucker exhibited severe blebbing on top of papillae ( fig.4c ) . there were wide and deep furrows between the transverse folds ; because of tegumental swelling ( fig.4 d ) . sems of adult p. microbothrium following 24 h incubation with 50 g / ml bae . ( a c ) sems of anterior and posterior ends reveal severe blebbing surrounding the oral aperture ( white arrow ) , and a number of pits resulted from disruption of some blebs ( black arrow ) . ( d ) sem of the mid - body region showing a few large blebs ( white arrow ) . os oral sucker , a acetabulum , p papilla sems of adult p. microbothrium following 24 h incubation with 100 g / ml bae . ( c ) sem of the tegumental surface showing severe blebbing on top of papillae ( arrow ) . ( d ) sem of the tegument showing wide and deep furrows between the transverse folds . os oral sucker , a acetabulum , p papilla with 200 g / ml bae , severe tegumental disruption was evident in all regions of the fluke ( fig.5a ) in the majority of examined specimens . the oral sucker was retracted and deformed , and the concentric tegumental rings encircling the oral aperture were no longer seen , but were substituted by tegumental ridges ( fig.5b ) . the tegument at the anterior third of the fluke s body was more swollen so that the papillae appeared to be submerged by the tegument , which gave the surface a smooth appearance . some areas of the tegument were characterized by a number of pits caused by rupture of papillae ( fig.5b , inset ) . in the mid - body region , severe damage was apparent in the form of focal erosions of the surface ; resulted from rupture of blebs ( fig.5 c ) . this disruption was more severe in the acetabular region , with large areas of tegument were completely removed ; exposing the basal lamina ( fig.5d ) . in this region , several flukes displayed a large swelling that was clearly visible to the naked eye . one fluke , however , displayed extreme disruption with a large hole that had penetrated completely through the tegument ( fig.5 a ) . sems of adult p. microbothrium following 24 h incubation with 200 g / ml bae . ( a ) entire worm revealing tegumental disruption in all regions of the fluke , with a large hole penetrated completely through the tegument ( arrow ) . ( b ) tegumental swelling at the anterior third of the fluke s body so that the papillae appeared to be submerged by the tegument ( arrow ) . inset shows a number of pits caused by rupture of papillae ( head arrow ) . ( c ) focal erosions of the surface resulted from rupture of blebs ( arrow ) . ( d ) large area of the tegument , at the acetabular region , is completely removed exposing the basal lamina ( arrow ) . adult p. microbothrium is pear - shaped with a broadly rounded posterior end and narrower anterior end . the oral aperture is terminally positioned surrounded by the oral sucker , and the acetabulum is subterminally positioned ( fig.2a ) . the anterior third of the body has clearly visible concentric aggregations of dome - shaped papillae ( fig.2b , inset ) . these papillae are very densely arranged towards the oral end , but decrease gradually toward the middle of the body before disappearance . around the acetabular aperture , the tegument has folds irregularly directed with prominent domed papillae arranged singly or in groups of a few between the folds . the tegument covering the body is transversely folded or ridged ( fig.2a , inset ) , the folds being closer and more numerous near the anterior end , decreasing in number posteriorly . no significant differences were observed between fresh and control flukes incubated for 24 h in solvent ( 0.2 % ( v / v ) mixture of liquid paraffin and tween 80 ) . scanning electron micrographs ( sems ) of adult p. microbothrium . ( a ) sem of entire fluke showing broadly rounded posterior end and narrower anterior end . the tegument shows folds or ridges , closer to each other near the anterior end of the fluke ( inset ) . ( b ) sem of anterior third of the body showing concentric aggregations of dome - shaped papillae ( inset ) arranged towards the oral end . ( c f ) following 24 h incubation with 10 g / ml bae . the anterior third and the mid - body regions of the fluke s body show slightly swollen tegument and papillae , with a few blebs covering their surfaces . after 24 h of incubation with 10 g / ml bae , the changes in adult flukes concerned the anterior end other than the posterior one , which retained its normal morphology ( fig.2c ) . at the anterior third of the fluke s body , the tegument appeared to be slightly more swollen than normal with contractions to sucker s opening ( fig.2d ) . the papillae surrounding the oral aperture were slightly swollen with a few blebs covering their surfaces ( fig.2e ) . swelling was also present towards the mid - body region of the fluke ( fig.2f ) . after 24 h of incubation with 50 g / ml bae , the tegumental alterations were similar to that described for 10 g / ml concentration , except that the blebbing became more pronounced ( fig.3a d ) . the papillae surrounding the oral aperture exhibited severe blebbing in the form of small bulbous structures at their surfaces ; some of them were disrupted causing a number of pits on the tegumental surface ( fig.3c ) . besides , a few large blebs extended towards the mid - body region of the fluke ( fig.3 d ) . the degrees of tegumental changes became more severe , on increasing the concentration of bae to 100 g / ml . both oral sucker and acetabulum were distorted in most of examined specimens ( fig.4a , b ) . the tegumental surface around the oral sucker exhibited severe blebbing on top of papillae ( fig.4c ) . there were wide and deep furrows between the transverse folds ; because of tegumental swelling ( fig.4 d ) . sems of adult p. microbothrium following 24 h incubation with 50 g / ml bae . ( a c ) sems of anterior and posterior ends reveal severe blebbing surrounding the oral aperture ( white arrow ) , and a number of pits resulted from disruption of some blebs ( black arrow ) . ( d ) sem of the mid - body region showing a few large blebs ( white arrow ) . os oral sucker , a acetabulum , p papilla sems of adult p. microbothrium following 24 h incubation with 100 g / ml bae . ( a , b ) sems revealing distortion of both oral sucker and acetabulum . ( c ) sem of the tegumental surface showing severe blebbing on top of papillae ( arrow ) . ( d ) sem of the tegument showing wide and deep furrows between the transverse folds . os oral sucker , a acetabulum , p papilla with 200 g / ml bae , severe tegumental disruption was evident in all regions of the fluke ( fig.5a ) in the majority of examined specimens . the oral sucker was retracted and deformed , and the concentric tegumental rings encircling the oral aperture were no longer seen , but were substituted by tegumental ridges ( fig.5b ) . the tegument at the anterior third of the fluke s body was more swollen so that the papillae appeared to be submerged by the tegument , which gave the surface a smooth appearance . some areas of the tegument were characterized by a number of pits caused by rupture of papillae ( fig.5b , inset ) . in the mid - body region , severe damage was apparent in the form of focal erosions of the surface ; resulted from rupture of blebs ( fig.5 c ) . this disruption was more severe in the acetabular region , with large areas of tegument were completely removed ; exposing the basal lamina ( fig.5d ) . in this region , several flukes displayed a large swelling that was clearly visible to the naked eye . one fluke , however , displayed extreme disruption with a large hole that had penetrated completely through the tegument ( fig.5 a ) . sems of adult p. microbothrium following 24 h incubation with 200 g / ml bae . ( a ) entire worm revealing tegumental disruption in all regions of the fluke , with a large hole penetrated completely through the tegument ( arrow ) . ( b ) tegumental swelling at the anterior third of the fluke s body so that the papillae appeared to be submerged by the tegument ( arrow ) . inset shows a number of pits caused by rupture of papillae ( head arrow ) . ( c ) focal erosions of the surface resulted from rupture of blebs ( arrow ) . ( d ) large area of the tegument , at the acetabular region , is completely removed exposing the basal lamina ( arrow ) . in this study , we have assessed , for the first time , the effect of bae on adult worms of p. microbothrium . the major target organ that was highly affected was the tegument , whose damages were observed by lm and sem studies . lm was used to observe changes in a limited area of the tegumental syncytium while tegumental surface changes , which reflected the changes in the tegument cytoplasm , could be observed over a much wider area by sem . these changes occurred in definite sequences in response to bae concentration , consisted of swelling , blebbing that was later disrupted , leading to erosion and desquamation of the tegument , resulting in the lesion , and finally the exposure and disruption of basal lamina . maximum anthelmintic activity was found with a dose of 200 g / ml bae , at which distinct damage to the whole body surface of the trematodes was very much distinct . this damage would undoubtedly disrupt many of the physiological processes associated with the tegument , including osmoregulation , protection , secretion and synthesis ( 17 ) . besides , the damage of the tegumental folds of the ace - tabular region might disrupt its function in drawing the rumen wall tissue of the host into the acetabular cavity ( 18 ) resulting in a weak hold for the parasite . surface changes observed in this study resembled that demonstrated on p. explanatum treated with methanolic extracts of leaves of bombax malabricum ( 19 ) and dregea volubilis ( 20 ) . besides , similar sequence of tegumental changes occurred in p. microbothrium treated with artemether ; which was obtained from the leaves of qinghao ( 16 ) as well as p. cervi treated with plumbagin ; a compound that was rich in the roots of plumbago indica / rosea ( 21 ) . moreover , the tegumental surface alterations induced by bae , in the present study , had also been observed in specimens of a biologically related trematode , fasciola gigantica , following incubation with a number of anthelmintics ( 22 ) . surface blebbing was a common feature of drug - treated parasites and had been described for other trematodes and flatworm parasites after exposure to anthelmintics ( 23 ) . it had been suggested that the blebbing occurred because of increased efforts on the part of the parasite to shed and replace outer tegumental membrane damaged by drug action ( 24 ) . it might be significant , then , that the process of blebbing appeared to start at the surfaces of papillae leading to their rupture . the rupture of sensory papillae induced by bae , in the present study , had also been observed following treatment with artemether ( 16 ) and in specimens of a closely related trematode , cotylophoron cotylophorum , treated in vitro with praziquantel , after 30 h of exposure ( 25 ) . the damage that bae caused to the papillae would undoubtedly disrupt many of their sensory functions . the disruption of the tegument became more severe , on increasing bae concentration and then more widespread sloughing occurred . in the highest bae concentration ( 200 g / ml ) , damage to one fluke was so severe that a hole had penetrated through its entire tegument . such damage had only been observed in specimens of fasciola gigantica treated in vitro with the sulphoxide metabolite of triclabendazole ( 23).the literature survey on b. aegyptiaca suggested that methanolic extracts of the plant s tissues had anthelmintic activity ( 9 ) , with saponins being one of the major secondary metabolites in plant tissues ( 26 ) . wiesman and chapagain ( 11 ) reported a strong correlation between the saponin content of methanolic extracts of b. aegyptiaca and mortality of larvae of the mosquito aedes aegypti . ( 27 ) who isolated a steroidal saponin with a high potential of anthelmintic activity , balanitin-7 . their surface - active properties were what distinguished these compounds from other glycosides ( 28 ) . most were haemolytic and toxic to cold - blooded animals , while certain saponins also displayed molluscicidal , anti - inflammatory , anti - fungal , anti - bacterial , anti - parasitic and anti - viral activities . numerous reports had also highlighted the highly cytotoxic properties of many saponins ( 28 ) . therefore , it might be speculated that phytochemicals , most probably saponins , found in bae might be responsible for its potent anthelmintic activity . the surface changes induced by this plant in vitro may serve to illustrate why it is so effective against a biologically related trematode , f. gigantica in vivo . the use of methanolic extract of b. aegyptiaca fruits offers a new dimension and potential for control of such a neglected infectious disease in ruminants , at a time when paramphistomosis has emerged as an important cause of productivity loss .

background : weak efficacy of different fasciolicidal compounds used for treatment of paramphistomosis has drawn the attention of many authors to alternative drugs . the purpose of this study was to assess , for the first time , the effect of the methanolic extract of balanites aegyptiaca fruits ( bae ) on adult paramphistomum microbothrium.methods:the effect of bae on adult p. microbothrium after 24 h incubating the parasites in rpmi 1640 culture medium containing 10 , 50 , 100 and 200 g / ml bae was determined by light and scanning electron microscopic studies.results:differences in response to bae action were concentration dependent.the major target organ that was highly affected was the tegument . maximum anthelmintic activity was found with a dose of 200 g / ml bae , at which distinct damage to the whole body surface of the trematodes was very much distinct . shape and structure of both suckers were deformed due to bae . this damage would undoubtedly disrupt many of the physiological processes associated with the tegument . besides , the damage of the tegumental folds of the acetabular region might disrupt its function in drawing the rumen wall tissue of the host into the acetabular cavity.conclusion:the use of methanolic extract of b. aegyptiaca fruits offers a new dimension and potential for control of such a neglected infectious disease in ruminants , at a time when paramphistomosis has emerged as an important cause of productivity loss .

Introduction Materials and Methods Plant extract Effect of B. aegyptiaca extract (BAE) on adult P. microbothrium Light microscopy Scanning electron microscopy (SEM) Results Light microscopic observations of the tegument cross section of adult P. microbothrium Normal fresh and control flukes Treated flukes Scanning electron microscopic observations of the adult flukes Normal fresh and control flukes Treated flukes Discussion Conclusion

paramphistomosis has been an ignored trematode infectious disease in ruminants however , it is distributed cosmopolitan and has appeared as an important cause of productivity loss ( 1 ) . adult paramphistomes are the main parasites in the rumen and reticulum of sheep , goats , cattle and water buffaloes . b. aegyptiaca fruits are commonly used to purge intestinal parasites , and have been found to be effective against fasciola gigantica , schistosoma japonicum ( 7 ) , trichinella spiralis ( 8) and toxocara vitulorum ( 9 ) . the fruit mesocarps are used for control of fresh water snails that act as intermediate host of bilharzia ( 10 ) and of water flea that acts as an alternate host of the guinea worm ( 6 ) . the methanolic extract of b. aegyptiaca revealed potent larvicidal effects against aedes aegypti mosquito larvae ( 11 ) . these findings motivated this study in order to assess the in vitro effects of methanolic extract of b. aegyptiaca fruits on attachment organs and tegument of adult p. microbothrium ; as the integrity of the trematode tegument is essential for nutritive , immunoprotective and osmoregulatory functions ( 12 ) . the methanolic extract of b. aegyptiaca fruits was obtained as described previously ( 8) . adult worms of p. microbothrium were collected from the rumen of cattle slaughtered in a cairo abattoir , and were identified according to the method of sey and abdel - rahman ( 13 ) . under sterile conditions in a laminar flow cabinet , worms were washed in several changes of warm ( 37c ) , sterile complete rpmi 1640 culture medium containing antibiotics ( penicillin , 50 iu / ml ; streptomycin , 50 mg / ml ) . they were subsequently transferred to fresh culture medium containing 50% ( v / v ) heat denatured rabbit serum ; 2% ( v / v ) rabbit red blood cells , as recommended by ibarra and jenkins ( 14 ) ; and bae at four different concentrations ; 10 , 50 , 100 and 200 g / ml . then the whole worms were incubated for 24 h at 37c in an atmosphere of 5% co 2 . the methanolic extract of b. aegyptiaca fruits was obtained as described previously ( 8) . adult worms of p. microbothrium were collected from the rumen of cattle slaughtered in a cairo abattoir , and were identified according to the method of sey and abdel - rahman ( 13 ) . under sterile conditions in a laminar flow cabinet , worms were washed in several changes of warm ( 37c ) , sterile complete rpmi 1640 culture medium containing antibiotics ( penicillin , 50 iu / ml ; streptomycin , 50 mg / ml ) . they were subsequently transferred to fresh culture medium containing 50% ( v / v ) heat denatured rabbit serum ; 2% ( v / v ) rabbit red blood cells , as recommended by ibarra and jenkins ( 14 ) ; and bae at four different concentrations ; 10 , 50 , 100 and 200 g / ml . solvent control worms were incubated for 24 h in rpmi1640 culture medium containing 0.2 % ( v / v ) mixture of liquid paraffin and tween 80 . the interior of the parasite was filled with the parenchyma composed of loose connective tissues and parenchymal cells ( fig.1a , b ) . ( c ) following 24 h incubation with 10 g / ml b. aegyptiaca extract ( bae ) . t tegument , bl basal lamina , m muscular layer , tc tegumental cell , p parenchyma flukes incubated in 10 g / ml bae for 24 h revealed only relatively slight swelling of tegument accompanied with corrugated appearance of its surface ( fig.1c ) . this swelling became pronounced , on increasing the concentration to 50 g / ml ( fig.1d ) , and appeared so severe , with folded tegumental surface and blebbing on increasing the concentration to 100 g / ml ( fig.1e ) . the strongest bae effects were reached with concentration of 200 g / ml ( fig.1f ) , where the treated flukes showed severe tegumental disruption , patches of tegument had been completely removed , exposing the basal lamina and severe damage was observed in muscle bundles and parenchymal tissues . adult p. microbothrium is pear - shaped with a broadly rounded posterior end and narrower anterior end . around the acetabular aperture , the tegument has folds irregularly directed with prominent domed papillae arranged singly or in groups of a few between the folds . the tegument covering the body is transversely folded or ridged ( fig.2a , inset ) , the folds being closer and more numerous near the anterior end , decreasing in number posteriorly . ( c f ) following 24 h incubation with 10 g / ml bae . the anterior third and the mid - body regions of the fluke s body show slightly swollen tegument and papillae , with a few blebs covering their surfaces . os oral sucker , a acetabulum , p papilla after 24 h of incubation with 10 g / ml bae , the changes in adult flukes concerned the anterior end other than the posterior one , which retained its normal morphology ( fig.2c ) . at the anterior third of the fluke s body , the tegument appeared to be slightly more swollen than normal with contractions to sucker s opening ( fig.2d ) . after 24 h of incubation with 50 g / ml bae , the tegumental alterations were similar to that described for 10 g / ml concentration , except that the blebbing became more pronounced ( fig.3a d ) . the degrees of tegumental changes became more severe , on increasing the concentration of bae to 100 g / ml . sems of adult p. microbothrium following 24 h incubation with 50 g / ml bae . os oral sucker , a acetabulum , p papilla sems of adult p. microbothrium following 24 h incubation with 100 g / ml bae . ( c ) sem of the tegumental surface showing severe blebbing on top of papillae ( arrow ) . ( d ) sem of the tegument showing wide and deep furrows between the transverse folds . os oral sucker , a acetabulum , p papilla with 200 g / ml bae , severe tegumental disruption was evident in all regions of the fluke ( fig.5a ) in the majority of examined specimens . the tegument at the anterior third of the fluke s body was more swollen so that the papillae appeared to be submerged by the tegument , which gave the surface a smooth appearance . some areas of the tegument were characterized by a number of pits caused by rupture of papillae ( fig.5b , inset ) . this disruption was more severe in the acetabular region , with large areas of tegument were completely removed ; exposing the basal lamina ( fig.5d ) . in this region , several flukes displayed a large swelling that was clearly visible to the naked eye . sems of adult p. microbothrium following 24 h incubation with 200 g / ml bae . ( a ) entire worm revealing tegumental disruption in all regions of the fluke , with a large hole penetrated completely through the tegument ( arrow ) . ( d ) large area of the tegument , at the acetabular region , is completely removed exposing the basal lamina ( arrow ) . ( c ) following 24 h incubation with 10 g / ml b. aegyptiaca extract ( bae ) . t tegument , bl basal lamina , m muscular layer , tc tegumental cell , p parenchyma flukes incubated in 10 g / ml bae for 24 h revealed only relatively slight swelling of tegument accompanied with corrugated appearance of its surface ( fig.1c ) . the strongest bae effects were reached with concentration of 200 g / ml ( fig.1f ) , where the treated flukes showed severe tegumental disruption , patches of tegument had been completely removed , exposing the basal lamina and severe damage was observed in muscle bundles and parenchymal tissues . the interior of the parasite was filled with the parenchyma composed of loose connective tissues and parenchymal cells ( fig.1a , b ) . ( c ) following 24 h incubation with 10 g / ml b. aegyptiaca extract ( bae ) . t tegument , bl basal lamina , m muscular layer , tc tegumental cell , p parenchyma flukes incubated in 10 g / ml bae for 24 h revealed only relatively slight swelling of tegument accompanied with corrugated appearance of its surface ( fig.1c ) . the strongest bae effects were reached with concentration of 200 g / ml ( fig.1f ) , where the treated flukes showed severe tegumental disruption , patches of tegument had been completely removed , exposing the basal lamina and severe damage was observed in muscle bundles and parenchymal tissues . adult p. microbothrium is pear - shaped with a broadly rounded posterior end and narrower anterior end . around the acetabular aperture , the tegument has folds irregularly directed with prominent domed papillae arranged singly or in groups of a few between the folds . the tegument covering the body is transversely folded or ridged ( fig.2a , inset ) , the folds being closer and more numerous near the anterior end , decreasing in number posteriorly . scanning electron micrographs ( sems ) of adult p. microbothrium . the tegument shows folds or ridges , closer to each other near the anterior end of the fluke ( inset ) . ( c f ) following 24 h incubation with 10 g / ml bae . the anterior third and the mid - body regions of the fluke s body show slightly swollen tegument and papillae , with a few blebs covering their surfaces . os oral sucker , a acetabulum , p papilla after 24 h of incubation with 10 g / ml bae , the changes in adult flukes concerned the anterior end other than the posterior one , which retained its normal morphology ( fig.2c ) . at the anterior third of the fluke s body , the tegument appeared to be slightly more swollen than normal with contractions to sucker s opening ( fig.2d ) . after 24 h of incubation with 50 g / ml bae , the tegumental alterations were similar to that described for 10 g / ml concentration , except that the blebbing became more pronounced ( fig.3a d ) . besides , a few large blebs extended towards the mid - body region of the fluke ( fig.3 d ) . the degrees of tegumental changes became more severe , on increasing the concentration of bae to 100 g / ml . sems of adult p. microbothrium following 24 h incubation with 50 g / ml bae . os oral sucker , a acetabulum , p papilla sems of adult p. microbothrium following 24 h incubation with 100 g / ml bae . ( c ) sem of the tegumental surface showing severe blebbing on top of papillae ( arrow ) . os oral sucker , a acetabulum , p papilla with 200 g / ml bae , severe tegumental disruption was evident in all regions of the fluke ( fig.5a ) in the majority of examined specimens . some areas of the tegument were characterized by a number of pits caused by rupture of papillae ( fig.5b , inset ) . this disruption was more severe in the acetabular region , with large areas of tegument were completely removed ; exposing the basal lamina ( fig.5d ) . in this region , several flukes displayed a large swelling that was clearly visible to the naked eye . one fluke , however , displayed extreme disruption with a large hole that had penetrated completely through the tegument ( fig.5 a ) . sems of adult p. microbothrium following 24 h incubation with 200 g / ml bae . ( a ) entire worm revealing tegumental disruption in all regions of the fluke , with a large hole penetrated completely through the tegument ( arrow ) . ( d ) large area of the tegument , at the acetabular region , is completely removed exposing the basal lamina ( arrow ) . adult p. microbothrium is pear - shaped with a broadly rounded posterior end and narrower anterior end . around the acetabular aperture , the tegument has folds irregularly directed with prominent domed papillae arranged singly or in groups of a few between the folds . the tegument covering the body is transversely folded or ridged ( fig.2a , inset ) , the folds being closer and more numerous near the anterior end , decreasing in number posteriorly . scanning electron micrographs ( sems ) of adult p. microbothrium . ( c f ) following 24 h incubation with 10 g / ml bae . the anterior third and the mid - body regions of the fluke s body show slightly swollen tegument and papillae , with a few blebs covering their surfaces . after 24 h of incubation with 10 g / ml bae , the changes in adult flukes concerned the anterior end other than the posterior one , which retained its normal morphology ( fig.2c ) . at the anterior third of the fluke s body , the tegument appeared to be slightly more swollen than normal with contractions to sucker s opening ( fig.2d ) . after 24 h of incubation with 50 g / ml bae , the tegumental alterations were similar to that described for 10 g / ml concentration , except that the blebbing became more pronounced ( fig.3a d ) . the degrees of tegumental changes became more severe , on increasing the concentration of bae to 100 g / ml . sems of adult p. microbothrium following 24 h incubation with 50 g / ml bae . os oral sucker , a acetabulum , p papilla sems of adult p. microbothrium following 24 h incubation with 100 g / ml bae . ( d ) sem of the tegument showing wide and deep furrows between the transverse folds . os oral sucker , a acetabulum , p papilla with 200 g / ml bae , severe tegumental disruption was evident in all regions of the fluke ( fig.5a ) in the majority of examined specimens . the tegument at the anterior third of the fluke s body was more swollen so that the papillae appeared to be submerged by the tegument , which gave the surface a smooth appearance . some areas of the tegument were characterized by a number of pits caused by rupture of papillae ( fig.5b , inset ) . this disruption was more severe in the acetabular region , with large areas of tegument were completely removed ; exposing the basal lamina ( fig.5d ) . one fluke , however , displayed extreme disruption with a large hole that had penetrated completely through the tegument ( fig.5 a ) . sems of adult p. microbothrium following 24 h incubation with 200 g / ml bae . ( a ) entire worm revealing tegumental disruption in all regions of the fluke , with a large hole penetrated completely through the tegument ( arrow ) . ( d ) large area of the tegument , at the acetabular region , is completely removed exposing the basal lamina ( arrow ) . in this study , we have assessed , for the first time , the effect of bae on adult worms of p. microbothrium . the major target organ that was highly affected was the tegument , whose damages were observed by lm and sem studies . lm was used to observe changes in a limited area of the tegumental syncytium while tegumental surface changes , which reflected the changes in the tegument cytoplasm , could be observed over a much wider area by sem . these changes occurred in definite sequences in response to bae concentration , consisted of swelling , blebbing that was later disrupted , leading to erosion and desquamation of the tegument , resulting in the lesion , and finally the exposure and disruption of basal lamina . maximum anthelmintic activity was found with a dose of 200 g / ml bae , at which distinct damage to the whole body surface of the trematodes was very much distinct . this damage would undoubtedly disrupt many of the physiological processes associated with the tegument , including osmoregulation , protection , secretion and synthesis ( 17 ) . besides , the damage of the tegumental folds of the ace - tabular region might disrupt its function in drawing the rumen wall tissue of the host into the acetabular cavity ( 18 ) resulting in a weak hold for the parasite . besides , similar sequence of tegumental changes occurred in p. microbothrium treated with artemether ; which was obtained from the leaves of qinghao ( 16 ) as well as p. cervi treated with plumbagin ; a compound that was rich in the roots of plumbago indica / rosea ( 21 ) . moreover , the tegumental surface alterations induced by bae , in the present study , had also been observed in specimens of a biologically related trematode , fasciola gigantica , following incubation with a number of anthelmintics ( 22 ) . the damage that bae caused to the papillae would undoubtedly disrupt many of their sensory functions . the disruption of the tegument became more severe , on increasing bae concentration and then more widespread sloughing occurred . in the highest bae concentration ( 200 g / ml ) , damage to one fluke was so severe that a hole had penetrated through its entire tegument . such damage had only been observed in specimens of fasciola gigantica treated in vitro with the sulphoxide metabolite of triclabendazole ( 23).the literature survey on b. aegyptiaca suggested that methanolic extracts of the plant s tissues had anthelmintic activity ( 9 ) , with saponins being one of the major secondary metabolites in plant tissues ( 26 ) . wiesman and chapagain ( 11 ) reported a strong correlation between the saponin content of methanolic extracts of b. aegyptiaca and mortality of larvae of the mosquito aedes aegypti . ( 27 ) who isolated a steroidal saponin with a high potential of anthelmintic activity , balanitin-7 . the use of methanolic extract of b. aegyptiaca fruits offers a new dimension and potential for control of such a neglected infectious disease in ruminants , at a time when paramphistomosis has emerged as an important cause of productivity loss .

the mhi biobank is a longitudinal cohort with the aim to recruit 30,000 patients of the mhi for clinical and genetic research . participants are recruited from different departments within the mhi and at its affiliated epic centre , the largest fitness centre in canada for coronary patients and research in primary and secondary prevention . the mhi ethics committee approves the project and informed consent is obtained from all participants . the mhi cohort collects data by using a 35-page questionnaire administered by a research nurse at baseline including demographics , personal and family medical history , physical activity , diet , tobacco , medication use , as well as depression and hostility questionnaires . vital signs ( heart rate , blood pressure , weight , height , waist circumference ) are obtained by the nurse and blood , dna , and plasma are collected and stored at the beaulieu - saucier pharmacogenomics center . patients health status is confirmed by the nurse by using the hospital s health record for retrospective and prospective follow - up . the cohort s database is updated frequently with patients medical information from the hospital s electronic records . blood cell counts and other related phenotypes were automatically generated with the unicel dxh 800 cellular analysis system from beckman coulter . the women s health initiative ( whi ) is one of the largest ( n=161,808 ) u.s . this project was approved by the ethics committee at the fred hutchinson cancer research center . the whi consists of two main components : ( 1 ) a clinical trial that enrolled 68,132 post - menopausal women ages 5079 and randomized them to one of three placebo - controlled clinical trials of hormone therapy , dietary modification , or supplementation with calcium and vitamin d ; and ( 2 ) an observational study that enrolled 93,676 women of the same age range into a parallel prospective study . of the women in whi who were eligible and consented to genetic research , 18,072 samples for blood count were collected at baseline by venipuncture into tubes containing ethylenediaminetetraaetic acid ( edta ) . blood counts were performed with automated hematology cell counters and standardized quality - assurance procedures . hemoglobin , hematocrit , wbc , and platelet count were the only complete blood count values recorded at data entry during the whi baseline examination , which was conducted nationwide during 19931998 . the study of health in pomerania consists of two independent prospectively collected population - based cohorts in west pomerania ( ship and ship - trend ) , a region in the northeast of germany , assessing the prevalence and incidence of common population - based diseases and their risk factors . the study protocol of ship was approved by the medical ethics committee of the university of greifswald . written informed consent was obtained from each of the study participants . briefly , a sample from the population aged 20 to 79 years was drawn from population registries . first , the three cities of the region ( with 17,076 to 65,977 inhabitants ) and the 12 towns ( with 1,516 to 3,044 inhabitants ) were selected , and then 17 out of 97 smaller towns ( with less than 1,500 inhabitants ) , were drawn at random . second , from each of the selected communities , subjects were drawn at random , proportional to the population size of each community and stratified by age and gender . only individuals with german citizenship and main residency in the study area baseline examinations were carried out from 1997 until 2001 , and the sample finally comprised 4,308 participants . baseline examinations for ship - trend were carried out between 2008 and 2012 , finally comprising 4420 participants . the samples were analyzed at the hospital laboratory in greifswald with a coulter max m analyzer ( coulter electronics , miami , usa ) and with a coulter t660 analyzer ( coulter electronics , miami , usa ) at the hospital laboratory in stralsund . quality control was performed internally as well as externally by participating in external proficiency testing programs . for this project , data of ship - trend and the first 5-year follow - up of ship were included . in all association analyses , we attempted to genotype 10,856 participants from the mhi biobank on the illumina exomechip array ( version infinium humanexome v1.0 dna analysis beadchip ) . we initially called genotypes with illumina s genomestudio software and , after data quality control ( see below ) , recalled missing genotypes with the zcall software . we carried out most quality control steps in plink , and developed additional custom scripts when needed . we excluded markers with genotyping success rate < 95% and hardy - weinberg equilibrium p<110 . we excluded samples with genotyping success rate < 95%% , abnormal heterozygosity ( inbreeding f values < -0.2 or > 0.1 ) and extensive low - level identity - by - descent ( ibd ) sharing with a large number of samples . genotype concordance calculated from samples genotyped in duplicate or samples also sequenced by the 1000 genomes project was > 99.99% . to identify population outliers , we used principal component analysis as implemented in eigensoft and anchor our analysis on continental populations from the 1000 genomes project . for this study , we only analyzed individuals of european ancestry . we used the gcta software to detect cryptic relatedness : we removed one individual from each pair of samples that share > 18% of their genome ( corresponding to duplicates and first- and second - degree relatives ) . blood cell phenotypes are available for 6,796 mhi biobank participants ( supplementary table 1 ) . dna samples from the whi clinical coordinating center were sent to the broad institute ( bi ) or the translational genomics research institute ( tgen ) for genotyping and were placed on 96-well plates for processing using the illumina humanexome v1.0 snp array . all genotypes were then merged into a master - file containing all genotypes from the bi and tgen . quality control was performed on this master - file using the plink and r computing platforms . we excluded samples with a genotyping success rate less than 98% . with the resulting sample set , we performed a principal component ( pc ) analysis as well as an analysis of relatedness using the plink ibs / ibd functionality . outlier samples on the pc plots were excluded as were samples that were determined to be contaminated via the relatedness analysis ( i.e. , they were apparently related to hundreds of other samples ) . for each related / duplicate pair of samples , we excluded the sample with the lower call - rate . unexpected duplicate samples were also filtered to prevent potential samples swaps from entering the analysis . for intentionally duplicated samples , we removed samples with low relatedness estimates as we expect them to be close to 1 . we excluded samples with hemoglobin levels greater than 20 g / dl or a hematocrit ( % ) to hemoglobin ( g / dl ) ratio greater than 5 . samples with wbc ( x10/l ) greater than 100 were also excluded from the analysis . the ship and ship - trend samples were genotyped using the illumina exomechip array ( version infinium humanexome v1.0 dna analysis beadchip ) . hybridisation of genomic dna was done in accordance with the manufacturer s standard recommendations at the helmholtz zentrum mnchen . initial genotypes were determined using the genomestudio genotyping module v1.0 ( gencall algorithm ) with the humanexome-12v1_a manifest file and the standard illumina cluster file ( humanexome-12v1.egt ) . contaminated samples , samples with a call rate < 90% , extreme heterozygosity , extensive estimated ibd sharing with a large number of samples , or mismatch between reported and genotyped gender were excluded . next , missing genotypes were re - called with the zcall software version 3.3 using the default values . in both cohorts together , 7366 individuals were successfully genotyped with an average call rate of 99.97% . we used untransformed hgb , hct and plt values and log10-transformed wbc values for association testing . because the analysis of rare variants is particularly sensitive to phenotypic outliers , we winsorized our data such that all individuals with a phenotype below the 0.5% or above the 99.5% of the trait distribution were respectively assigned the phenotype corresponding to 0.5% and 99.5% values . in our analysis , phenotype winsorisation reduced inflation while maintaining phenotype scale . in mhi , we used sex , age , age - squared and the first ten principal components as covariates . in whi , we used age , and the first two principal components as covariates . we also included a term in the linear model to account for the different whi sub - studies that contributed to this project . we used plink to test association between phenotypes and single variant genotypes ( including single variant conditional analyses ) under an additive genetic model . for the gene - based analyses , each cohort ran an analysis with the rvtests software . this software calculates a score statistic for each variant and a covariance matrix for markers within sliding windows . for the gene - based tests , we combined score test results from rvtests with the raremetal software using default parameters . a priori , we decided to focus exclusively on missense and nonsense variants as well as variants within donor or acceptor splice sites for these analyses . for each trait , we ran two gene - based test : a simple burden test with a minor allele frequency ( maf ) cutoff of < 1% ( burden t1 ) and the sequence kernel association test ( skat ) with a maf cutoff of < 5% . for single variant analyses , we tested 183,585 dna sequence variants and four phenotypes : = 0.05 / 183,585 variants / 4 phenotypes = 6.810 . for gene - based analyses , we tested 15,930 genes ( genes with no or only one missense / nonsense / splice site variants were not tested ) , four phenotypes and two different statistical tests : = 0.05 / 15,930 genes / 4 phenotypes / 2 tests = 3.910 . a previously described family in which two sisters were affected with isolated myelokathexis the clinical manifestations in this family included neutropenia without lymphopenia or warts leading to recurrent bacterial infections including septic thrombophlebitis and subacute bacterial endocarditis . at age 43 , one of the siblings her younger sister had fewer infectious episodes , which was speculated to result from a more robust transient release of neutrophils into the peripheral blood during infection . the family was lost to follow up and not available for further functional studies on primary cells . human cxcr2 is encoded by a single exon with multiple upstream non - coding exons giving rise to a number of different splice isoforms . the coding sequence of the gene was amplified on three overlapping pcr fragments and analyzed by automated sequencing . exonic boundaries were identified using publicly available genomic database information and primers were designed to amplify coding exons and flanking intronic sequences using the primer3 software . confirmatory restriction enzyme analysis was performed using ncoi ( new england biolabs ) under standard conditions . image clone 5752441 containing the full - length cxcr2 open reading frame was obtained from invitrogen . the insert was confirmed by sequencing the clone using cxcr2-specific primers . the 968dela ( cxcr2fs ) , c967t / t969 g ( h323x ) , and 968dela/986insc ( cxcr2fs - wt ) mutations were introduced by site - directed mutagenesis primers and amplified by pfuturbo ( stratagene ) . the resulting clones were tagged with epitopes on the c - terminal tail of the receptor by pcr amplification of the cxcr2 open reading frame using the image clone as a template . the amplification primers contained exogenous restriction sites at the 3 and 5 ends and were digested using ecori / xhoi ( flag ) or ecori / sali ( yfp ) . after digestion with the appropriate enzymes , fragments were ligated in - frame upstream of the 3xflag epitope in pires-3xflag - hrgfp ( invitrogen ) or the yfp full - length protein in peyfp - n1 ( clontech ) . the full - length orfs for flag - tagged and yfp - fusion proteins were sequence verified . hek293 and hela cell cultures were maintained in 5% co2 at 37c in dmem ( cellgro ) supplemented with 10% fetal bovine serum ( gibco ) , 2 mm l - glutamine , 100 u / ml penicillin and 100 g / ml streptomycin ( cellgro ) . transgene expression was evaluated 3672 hrs post - transfection by indirect immunofluorescence or western blot . primary antibodies used in these studies - anti - flag ( sigma ) , anti - cxcr2 ( santa cruz biotechnology ) , anti - gfp ( molecular probes ) , anti - calnexin ( chemicon ) , anti - phospho - erk/-erk ( santa cruz biotechnology ) - were used at dilutions as suggested by manufacturer . anti - rabbit and anti - mouse hrp conjugates ( pierce ) were used as secondary antibodies for western blots and fitc - labeled anti - mouse ( scbt ) , rabbit - af594- and af488-labeled anti - rabbit ( invitrogen ) and direct - labeled phalloidin - af594 ( molecular probes ) were used for immunofluorescence microscopy . fluorescein - labeled monoclonal 48311 anti - cxcr2 ( r&d systems ) and phycoerythrin - labeled 12g5 monoclonal anti - cxcr4 ( pharmingen ) and labeled isotype control antibodies were used for flow cytometry experiments . transfected hela cells were made quiescent in serum - free , antibiotic - free dmem overnight prior to the addition of ligand . cells ( 1 10 ) were stimulated with 100 ng / ml cxcl8 , 100 ng / ml cxcl12 ( cell sciences ) or kept in serum - free medium for times as described in figure legends . prior to harvesting , cells were rinsed with ice cold pbs , lysed on ice in ripa lysis buffer ( 50 mm tris - hcl ph 7.4 , 150 mm nacl , 1.0% triton x-100 , 0.5% doc , 0.1% sds , 0.025% nan3 ) supplemented with complete protease inhibitor cocktail ( roche ) for a minimum of 30 minutes . after determination of protein concentration by bradford assay , lysates were denatured in 1x laemmli sample buffer with 100 mm dtt and equivalent amounts run on 10% sds - page gels . proteins were transferred to nitrocellulose ( pall / ge ) using the bio - rad semi - dry trans - blot system for 1 hour , blocked in 5% milk in pbs ( w / v ) for 1 hour , then incubated overnight at 4c in primary antibody . blots were washed three times for 10 minutes in pbs - tween-20 ( 0.05% ) . incubation with species - specific hrp - conjugated antibody for 1 hour was followed by three more 10 min washes in pbst . membranes were developed using supersignal west pico and chemiluminescent substrate ( pierce ) on hyblot cl film ( denville ) . for deglycosylation experiments , 30 g of total protein was incubated with endo hf ( neb ) , pngase f ( neb ) or buffer alone for 1 hour at 37c as recommended by manufacturer . cells were transfected with cxcr2 constructs as described below and an aliquot of cells resuspended by trypsin treatment were fixed for 15 minutes using 2.2% paraformaldehyde ( electron microscopy sciences ) in pbs ( v / v ) then spun down at 500 g for 5 minutes . cells were washed twice in flow buffer ( pbs with 0.5% bsa ( w / v ) , 5 mm edta ) to inhibit clumping of cells . flow cytometric data for quantitation of cxcr2 construct expression were collected using a facs lsr ii ( becton dickson ) . all data were analyzed using flowjo software ( tree star , inc , ashland , or 97520 ) . hela cells at 70% confluence in 10 cm dishes were transfected with either peyfp - n1-cxcr2wt or peyfp - n1-cxcr2wt and cultured for 3 days . after overnight serum - starvation , cells were harvested on the fourth day and resuspended in dmem/0.5% bsa at a density of 510 cells/100 l . 100 l aliquots were added to the upper chamber of 24-well transwell plates ( corning - costar ) with collagen - coated 8.0 m pore polycarbonate membranes . chemotaxis was assayed by addition of cxcl8 ( 100ng / ml ) to dmem in the bottom chamber of the plates . after 2 hours in a 37 c , 5% co2 tissue culture incubator , inserts were removed , loose cells scraped off and transmigrated cells fixed and stained in crystal violet . cells migrated onto the lower surface of the membrane were counted for five low power ( 10x ) fields using a brightfield inverted microscope to determine the average cell number migrated per field . chemotactic index was calculated by dividing the average number of cells migrated per field under conditions of chemokine addition by the average number under conditions of saline addition . the mhi biobank is a longitudinal cohort with the aim to recruit 30,000 patients of the mhi for clinical and genetic research . participants are recruited from different departments within the mhi and at its affiliated epic centre , the largest fitness centre in canada for coronary patients and research in primary and secondary prevention . the mhi ethics committee approves the project and informed consent is obtained from all participants . the mhi cohort collects data by using a 35-page questionnaire administered by a research nurse at baseline including demographics , personal and family medical history , physical activity , diet , tobacco , medication use , as well as depression and hostility questionnaires . vital signs ( heart rate , blood pressure , weight , height , waist circumference ) are obtained by the nurse and blood , dna , and plasma are collected and stored at the beaulieu - saucier pharmacogenomics center . patients health status is confirmed by the nurse by using the hospital s health record for retrospective and prospective follow - up . the cohort s database is updated frequently with patients medical information from the hospital s electronic records . blood cell counts and other related phenotypes were automatically generated with the unicel dxh 800 cellular analysis system from beckman coulter . the women s health initiative ( whi ) is one of the largest ( n=161,808 ) u.s . this project was approved by the ethics committee at the fred hutchinson cancer research center . the whi consists of two main components : ( 1 ) a clinical trial that enrolled 68,132 post - menopausal women ages 5079 and randomized them to one of three placebo - controlled clinical trials of hormone therapy , dietary modification , or supplementation with calcium and vitamin d ; and ( 2 ) an observational study that enrolled 93,676 women of the same age range into a parallel prospective study . of the women in whi who were eligible and consented to genetic research , 18,072 samples for blood count were collected at baseline by venipuncture into tubes containing ethylenediaminetetraaetic acid ( edta ) . blood counts were performed with automated hematology cell counters and standardized quality - assurance procedures . hemoglobin , hematocrit , wbc , and platelet count were the only complete blood count values recorded at data entry during the whi baseline examination , which was conducted nationwide during 19931998 . the study of health in pomerania consists of two independent prospectively collected population - based cohorts in west pomerania ( ship and ship - trend ) , a region in the northeast of germany , assessing the prevalence and incidence of common population - based diseases and their risk factors . the study protocol of ship was approved by the medical ethics committee of the university of greifswald . written informed consent was obtained from each of the study participants . briefly , a sample from the population aged 20 to 79 years was drawn from population registries . first , the three cities of the region ( with 17,076 to 65,977 inhabitants ) and the 12 towns ( with 1,516 to 3,044 inhabitants ) were selected , and then 17 out of 97 smaller towns ( with less than 1,500 inhabitants ) , were drawn at random . second , from each of the selected communities , subjects were drawn at random , proportional to the population size of each community and stratified by age and gender . only individuals with german citizenship and main residency in the study area baseline examinations were carried out from 1997 until 2001 , and the sample finally comprised 4,308 participants . baseline examinations for ship - trend were carried out between 2008 and 2012 , finally comprising 4420 participants . the samples were analyzed at the hospital laboratory in greifswald with a coulter max m analyzer ( coulter electronics , miami , usa ) and with a coulter t660 analyzer ( coulter electronics , miami , usa ) at the hospital laboratory in stralsund . quality control was performed internally as well as externally by participating in external proficiency testing programs . for this project , data of ship - trend and the first 5-year follow - up of ship were included . in all association analyses , we attempted to genotype 10,856 participants from the mhi biobank on the illumina exomechip array ( version infinium humanexome v1.0 dna analysis beadchip ) . we initially called genotypes with illumina s genomestudio software and , after data quality control ( see below ) , recalled missing genotypes with the zcall software . we carried out most quality control steps in plink , and developed additional custom scripts when needed . we excluded markers with genotyping success rate < 95% and hardy - weinberg equilibrium p<110 . we excluded samples with genotyping success rate < 95%% , abnormal heterozygosity ( inbreeding f values < -0.2 or > 0.1 ) and extensive low - level identity - by - descent ( ibd ) sharing with a large number of samples . genotype concordance calculated from samples genotyped in duplicate or samples also sequenced by the 1000 genomes project was > 99.99% . to identify population outliers , we used principal component analysis as implemented in eigensoft and anchor our analysis on continental populations from the 1000 genomes project . for this study , we only analyzed individuals of european ancestry . we used the gcta software to detect cryptic relatedness : we removed one individual from each pair of samples that share > 18% of their genome ( corresponding to duplicates and first- and second - degree relatives ) . blood cell phenotypes are available for 6,796 mhi biobank participants ( supplementary table 1 ) . dna samples from the whi clinical coordinating center were sent to the broad institute ( bi ) or the translational genomics research institute ( tgen ) for genotyping and were placed on 96-well plates for processing using the illumina humanexome v1.0 snp array . all genotypes were then merged into a master - file containing all genotypes from the bi and tgen . quality control was performed on this master - file using the plink and r computing platforms . we excluded samples with a genotyping success rate less than 98% . with the resulting sample set , we performed a principal component ( pc ) analysis as well as an analysis of relatedness using the plink ibs / ibd functionality . outlier samples on the pc plots were excluded as were samples that were determined to be contaminated via the relatedness analysis ( i.e. , they were apparently related to hundreds of other samples ) . for each related / duplicate pair of samples , we excluded the sample with the lower call - rate . unexpected duplicate samples were also filtered to prevent potential samples swaps from entering the analysis . for intentionally duplicated samples , we removed samples with low relatedness estimates as we expect them to be close to 1 . we excluded samples with hemoglobin levels greater than 20 g / dl or a hematocrit ( % ) to hemoglobin ( g / dl ) ratio greater than 5 . samples with wbc ( x10/l ) greater than 100 were also excluded from the analysis . the ship and ship - trend samples were genotyped using the illumina exomechip array ( version infinium humanexome v1.0 dna analysis beadchip ) . hybridisation of genomic dna was done in accordance with the manufacturer s standard recommendations at the helmholtz zentrum mnchen . initial genotypes were determined using the genomestudio genotyping module v1.0 ( gencall algorithm ) with the humanexome-12v1_a manifest file and the standard illumina cluster file ( humanexome-12v1.egt ) . contaminated samples , samples with a call rate < 90% , extreme heterozygosity , extensive estimated ibd sharing with a large number of samples , or mismatch between reported and genotyped gender were excluded . next , missing genotypes were re - called with the zcall software version 3.3 using the default values . in both cohorts together , 7366 individuals were successfully genotyped with an average call rate of 99.97% . we used untransformed hgb , hct and plt values and log10-transformed wbc values for association testing . because the analysis of rare variants is particularly sensitive to phenotypic outliers , we winsorized our data such that all individuals with a phenotype below the 0.5% or above the 99.5% of the trait distribution were respectively assigned the phenotype corresponding to 0.5% and 99.5% values . in our analysis , phenotype winsorisation reduced inflation while maintaining phenotype scale . in mhi , we used sex , age , age - squared and the first ten principal components as covariates . in whi , we used age , and the first two principal components as covariates . we also included a term in the linear model to account for the different whi sub - studies that contributed to this project . we used plink to test association between phenotypes and single variant genotypes ( including single variant conditional analyses ) under an additive genetic model . for the gene - based analyses , each cohort ran an analysis with the rvtests software . this software calculates a score statistic for each variant and a covariance matrix for markers within sliding windows . we combined single variant results with metal using the inverse variance method . for the gene - based tests , we combined score test results from rvtests with the raremetal software using default parameters . a priori , we decided to focus exclusively on missense and nonsense variants as well as variants within donor or acceptor splice sites for these analyses . for each trait , we ran two gene - based test : a simple burden test with a minor allele frequency ( maf ) cutoff of < 1% ( burden t1 ) and the sequence kernel association test ( skat ) with a maf cutoff of < 5% . for single variant analyses , we tested 183,585 dna sequence variants and four phenotypes : = 0.05 / 183,585 variants / 4 phenotypes = 6.810 . for gene - based analyses , we tested 15,930 genes ( genes with no or only one missense / nonsense / splice site variants were not tested ) , four phenotypes and two different statistical tests : = 0.05 / 15,930 genes / 4 phenotypes / 2 tests = 3.910 . a previously described family in which two sisters were affected with isolated myelokathexis was evaluated for mutations in cxcr2 . the clinical manifestations in this family included neutropenia without lymphopenia or warts leading to recurrent bacterial infections including septic thrombophlebitis and subacute bacterial endocarditis . at age 43 , one of the siblings her younger sister had fewer infectious episodes , which was speculated to result from a more robust transient release of neutrophils into the peripheral blood during infection . the family was lost to follow up and not available for further functional studies on primary cells . human cxcr2 is encoded by a single exon with multiple upstream non - coding exons giving rise to a number of different splice isoforms . the coding sequence of the gene was amplified on three overlapping pcr fragments and analyzed by automated sequencing . exonic boundaries were identified using publicly available genomic database information and primers were designed to amplify coding exons and flanking intronic sequences using the primer3 software . confirmatory restriction enzyme analysis was performed using ncoi ( new england biolabs ) under standard conditions . image clone 5752441 containing the full - length cxcr2 open reading frame was obtained from invitrogen . the insert was confirmed by sequencing the clone using cxcr2-specific primers . the 968dela ( cxcr2fs ) , c967t / t969 g ( h323x ) , and 968dela/986insc ( cxcr2fs - wt ) mutations were introduced by site - directed mutagenesis primers and amplified by pfuturbo ( stratagene ) . the resulting clones were tagged with epitopes on the c - terminal tail of the receptor by pcr amplification of the cxcr2 open reading frame using the image clone as a template . the amplification primers contained exogenous restriction sites at the 3 and 5 ends and were digested using ecori / xhoi ( flag ) or ecori / sali ( yfp ) . after digestion with the appropriate enzymes , fragments were ligated in - frame upstream of the 3xflag epitope in pires-3xflag - hrgfp ( invitrogen ) or the yfp full - length protein in peyfp - n1 ( clontech ) . the full - length orfs for flag - tagged and yfp - fusion proteins were sequence verified . hek293 and hela cell cultures were maintained in 5% co2 at 37c in dmem ( cellgro ) supplemented with 10% fetal bovine serum ( gibco ) , 2 mm l - glutamine , 100 u / ml penicillin and 100 g / ml streptomycin ( cellgro ) . transgene expression was evaluated 3672 hrs post - transfection by indirect immunofluorescence or western blot . primary antibodies used in these studies - anti - flag ( sigma ) , anti - cxcr2 ( santa cruz biotechnology ) , anti - gfp ( molecular probes ) , anti - calnexin ( chemicon ) , anti - phospho - erk/-erk ( santa cruz biotechnology ) - were used at dilutions as suggested by manufacturer . anti - rabbit and anti - mouse hrp conjugates ( pierce ) were used as secondary antibodies for western blots and fitc - labeled anti - mouse ( scbt ) , rabbit - af594- and af488-labeled anti - rabbit ( invitrogen ) and direct - labeled phalloidin - af594 ( molecular probes ) were used for immunofluorescence microscopy . fluorescein - labeled monoclonal 48311 anti - cxcr2 ( r&d systems ) and phycoerythrin - labeled 12g5 monoclonal anti - cxcr4 ( pharmingen ) and labeled isotype control antibodies were used for flow cytometry experiments . transfected hela cells were made quiescent in serum - free , antibiotic - free dmem overnight prior to the addition of ligand . cells ( 1 10 ) were stimulated with 100 ng / ml cxcl8 , 100 ng / ml cxcl12 ( cell sciences ) or kept in serum - free medium for times as described in figure legends . prior to harvesting , cells were rinsed with ice cold pbs , lysed on ice in ripa lysis buffer ( 50 mm tris - hcl ph 7.4 , 150 mm nacl , 1.0% triton x-100 , 0.5% doc , 0.1% sds , 0.025% nan3 ) supplemented with complete protease inhibitor cocktail ( roche ) for a minimum of 30 minutes . after determination of protein concentration by bradford assay , lysates were denatured in 1x laemmli sample buffer with 100 mm dtt and equivalent amounts run on 10% sds - page gels . proteins were transferred to nitrocellulose ( pall / ge ) using the bio - rad semi - dry trans - blot system for 1 hour , blocked in 5% milk in pbs ( w / v ) for 1 hour , then incubated overnight at 4c in primary antibody . blots were washed three times for 10 minutes in pbs - tween-20 ( 0.05% ) . incubation with species - specific hrp - conjugated antibody for 1 hour was followed by three more 10 min washes in pbst . membranes were developed using supersignal west pico and chemiluminescent substrate ( pierce ) on hyblot cl film ( denville ) . for deglycosylation experiments , 30 g of total protein was incubated with endo hf ( neb ) , pngase f ( neb ) or buffer alone for 1 hour at 37c as recommended by manufacturer . cells were transfected with cxcr2 constructs as described below and an aliquot of cells resuspended by trypsin treatment were fixed for 15 minutes using 2.2% paraformaldehyde ( electron microscopy sciences ) in pbs ( v / v ) then spun down at 500 g for 5 minutes . cells were washed twice in flow buffer ( pbs with 0.5% bsa ( w / v ) , 5 mm edta ) to inhibit clumping of cells . flow cytometric data for quantitation of cxcr2 construct expression were collected using a facs lsr ii ( becton dickson ) . all data were analyzed using flowjo software ( tree star , inc , ashland , or 97520 ) . hela cells at 70% confluence in 10 cm dishes were transfected with either peyfp - n1-cxcr2wt or peyfp - n1-cxcr2wt and cultured for 3 days . after overnight serum - starvation , cells were harvested on the fourth day and resuspended in dmem/0.5% bsa at a density of 510 cells/100 l . 100 l aliquots were added to the upper chamber of 24-well transwell plates ( corning - costar ) with collagen - coated 8.0 m pore polycarbonate membranes . chemotaxis was assayed by addition of cxcl8 ( 100ng / ml ) to dmem in the bottom chamber of the plates . after 2 hours in a 37 c , 5% co2 tissue culture incubator , inserts were removed , loose cells scraped off and transmigrated cells fixed and stained in crystal violet . cells migrated onto the lower surface of the membrane were counted for five low power ( 10x ) fields using a brightfield inverted microscope to determine the average cell number migrated per field . chemotactic index was calculated by dividing the average number of cells migrated per field under conditions of chemokine addition by the average number under conditions of saline addition .

hematological traits are important clinical parameters . to test the role of rare and low - frequency coding variants on hematological traits , we analyzed hemoglobin , hematocrit , white blood cell ( wbc ) and platelet count in 31,340 individuals genotyped on an exome array . we identified several missense variants of cxcr2 associated with reduced wbc count ( gene - based p=2.61013 ) . in a separate family - based re - sequencing study , we identified a novel loss - of - function cxcr2 frameshift mutation in a pedigree with congenital neutropenia that abolished ligand - induced cxcr2 signal transduction and chemotaxis . we also identified novel missense or splice site variants in key hematopoiesis regulators ( epo , trf2 , hbb , tubb1 , sh2b3 ) associated with blood cell traits . finally , we were able to detect associations between the rare somatic jak2 p.val617phe mutation and platelet count ( p=3.91022 ) as well as hemoglobin ( p=0.002 ) , hematocrit ( p=9.5107 ) and wbc ( p=3.1105 ) . in conclusion , exome arrays complement gwas in identifying new variants that contribute to complex human traits .

Methods Study participants and phenotypes Genotyping and quality-control steps Trait modeling and study-level association testing Meta-analysis and statistical significance Functional characterization of CXCR2 Supplementary Material

participants are recruited from different departments within the mhi and at its affiliated epic centre , the largest fitness centre in canada for coronary patients and research in primary and secondary prevention . the mhi cohort collects data by using a 35-page questionnaire administered by a research nurse at baseline including demographics , personal and family medical history , physical activity , diet , tobacco , medication use , as well as depression and hostility questionnaires . vital signs ( heart rate , blood pressure , weight , height , waist circumference ) are obtained by the nurse and blood , dna , and plasma are collected and stored at the beaulieu - saucier pharmacogenomics center . blood cell counts and other related phenotypes were automatically generated with the unicel dxh 800 cellular analysis system from beckman coulter . hemoglobin , hematocrit , wbc , and platelet count were the only complete blood count values recorded at data entry during the whi baseline examination , which was conducted nationwide during 19931998 . the study of health in pomerania consists of two independent prospectively collected population - based cohorts in west pomerania ( ship and ship - trend ) , a region in the northeast of germany , assessing the prevalence and incidence of common population - based diseases and their risk factors . first , the three cities of the region ( with 17,076 to 65,977 inhabitants ) and the 12 towns ( with 1,516 to 3,044 inhabitants ) were selected , and then 17 out of 97 smaller towns ( with less than 1,500 inhabitants ) , were drawn at random . the samples were analyzed at the hospital laboratory in greifswald with a coulter max m analyzer ( coulter electronics , miami , usa ) and with a coulter t660 analyzer ( coulter electronics , miami , usa ) at the hospital laboratory in stralsund . quality control was performed internally as well as externally by participating in external proficiency testing programs . in all association analyses , we attempted to genotype 10,856 participants from the mhi biobank on the illumina exomechip array ( version infinium humanexome v1.0 dna analysis beadchip ) . we initially called genotypes with illumina s genomestudio software and , after data quality control ( see below ) , recalled missing genotypes with the zcall software . we excluded samples with genotyping success rate < 95%% , abnormal heterozygosity ( inbreeding f values < -0.2 or > 0.1 ) and extensive low - level identity - by - descent ( ibd ) sharing with a large number of samples . genotype concordance calculated from samples genotyped in duplicate or samples also sequenced by the 1000 genomes project was > 99.99% . to identify population outliers , we used principal component analysis as implemented in eigensoft and anchor our analysis on continental populations from the 1000 genomes project . for this study , we only analyzed individuals of european ancestry . we used the gcta software to detect cryptic relatedness : we removed one individual from each pair of samples that share > 18% of their genome ( corresponding to duplicates and first- and second - degree relatives ) . blood cell phenotypes are available for 6,796 mhi biobank participants ( supplementary table 1 ) . with the resulting sample set , we performed a principal component ( pc ) analysis as well as an analysis of relatedness using the plink ibs / ibd functionality . outlier samples on the pc plots were excluded as were samples that were determined to be contaminated via the relatedness analysis ( i.e. for each related / duplicate pair of samples , we excluded the sample with the lower call - rate . for intentionally duplicated samples , we removed samples with low relatedness estimates as we expect them to be close to 1 . we excluded samples with hemoglobin levels greater than 20 g / dl or a hematocrit ( % ) to hemoglobin ( g / dl ) ratio greater than 5 . samples with wbc ( x10/l ) greater than 100 were also excluded from the analysis . hybridisation of genomic dna was done in accordance with the manufacturer s standard recommendations at the helmholtz zentrum mnchen . contaminated samples , samples with a call rate < 90% , extreme heterozygosity , extensive estimated ibd sharing with a large number of samples , or mismatch between reported and genotyped gender were excluded . next , missing genotypes were re - called with the zcall software version 3.3 using the default values . we used untransformed hgb , hct and plt values and log10-transformed wbc values for association testing . because the analysis of rare variants is particularly sensitive to phenotypic outliers , we winsorized our data such that all individuals with a phenotype below the 0.5% or above the 99.5% of the trait distribution were respectively assigned the phenotype corresponding to 0.5% and 99.5% values . in our analysis , phenotype winsorisation reduced inflation while maintaining phenotype scale . in mhi , we used sex , age , age - squared and the first ten principal components as covariates . in whi , we used age , and the first two principal components as covariates . we also included a term in the linear model to account for the different whi sub - studies that contributed to this project . we used plink to test association between phenotypes and single variant genotypes ( including single variant conditional analyses ) under an additive genetic model . for the gene - based analyses , each cohort ran an analysis with the rvtests software . for the gene - based tests , we combined score test results from rvtests with the raremetal software using default parameters . a priori , we decided to focus exclusively on missense and nonsense variants as well as variants within donor or acceptor splice sites for these analyses . for each trait , we ran two gene - based test : a simple burden test with a minor allele frequency ( maf ) cutoff of < 1% ( burden t1 ) and the sequence kernel association test ( skat ) with a maf cutoff of < 5% . for single variant analyses , we tested 183,585 dna sequence variants and four phenotypes : = 0.05 / 183,585 variants / 4 phenotypes = 6.810 . for gene - based analyses , we tested 15,930 genes ( genes with no or only one missense / nonsense / splice site variants were not tested ) , four phenotypes and two different statistical tests : = 0.05 / 15,930 genes / 4 phenotypes / 2 tests = 3.910 . the family was lost to follow up and not available for further functional studies on primary cells . the coding sequence of the gene was amplified on three overlapping pcr fragments and analyzed by automated sequencing . the 968dela ( cxcr2fs ) , c967t / t969 g ( h323x ) , and 968dela/986insc ( cxcr2fs - wt ) mutations were introduced by site - directed mutagenesis primers and amplified by pfuturbo ( stratagene ) . the full - length orfs for flag - tagged and yfp - fusion proteins were sequence verified . hek293 and hela cell cultures were maintained in 5% co2 at 37c in dmem ( cellgro ) supplemented with 10% fetal bovine serum ( gibco ) , 2 mm l - glutamine , 100 u / ml penicillin and 100 g / ml streptomycin ( cellgro ) . primary antibodies used in these studies - anti - flag ( sigma ) , anti - cxcr2 ( santa cruz biotechnology ) , anti - gfp ( molecular probes ) , anti - calnexin ( chemicon ) , anti - phospho - erk/-erk ( santa cruz biotechnology ) - were used at dilutions as suggested by manufacturer . anti - rabbit and anti - mouse hrp conjugates ( pierce ) were used as secondary antibodies for western blots and fitc - labeled anti - mouse ( scbt ) , rabbit - af594- and af488-labeled anti - rabbit ( invitrogen ) and direct - labeled phalloidin - af594 ( molecular probes ) were used for immunofluorescence microscopy . fluorescein - labeled monoclonal 48311 anti - cxcr2 ( r&d systems ) and phycoerythrin - labeled 12g5 monoclonal anti - cxcr4 ( pharmingen ) and labeled isotype control antibodies were used for flow cytometry experiments . transfected hela cells were made quiescent in serum - free , antibiotic - free dmem overnight prior to the addition of ligand . proteins were transferred to nitrocellulose ( pall / ge ) using the bio - rad semi - dry trans - blot system for 1 hour , blocked in 5% milk in pbs ( w / v ) for 1 hour , then incubated overnight at 4c in primary antibody . for deglycosylation experiments , 30 g of total protein was incubated with endo hf ( neb ) , pngase f ( neb ) or buffer alone for 1 hour at 37c as recommended by manufacturer . cells were transfected with cxcr2 constructs as described below and an aliquot of cells resuspended by trypsin treatment were fixed for 15 minutes using 2.2% paraformaldehyde ( electron microscopy sciences ) in pbs ( v / v ) then spun down at 500 g for 5 minutes . cells were washed twice in flow buffer ( pbs with 0.5% bsa ( w / v ) , 5 mm edta ) to inhibit clumping of cells . flow cytometric data for quantitation of cxcr2 construct expression were collected using a facs lsr ii ( becton dickson ) . after 2 hours in a 37 c , 5% co2 tissue culture incubator , inserts were removed , loose cells scraped off and transmigrated cells fixed and stained in crystal violet . the mhi cohort collects data by using a 35-page questionnaire administered by a research nurse at baseline including demographics , personal and family medical history , physical activity , diet , tobacco , medication use , as well as depression and hostility questionnaires . vital signs ( heart rate , blood pressure , weight , height , waist circumference ) are obtained by the nurse and blood , dna , and plasma are collected and stored at the beaulieu - saucier pharmacogenomics center . patients health status is confirmed by the nurse by using the hospital s health record for retrospective and prospective follow - up . blood cell counts and other related phenotypes were automatically generated with the unicel dxh 800 cellular analysis system from beckman coulter . hemoglobin , hematocrit , wbc , and platelet count were the only complete blood count values recorded at data entry during the whi baseline examination , which was conducted nationwide during 19931998 . the study of health in pomerania consists of two independent prospectively collected population - based cohorts in west pomerania ( ship and ship - trend ) , a region in the northeast of germany , assessing the prevalence and incidence of common population - based diseases and their risk factors . first , the three cities of the region ( with 17,076 to 65,977 inhabitants ) and the 12 towns ( with 1,516 to 3,044 inhabitants ) were selected , and then 17 out of 97 smaller towns ( with less than 1,500 inhabitants ) , were drawn at random . the samples were analyzed at the hospital laboratory in greifswald with a coulter max m analyzer ( coulter electronics , miami , usa ) and with a coulter t660 analyzer ( coulter electronics , miami , usa ) at the hospital laboratory in stralsund . quality control was performed internally as well as externally by participating in external proficiency testing programs . in all association analyses , we attempted to genotype 10,856 participants from the mhi biobank on the illumina exomechip array ( version infinium humanexome v1.0 dna analysis beadchip ) . we initially called genotypes with illumina s genomestudio software and , after data quality control ( see below ) , recalled missing genotypes with the zcall software . we excluded samples with genotyping success rate < 95%% , abnormal heterozygosity ( inbreeding f values < -0.2 or > 0.1 ) and extensive low - level identity - by - descent ( ibd ) sharing with a large number of samples . genotype concordance calculated from samples genotyped in duplicate or samples also sequenced by the 1000 genomes project was > 99.99% . to identify population outliers , we used principal component analysis as implemented in eigensoft and anchor our analysis on continental populations from the 1000 genomes project . for this study , we only analyzed individuals of european ancestry . we used the gcta software to detect cryptic relatedness : we removed one individual from each pair of samples that share > 18% of their genome ( corresponding to duplicates and first- and second - degree relatives ) . blood cell phenotypes are available for 6,796 mhi biobank participants ( supplementary table 1 ) . all genotypes were then merged into a master - file containing all genotypes from the bi and tgen . quality control was performed on this master - file using the plink and r computing platforms . with the resulting sample set , we performed a principal component ( pc ) analysis as well as an analysis of relatedness using the plink ibs / ibd functionality . for each related / duplicate pair of samples , we excluded the sample with the lower call - rate . for intentionally duplicated samples , we removed samples with low relatedness estimates as we expect them to be close to 1 . we excluded samples with hemoglobin levels greater than 20 g / dl or a hematocrit ( % ) to hemoglobin ( g / dl ) ratio greater than 5 . samples with wbc ( x10/l ) greater than 100 were also excluded from the analysis . next , missing genotypes were re - called with the zcall software version 3.3 using the default values . we used untransformed hgb , hct and plt values and log10-transformed wbc values for association testing . because the analysis of rare variants is particularly sensitive to phenotypic outliers , we winsorized our data such that all individuals with a phenotype below the 0.5% or above the 99.5% of the trait distribution were respectively assigned the phenotype corresponding to 0.5% and 99.5% values . in our analysis , phenotype winsorisation reduced inflation while maintaining phenotype scale . in mhi , we used sex , age , age - squared and the first ten principal components as covariates . in whi , we used age , and the first two principal components as covariates . we also included a term in the linear model to account for the different whi sub - studies that contributed to this project . we used plink to test association between phenotypes and single variant genotypes ( including single variant conditional analyses ) under an additive genetic model . for the gene - based analyses , each cohort ran an analysis with the rvtests software . we combined single variant results with metal using the inverse variance method . for the gene - based tests , we combined score test results from rvtests with the raremetal software using default parameters . a priori , we decided to focus exclusively on missense and nonsense variants as well as variants within donor or acceptor splice sites for these analyses . for each trait , we ran two gene - based test : a simple burden test with a minor allele frequency ( maf ) cutoff of < 1% ( burden t1 ) and the sequence kernel association test ( skat ) with a maf cutoff of < 5% . for single variant analyses , we tested 183,585 dna sequence variants and four phenotypes : = 0.05 / 183,585 variants / 4 phenotypes = 6.810 . for gene - based analyses , we tested 15,930 genes ( genes with no or only one missense / nonsense / splice site variants were not tested ) , four phenotypes and two different statistical tests : = 0.05 / 15,930 genes / 4 phenotypes / 2 tests = 3.910 . the clinical manifestations in this family included neutropenia without lymphopenia or warts leading to recurrent bacterial infections including septic thrombophlebitis and subacute bacterial endocarditis . the 968dela ( cxcr2fs ) , c967t / t969 g ( h323x ) , and 968dela/986insc ( cxcr2fs - wt ) mutations were introduced by site - directed mutagenesis primers and amplified by pfuturbo ( stratagene ) . the resulting clones were tagged with epitopes on the c - terminal tail of the receptor by pcr amplification of the cxcr2 open reading frame using the image clone as a template . hek293 and hela cell cultures were maintained in 5% co2 at 37c in dmem ( cellgro ) supplemented with 10% fetal bovine serum ( gibco ) , 2 mm l - glutamine , 100 u / ml penicillin and 100 g / ml streptomycin ( cellgro ) . primary antibodies used in these studies - anti - flag ( sigma ) , anti - cxcr2 ( santa cruz biotechnology ) , anti - gfp ( molecular probes ) , anti - calnexin ( chemicon ) , anti - phospho - erk/-erk ( santa cruz biotechnology ) - were used at dilutions as suggested by manufacturer . anti - rabbit and anti - mouse hrp conjugates ( pierce ) were used as secondary antibodies for western blots and fitc - labeled anti - mouse ( scbt ) , rabbit - af594- and af488-labeled anti - rabbit ( invitrogen ) and direct - labeled phalloidin - af594 ( molecular probes ) were used for immunofluorescence microscopy . fluorescein - labeled monoclonal 48311 anti - cxcr2 ( r&d systems ) and phycoerythrin - labeled 12g5 monoclonal anti - cxcr4 ( pharmingen ) and labeled isotype control antibodies were used for flow cytometry experiments . after determination of protein concentration by bradford assay , lysates were denatured in 1x laemmli sample buffer with 100 mm dtt and equivalent amounts run on 10% sds - page gels . for deglycosylation experiments , 30 g of total protein was incubated with endo hf ( neb ) , pngase f ( neb ) or buffer alone for 1 hour at 37c as recommended by manufacturer . cells were transfected with cxcr2 constructs as described below and an aliquot of cells resuspended by trypsin treatment were fixed for 15 minutes using 2.2% paraformaldehyde ( electron microscopy sciences ) in pbs ( v / v ) then spun down at 500 g for 5 minutes . cells were washed twice in flow buffer ( pbs with 0.5% bsa ( w / v ) , 5 mm edta ) to inhibit clumping of cells . flow cytometric data for quantitation of cxcr2 construct expression were collected using a facs lsr ii ( becton dickson ) . after 2 hours in a 37 c , 5% co2 tissue culture incubator , inserts were removed , loose cells scraped off and transmigrated cells fixed and stained in crystal violet .

cardiac resynchronisation therapy ( crt ) has become an important treatment for patients with heart failure and left ventricular ( lv ) dyssynchrony [ 13 ] . however , clinical non - response to crt is reported in 2535 % of patients . although a large variety of causes for a suboptimal response have been cited , most attention has been focused on the selection of patients eligible for crt , and less on inadequate delivery of crt therapy . however , even with growing experience and improved materials and tools , the optimal position can not always be reached in one of the tributaries of the coronary sinus . this can be due to the absence of suitable side branches in the posterolateral area , coronary vein stenosis , lead instability , high stimulation threshold , phrenic nerve stimulation , or a combination of the above [ 57 ] . we studied the acute haemodynamic response ( ahr ) of the implanted system and alternative left ventricular endocardial pacing sites in patients clinically not responding to crt . we asked patients who remained in new york heart failure class iii or iv despite at least 6 months of crt and adequate medical therapy and with no therapeutic options left to undergo an acute haemodynamic study in search of improving existing crt , as previously described [ 8 , 9 ] . twenty - four non - consecutive patients , 23 males , age 72.8 9.1 years , mean ejection fraction 22.5 7.1 % , agreed to undergo this test ( table 1 ) . the ecg prior to implantation showed left bundle branch block ( lbbb ) in 12 , non - lbbb in 4 , and right ventricular ( rv ) pacing in 8 patients . table 1characteristics of clinical non - responders ( 24 patients)patientgenderage ( years)nyha classicm / dcmejection fraction ( % ) qrs morphologyqrs width ( ms)pt . 01m56ivdcm13lbbb135pt . ivicm17rvp210average23m/1f72.8 9.13.2 0.36dcm22.5 7.112lbbb/8rvp175 22patient characteristics of 24 clinical non - responders to crt . nyha class new york heart association class , dcm dilated cardiomyopathy , icm ischaemic cardiomyopathy , lbbb left bundle branch block , non - lbbb non left bundle branch block , pt . characteristics of clinical non - responders ( 24 patients ) patient characteristics of 24 clinical non - responders to crt . nyha class new york heart association class , dcm dilated cardiomyopathy , icm ischaemic cardiomyopathy , lbbb left bundle branch block , non - lbbb non left bundle branch block , pt . a 6f multipurpose angioplasty guiding catheter was introduced into the lv after a standard transseptal catheterisation . in one patient , the guiding catheter was introduced through the radial artery following coronary angiography . through this guiding catheter , endocardial pacing and measurements of lvdp / dtmax were accomplished by roving a medtronic 6416 temporary bipolar screw - in lead ( medtronic inc . minneapolis , mn . ) and a radi pressure wire ( radi medical , a st jude medical company , st . initially the atrioventricular and interventricular intervals of the implanted system were optimised to obtain the maximal lvdp / dtmax . lv pacing was performed from basal posterolateral , mid - posterolateral , lv apical and lv septal locations , and optimal ahr obtained after optimisation of the crt system for all positions . if none of the 4 endocardial positions were opposite the position of the coronary sinus lead , an additional measurement was done at this location . a rise in lvdp / dtmax 15 % from baseline was considered to be a positive haemodynamic response . in all patients with an ahr of 15 % during lv endocardial pacing , the interval between the onset of the qrs complex and the intrinsic activation at the lv electrode ( q - lv interval ) was measured and the ratio between q - lv / qrs width interval calculated . this ratio expresses the relation between q - lv interval and qrs width , which is a better indicator for late or early lv sensing than the absolute value of q - lv . notwithstanding that all patients were clinical non - responders , in 5 patients lvdp / dtmax with the implanted coronary sinus system increased 15 % after optimisation : average 30.2 % , 15.644.5 % ( table 2 ) . in 3 out of these 5 patients with the coronary sinus lead in a posterolateral position , endocardial pacing did not increase the lvdp / dtmax substantially ( ahr less than 3 % and even an adverse effect from lv endocardial pacing was observed in one patient ) . in one of the two remaining patients with an apical position of the coronary sinus lead , the ahr could be increased during lv endocardial pacing from 19.766 % and we considered the increase sufficient to justify the upgrade to lv endocardial pacing ( fig . 1 ) . table 2individual haemodynamic results of all clinical non - responders ( 24 patients)patientqrs morphologybaseline lvdp / dtmax ( mmhg / s)position cs leadahr cs lead ( % ) ahr cs - level - endo(%)lv endo optimal positionahr lvendo ( % ) pt . 07non - lbbb893pl - mid 23.4lv septal 21.5pt . 08lbbb827pl - bas44.951.0pl - bas51.0pt . 24rvp489lv apical12.717.2pl - mid24.1average891 2478.7 15.810.5 8.919.6 17.5haemodynamic measurements of 24 clinical nonresponders to crt . baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . grey shaded patients have an ahr from the cs lead 15 % and are considered haemodynamic responders , pl - bas basal posterolateral , pl - mid mid - posterolateral , rvp right ventricular pacing . individual haemodynamic results of all clinical non - responders ( 24 patients ) haemodynamic measurements of 24 clinical nonresponders to crt . baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . grey shaded patients have an ahr from the cs lead 15 % and are considered haemodynamic responders , pl - bas basal posterolateral , pl - mid mid - posterolateral , rvp right ventricular pacing . all views are in left anterior oblique ( lao ) showing the haemodynamic effects of stimulation from the coronary sinus lead and lv endocardial stimulation in the mid - posterolateral area ( left upper panel ) , basal posterolateral area ( left lower panel ) , lv septum ( right upper panel ) and lead positions after lv endocardial implantation of a permanent lead in the lv basal posterolateral segment . arrows indicate the position of the bipolar pacing lead angiographic pictures of the haemodynamic study in patient no . all views are in left anterior oblique ( lao ) showing the haemodynamic effects of stimulation from the coronary sinus lead and lv endocardial stimulation in the mid - posterolateral area ( left upper panel ) , basal posterolateral area ( left lower panel ) , lv septum ( right upper panel ) and lead positions after lv endocardial implantation of a permanent lead in the lv basal posterolateral segment . arrows indicate the position of the bipolar pacing lead in the 19 patients with an ahr from the implanted system < 15 % , 9 patients had an increase in ahr above the 15 % limit . the results of the total group of 24 patients showed a positive response in 11 patients with an apical lead position in 7 , anterolateral in 2 and mid - posterolateral in 2 patients . a negative response of endocardial pacing was observed in 13 patients with a basal posterolateral lead position in 3 , mid - posterolateral in 8 and apical in 2 patients . in 5 out of the 9 acute haemodynamic non - responders ( ahr < 15 % ) with lbbb , the ahr could be increased above the 15 % level by lv endocardial pacing . the average ahr with the coronary sinus system was 9.6 % ( 5.814.9 % ) and at the lv endocardial optimal position 25.8 % ( 19.034.3 % ) . the average qrs width in these patients was 169 ms ( 135198 ms ) and the average q - lv at the optimal lv endocardial pacing site 157 ms ( 128176 ms ) , resulting in a q - lv / qrs ratio of 93 % . the position of the original coronary sinus lead was apical in 3 , anterolateral in 1 and mid - posterolateral in 1 patient ( fig . 2 ) . 2recordings of the temporary study of patient no . 22 with a lbbb showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions recordings of the temporary study of patient no . 22 with a lbbb showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions in the remaining 4 patients , the average ahr at the optimal endocardial position increased < 15 % , with an average of 7.3 % ( 1.79.9 % ) . the average qrs width in these patients was 176 ms ( 149209 ms ) and the average q - lv 136 ms ( 100174 ms ) , resulting in a q - lv / qrs ratio of 83 % . of these , only one patient with rbbb , combined with left anterior hemiblock , showed an increase in the ahr , from 9.1 % ( apical coronary sinus position ) to 25.8 % at an endocardial basal posterolateral position . 3 ) ; one had a non - specific intraventricular conduction delay with a qrs complex of 175 ms . the coronary sinus lead position was mid - posterolateral in 2 and apical in 1 patient . 3recordings of a temporary study of patient no.18 with non - lbbb , showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions . this recording illustrates that there is no conduction delay in the lv and a minimal haemodynamic effect recordings of a temporary study of patient no.18 with non - lbbb , showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions . this recording illustrates that there is no conduction delay in the lv and a minimal haemodynamic effect three out of the 6 patients with rv pacing became haemodynamic responders ( lv dp / dtmax from average 9.7 % , ( 9.312.7 % ) increased to 21.8 % ( 16.624.8 % ) ) . in the other 3 patients the average ahr increased from 2.9 % ( 1.14.0 % ) to 9.7 % ( 7.811.4 % ) . in 2 patients the coronary sinus lead was in a mid - posterolateral position , one in an apical position . individual details are summarised in table 2 and a flow chart overview in fig . 4 . 4flow chart of the study showing the haemodynamic results in relation to the coronary sinus lead positions . ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular flow chart of the study showing the haemodynamic results in relation to the coronary sinus lead positions . ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular when pacing endocardially , opposite the epicardial coronary sinus lead , we found that lvdp / dtmax did not differ significantly for any of the patients : lvdp / dtmax from the coronary sinus averaged 1046 292 mmhg / s vs. 1068 296 mmhg / s from the corresponding lv endocardial site ( p = 0.37 ) . we found that basal or mid - posterolateral segments had the highest ahr of the tested endocardial sites in all 14 patients who showed an ahr 15 % rise in lvdp / dtmax compared with baseline . because either the basal or mid - posterolateral segment had the highest ahr , we also calculated the combined best results from basal posterolateral and mid - posterolateral ( pl - best ) . pl - best had a rise to 30.2 13.6 % in lvdp / dtmax , basal posterolateral 27.7 15.2 % , mid - posterolateral 24.6 12.6 % , lv apical 14.8 9.1 % and lv septal 17.0 11.2 % . we also found that in 12 out of the 14 patients ( 85 % ) with an ahr 15 % , the lv endocardial site with the longest time interval between the onset of the qrs complex and lv sensing ( q - lv interval ) resulted in the best haemodynamic response . the average qrs width was 133 22 ms ( range 120205 ms ) and average q - lv interval 155 26 ms ( 128205 ms ) . the average q - lv / qrs width ratio , which is q - lv expressed as a percentage of qrs width , was 90 % . table 3haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax 15 % from endocardial pacingpts.ahr pl - best ( % ) ahr pl - basal ( % ) ahr pl - mid ( % ) ahr lv apical ( % ) ahr lv septal ( % ) qrs width ( ms)max q - lv interval ( ms)location longest q - lvpt.0124.224.22.71.72.1135128pl - baspt.0331.631.625.84.826.1174165pl - baspt.0424.818.724.814.219.6196205pl - midpt.0520.120.118.018.111.8165163pl - midpt.0851.051.046.022.029.2154138pl - baspt.1219.013.519.017.613.7198176pl - midpt.1316.55.316.68.89.6152128pl - midpt.1566.066.050.231.347.3200181pl - midpt.1625.825.814.1 0.612.2175147pl - baspt.2019.719.716.78.916.3156124pl - midpt.2134.928.034.916.96.9174132pl - midpt.2234.334.320.315.614.4175155pl - baspt.2331.026.231.026.718.6160138pl - midpt.2424.122.924.117.211.5210192pl - mid30.2 13.627.7 15.224.6 12.614.8 9.117.1 11.2173 22155 26 ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax 15 % from endocardial pacing ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . ten patients were considered possible candidates for lv endocardial pacing : 9 patients in whom a rise in lvdp / dtmax 15 % level was only obtained by endocardial pacing , and the patient that showed a substantial additional rise in ahr with lv pacing compared with coronary sinus pacing ( patient 15 ) . of the remaining 9 patients , and after deliberation with the patients about the possible benefits and risks , 5 of them finally agreed to lv endocardial implant at the optimal lv site as indicated from the acute study . all five patients improved clinically with a reduction of at least 1 class in the nyha score after 6 months of follow - up . two patients with a positive response in the temporary study refrained from implantation of an lv endocardial lead . one patient with a positive ahr from endocardial pacing also improved significantly after changing stimulation from the distal to the proximal electrode with a more basal position , and became a clinical responder after optimisation of the atrioventricular and interventricular interval . in one patient , the deterioration of his condition after initial improvement proved to be the result of a blunted chronotropic response after increasing beta - blocker dosage following an episode of atrial fibrillation resulting in an inappropriate icd shock . notwithstanding that all patients were clinical non - responders , in 5 patients lvdp / dtmax with the implanted coronary sinus system increased 15 % after optimisation : average 30.2 % , 15.644.5 % ( table 2 ) . in 3 out of these 5 patients with the coronary sinus lead in a posterolateral position , endocardial pacing did not increase the lvdp / dtmax substantially ( ahr less than 3 % and even an adverse effect from lv endocardial pacing was observed in one patient ) . in one of the two remaining patients with an apical position of the coronary sinus lead , the ahr could be increased during lv endocardial pacing from 19.766 % and we considered the increase sufficient to justify the upgrade to lv endocardial pacing ( fig . 1 ) . table 2individual haemodynamic results of all clinical non - responders ( 24 patients)patientqrs morphologybaseline lvdp / dtmax ( mmhg / s)position cs leadahr cs lead ( % ) ahr cs - level - endo(%)lv endo optimal positionahr lvendo ( % ) pt . 07non - lbbb893pl - mid 23.4lv septal 21.5pt . 08lbbb827pl - bas44.951.0pl - bas51.0pt . 24rvp489lv apical12.717.2pl - mid24.1average891 2478.7 15.810.5 8.919.6 17.5haemodynamic measurements of 24 clinical nonresponders to crt . baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . grey shaded patients have an ahr from the cs lead 15 % and are considered haemodynamic responders , pl - bas basal posterolateral , pl - mid mid - posterolateral , rvp right ventricular pacing . individual haemodynamic results of all clinical non - responders ( 24 patients ) haemodynamic measurements of 24 clinical nonresponders to crt . baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . grey shaded patients have an ahr from the cs lead 15 % and are considered haemodynamic responders , pl - bas basal posterolateral , pl - mid mid - posterolateral , rvp right ventricular pacing . all views are in left anterior oblique ( lao ) showing the haemodynamic effects of stimulation from the coronary sinus lead and lv endocardial stimulation in the mid - posterolateral area ( left upper panel ) , basal posterolateral area ( left lower panel ) , lv septum ( right upper panel ) and lead positions after lv endocardial implantation of a permanent lead in the lv basal posterolateral segment . arrows indicate the position of the bipolar pacing lead angiographic pictures of the haemodynamic study in patient no . all views are in left anterior oblique ( lao ) showing the haemodynamic effects of stimulation from the coronary sinus lead and lv endocardial stimulation in the mid - posterolateral area ( left upper panel ) , basal posterolateral area ( left lower panel ) , lv septum ( right upper panel ) and lead positions after lv endocardial implantation of a permanent lead in the lv basal posterolateral segment . arrows indicate the position of the bipolar pacing lead in the 19 patients with an ahr from the implanted system < 15 % , 9 patients had an increase in ahr above the 15 % limit . the results of the total group of 24 patients showed a positive response in 11 patients with an apical lead position in 7 , anterolateral in 2 and mid - posterolateral in 2 patients . a negative response of endocardial pacing was observed in 13 patients with a basal posterolateral lead position in 3 , mid - posterolateral in 8 and apical in 2 patients . in 5 out of the 9 acute haemodynamic non - responders ( ahr < 15 % ) with lbbb , the ahr could be increased above the 15 % level by lv endocardial pacing . the average ahr with the coronary sinus system was 9.6 % ( 5.814.9 % ) and at the lv endocardial optimal position 25.8 % ( 19.034.3 % ) . the average qrs width in these patients was 169 ms ( 135198 ms ) and the average q - lv at the optimal lv endocardial pacing site 157 ms ( 128176 ms ) , resulting in a q - lv / qrs ratio of 93 % . the position of the original coronary sinus lead was apical in 3 , anterolateral in 1 and mid - posterolateral in 1 patient ( fig . 2 ) . 2recordings of the temporary study of patient no . 22 with a lbbb showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions recordings of the temporary study of patient no . 22 with a lbbb showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions in the remaining 4 patients , the average ahr at the optimal endocardial position increased < 15 % , with an average of 7.3 % ( 1.79.9 % ) . the average qrs width in these patients was 176 ms ( 149209 ms ) and the average q - lv 136 ms ( 100174 ms ) , resulting in a q - lv / qrs ratio of 83 % . four patients did not have lbbb . of these , only one patient with rbbb , combined with left anterior hemiblock , showed an increase in the ahr , from 9.1 % ( apical coronary sinus position ) to 25.8 % at an endocardial basal posterolateral position . 3 ) ; one had a non - specific intraventricular conduction delay with a qrs complex of 175 ms . the coronary sinus lead position was mid - posterolateral in 2 and apical in 1 patient . 3recordings of a temporary study of patient no.18 with non - lbbb , showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions . this recording illustrates that there is no conduction delay in the lv and a minimal haemodynamic effect recordings of a temporary study of patient no.18 with non - lbbb , showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions . this recording illustrates that there is no conduction delay in the lv and a minimal haemodynamic effect three out of the 6 patients with rv pacing became haemodynamic responders ( lv dp / dtmax from average 9.7 % , ( 9.312.7 % ) increased to 21.8 % ( 16.624.8 % ) ) . in the other 3 patients the average ahr increased from 2.9 % ( 1.14.0 % ) to 9.7 % ( 7.811.4 % ) . in 2 patients the coronary sinus lead was in a mid - posterolateral position , one in an apical position . individual details are summarised in table 2 and a flow chart overview in fig . 4 . 4flow chart of the study showing the haemodynamic results in relation to the coronary sinus lead positions . ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular flow chart of the study showing the haemodynamic results in relation to the coronary sinus lead positions . ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular when pacing endocardially , opposite the epicardial coronary sinus lead , we found that lvdp / dtmax did not differ significantly for any of the patients : lvdp / dtmax from the coronary sinus averaged 1046 292 mmhg / s vs. 1068 296 mmhg / s from the corresponding lv endocardial site ( p = 0.37 ) . we found that basal or mid - posterolateral segments had the highest ahr of the tested endocardial sites in all 14 patients who showed an ahr 15 % rise in lvdp / dtmax compared with baseline . because either the basal or mid - posterolateral segment had the highest ahr , we also calculated the combined best results from basal posterolateral and mid - posterolateral ( pl - best ) . pl - best had a rise to 30.2 13.6 % in lvdp / dtmax , basal posterolateral 27.7 15.2 % , mid - posterolateral 24.6 12.6 % , lv apical 14.8 9.1 % and lv septal 17.0 11.2 % . we also found that in 12 out of the 14 patients ( 85 % ) with an ahr 15 % , the lv endocardial site with the longest time interval between the onset of the qrs complex and lv sensing ( q - lv interval ) resulted in the best haemodynamic response . the average qrs width was 133 22 ms ( range 120205 ms ) and average q - lv interval 155 26 ms ( 128205 ms ) . the average q - lv / qrs width ratio , which is q - lv expressed as a percentage of qrs width , was 90 % . table 3haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax 15 % from endocardial pacingpts.ahr pl - best ( % ) ahr pl - basal ( % ) ahr pl - mid ( % ) ahr lv apical ( % ) ahr lv septal ( % ) qrs width ( ms)max q - lv interval ( ms)location longest q - lvpt.0124.224.22.71.72.1135128pl - baspt.0331.631.625.84.826.1174165pl - baspt.0424.818.724.814.219.6196205pl - midpt.0520.120.118.018.111.8165163pl - midpt.0851.051.046.022.029.2154138pl - baspt.1219.013.519.017.613.7198176pl - midpt.1316.55.316.68.89.6152128pl - midpt.1566.066.050.231.347.3200181pl - midpt.1625.825.814.1 0.612.2175147pl - baspt.2019.719.716.78.916.3156124pl - midpt.2134.928.034.916.96.9174132pl - midpt.2234.334.320.315.614.4175155pl - baspt.2331.026.231.026.718.6160138pl - midpt.2424.122.924.117.211.5210192pl - mid30.2 13.627.7 15.224.6 12.614.8 9.117.1 11.2173 22155 26 ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax 15 % from endocardial pacing ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . ten patients were considered possible candidates for lv endocardial pacing : 9 patients in whom a rise in lvdp / dtmax 15 % level was only obtained by endocardial pacing , and the patient that showed a substantial additional rise in ahr with lv pacing compared with coronary sinus pacing ( patient 15 ) . of the remaining 9 patients , and after deliberation with the patients about the possible benefits and risks , 5 of them finally agreed to lv endocardial implant at the optimal lv site as indicated from the acute study . all five patients improved clinically with a reduction of at least 1 class in the nyha score after 6 months of follow - up . two patients with a positive response in the temporary study refrained from implantation of an lv endocardial lead . one patient with a positive ahr from endocardial pacing also improved significantly after changing stimulation from the distal to the proximal electrode with a more basal position , and became a clinical responder after optimisation of the atrioventricular and interventricular interval . in one patient , the deterioration of his condition after initial improvement proved to be the result of a blunted chronotropic response after increasing beta - blocker dosage following an episode of atrial fibrillation resulting in an inappropriate icd shock . this observational study showed that endocardial lv pacing , guided by acute haemodynamic studies , could improve clinical outcome in patients not responding to conventional crt . it also demonstrated the importance of lead location , as the clinical response improved with a posterolateral position of the lead instead of the original apical or anterolateral lead location . conversely , none of the patients who already had a coronary sinus lead in a posterolateral position improved sufficiently with endocardial pacing at any site during the acute haemodynamic study to justify endocardial lead implant . this is in line with the madit - crt trial , where the apical and anterior positions of the lv lead had a lower clinical response . noteworthy , and similar to what has previously been reported , endocardial pacing opposite the coronary sinus pacing site did not improve the ahr in our study [ 1113 ] . this is in contrast with animal experiments that show significantly better haemodynamic results with endocardial vs. epicardial pacing at the same location . there is no clear single cause for this difference and it is most probably related to variance in the anatomical and electrophysiological substrate . besides this , in animal experience the lead configuration guarantees an exact position of the endocardial lead opposite to the epicardial position ; this in contrast with human studies in which the endocardial lead is placed as close as possible to its epicardial counterpart . therefore , although not examined in this and other studies , one either might speculate that epicardial pacing opposite the optimal endocardial sites , via the coronary sinus or surgically by thoracoscopy , might have resulted in similar benefits . if feasible , this might be a good alternative to avoid the uncertainty of thromboembolic complications with lv endocardial pacing . our observational study also showed some limitations in the use of acute haemodynamic studies to predict clinical outcome . first , five clinical non - responders showed a clear ahr according to the accepted criteria ( even with the higher than usual cut - off value of 15 % increase in lvdp / dtmax to exclude borderline cases ) but failed to respond clinically . still , when a substantial further increase in lvdp / dtmax from endocardial pacing at a posterolateral position compared with the apical coronary sinus lead ( 66 vs. 19.7 % ) was obtained in one patient , endocardial lead implantation resulted in a positive clinical response . this illustrates the limitation of cut - off values in crt studies of the 15 % level as surrogate for differentiating between clinical response and non - response , and that what is considered a positive haemodynamic response can still be significantly improved in the presence of an anterior or apical lead position . a similar discrepancy is found with echocardiography as the observed presence or absence of decrease in lv end - diastolic volume or end - systolic volume and clinical response does not always correlate . to err on the safe side , we therefore proceeded to a permanent lv endocardial implantation when the final increase in ahr was at least 25 % . the best ahr correlates well with the longest time interval between onset qrs and lv sensing in relation to the qrs width in the individual patient . the results of the group of patients with non - lbbb are in line with what could be expected according to the literature . the absence of response correlated well to the lack of conduction delay in any of the lv segments on the intracardiac lv electrogram . the only responder to endocardial pacing in this group had a significant lv conduction delay due to the left anterior hemiblock that accompanied rbbb . it is therefore questionable if one should proceed with endocardial mapping in this cohort of patients given the disappointing results . this observational study showed that a temporary acute haemodynamic study could be useful in non - responders to establish the chance of improvement with alternative pacing sites before embarking on complex procedures as endocardial lv pacing . it remains , however , uncertain what the optimal cut - off value for increase in lvdp / dtmax is before the improvement justifies intervention . the longest q - lv interval is a reliable indicator for the optimal electrode position in the individual patient . endocardial pacing opposite epicardial sites does not show acute haemodynamic improvement , so epicardial pacing at the optimal site may be a good alternative for endocardial lv lead placement . although all patients who had a lv endocardial system implanted after a positive ahr improved clinically , larger randomised series are necessary to justify this technique before definitive conclusions can be drawn . van gelder has received training and education on pacing and crt from st jude medical , is a consultant with radi pressure wire systems , a st jude medical company , and consultant for pacing and crt with sorin group crm sas . b.m . van gelder has received training and education on pacing and crt from st jude medical , is a consultant with radi pressure wire systems , a st jude medical company , and consultant for pacing and crt with sorin group crm sas .

introductionnon response to cardiac resynchronisation therapy ( crt ) may be related to the position of the coronary sinus lead.methodswe studied the acute haemodynamic response ( ahr ) from alternative left ventricular ( lv ) endocardial pacing sites in clinical non - responders to crt . ahr and the interval from qrs onset to lv sensing ( q - lv interval ) from four different endocardial pacing sites were evaluated in 24 clinical non - responders . a rise in lvdp / dtmax 15 % from baseline was considered a positive ahr . we also compared the ahr from endocardial with the corresponding epicardial lead position.resultsthe implanted system showed an ahr 15 % in 5 patients . in 9 of the 19 remaining patients , ahr could be elevated to 15 % by endocardial lv pacing . the optimal endocardial pacing site was posterolateral . there was no significant difference in ahr between the epicardial and the corresponding endocardial position . the longest q - lv interval corresponded with the best ahr in 12 out of the 14 patients with a positive ahr , with an average q - lv / qrs width ratio of 90 % .conclusionsacute haemodynamic testing may indicate an alternative endocardial pacing site with a positive ahr in clinical non - responders . the q - lv interval is a strongly correlated with the optimal endocardial pacing site . endocardial pacing opposite epicardial sites does not result in a better ahr .

Introduction Patients and methods Results Acute haemodynamic response of the total study population Non-responders with LBBB Non-responders with non-LBBB Non-responders with right ventricular pacing Epicardial vs endocardial haemodynamics Haemodynamics during endocardial pacing from different locations Haemodynamic effect and Q-LV interval Follow-up Discussion Conclusions Disclosures

cardiac resynchronisation therapy ( crt ) has become an important treatment for patients with heart failure and left ventricular ( lv ) dyssynchrony [ 13 ] . we studied the acute haemodynamic response ( ahr ) of the implanted system and alternative left ventricular endocardial pacing sites in patients clinically not responding to crt . a rise in lvdp / dtmax 15 % from baseline was considered to be a positive haemodynamic response . in all patients with an ahr of 15 % during lv endocardial pacing , the interval between the onset of the qrs complex and the intrinsic activation at the lv electrode ( q - lv interval ) was measured and the ratio between q - lv / qrs width interval calculated . notwithstanding that all patients were clinical non - responders , in 5 patients lvdp / dtmax with the implanted coronary sinus system increased 15 % after optimisation : average 30.2 % , 15.644.5 % ( table 2 ) . in 3 out of these 5 patients with the coronary sinus lead in a posterolateral position , endocardial pacing did not increase the lvdp / dtmax substantially ( ahr less than 3 % and even an adverse effect from lv endocardial pacing was observed in one patient ) . in one of the two remaining patients with an apical position of the coronary sinus lead , the ahr could be increased during lv endocardial pacing from 19.766 % and we considered the increase sufficient to justify the upgrade to lv endocardial pacing ( fig . baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . arrows indicate the position of the bipolar pacing lead in the 19 patients with an ahr from the implanted system < 15 % , 9 patients had an increase in ahr above the 15 % limit . in 5 out of the 9 acute haemodynamic non - responders ( ahr < 15 % ) with lbbb , the ahr could be increased above the 15 % level by lv endocardial pacing . the average qrs width in these patients was 169 ms ( 135198 ms ) and the average q - lv at the optimal lv endocardial pacing site 157 ms ( 128176 ms ) , resulting in a q - lv / qrs ratio of 93 % . 22 with a lbbb showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions in the remaining 4 patients , the average ahr at the optimal endocardial position increased < 15 % , with an average of 7.3 % ( 1.79.9 % ) . the average qrs width in these patients was 176 ms ( 149209 ms ) and the average q - lv 136 ms ( 100174 ms ) , resulting in a q - lv / qrs ratio of 83 % . ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular flow chart of the study showing the haemodynamic results in relation to the coronary sinus lead positions . ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular when pacing endocardially , opposite the epicardial coronary sinus lead , we found that lvdp / dtmax did not differ significantly for any of the patients : lvdp / dtmax from the coronary sinus averaged 1046 292 mmhg / s vs. 1068 296 mmhg / s from the corresponding lv endocardial site ( p = 0.37 ) . we found that basal or mid - posterolateral segments had the highest ahr of the tested endocardial sites in all 14 patients who showed an ahr 15 % rise in lvdp / dtmax compared with baseline . we also found that in 12 out of the 14 patients ( 85 % ) with an ahr 15 % , the lv endocardial site with the longest time interval between the onset of the qrs complex and lv sensing ( q - lv interval ) resulted in the best haemodynamic response . the average q - lv / qrs width ratio , which is q - lv expressed as a percentage of qrs width , was 90 % . table 3haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax 15 % from endocardial pacingpts.ahr pl - best ( % ) ahr pl - basal ( % ) ahr pl - mid ( % ) ahr lv apical ( % ) ahr lv septal ( % ) qrs width ( ms)max q - lv interval ( ms)location longest q - lvpt.0124.224.22.71.72.1135128pl - baspt.0331.631.625.84.826.1174165pl - baspt.0424.818.724.814.219.6196205pl - midpt.0520.120.118.018.111.8165163pl - midpt.0851.051.046.022.029.2154138pl - baspt.1219.013.519.017.613.7198176pl - midpt.1316.55.316.68.89.6152128pl - midpt.1566.066.050.231.347.3200181pl - midpt.1625.825.814.1 0.612.2175147pl - baspt.2019.719.716.78.916.3156124pl - midpt.2134.928.034.916.96.9174132pl - midpt.2234.334.320.315.614.4175155pl - baspt.2331.026.231.026.718.6160138pl - midpt.2424.122.924.117.211.5210192pl - mid30.2 13.627.7 15.224.6 12.614.8 9.117.1 11.2173 22155 26 ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax 15 % from endocardial pacing ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . ten patients were considered possible candidates for lv endocardial pacing : 9 patients in whom a rise in lvdp / dtmax 15 % level was only obtained by endocardial pacing , and the patient that showed a substantial additional rise in ahr with lv pacing compared with coronary sinus pacing ( patient 15 ) . one patient with a positive ahr from endocardial pacing also improved significantly after changing stimulation from the distal to the proximal electrode with a more basal position , and became a clinical responder after optimisation of the atrioventricular and interventricular interval . notwithstanding that all patients were clinical non - responders , in 5 patients lvdp / dtmax with the implanted coronary sinus system increased 15 % after optimisation : average 30.2 % , 15.644.5 % ( table 2 ) . in 3 out of these 5 patients with the coronary sinus lead in a posterolateral position , endocardial pacing did not increase the lvdp / dtmax substantially ( ahr less than 3 % and even an adverse effect from lv endocardial pacing was observed in one patient ) . in one of the two remaining patients with an apical position of the coronary sinus lead , the ahr could be increased during lv endocardial pacing from 19.766 % and we considered the increase sufficient to justify the upgrade to lv endocardial pacing ( fig . baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . baseline lvdp / dtmax lvdp / dtmax with intrinsic rhythm or right ventricular pacing , ahr cs lead acute haemodynamic response from the coronary sinus ( cs ) lead expressed as percentage rise in lvdp / dtmax from baseline , ahr cs level endo acute haemodynamic response at an endocardial location opposite the cs lead , lv endo optimal position anatomic site with the highest ahr , ahr lv endo acute haemodynamic response from the optimal endocardial position . arrows indicate the position of the bipolar pacing lead in the 19 patients with an ahr from the implanted system < 15 % , 9 patients had an increase in ahr above the 15 % limit . in 5 out of the 9 acute haemodynamic non - responders ( ahr < 15 % ) with lbbb , the ahr could be increased above the 15 % level by lv endocardial pacing . the average qrs width in these patients was 169 ms ( 135198 ms ) and the average q - lv at the optimal lv endocardial pacing site 157 ms ( 128176 ms ) , resulting in a q - lv / qrs ratio of 93 % . 22 with a lbbb showing the haemodynamic effects and the timing of the lv endocardial electrogram from the different positions in the remaining 4 patients , the average ahr at the optimal endocardial position increased < 15 % , with an average of 7.3 % ( 1.79.9 % ) . the average qrs width in these patients was 176 ms ( 149209 ms ) and the average q - lv 136 ms ( 100174 ms ) , resulting in a q - lv / qrs ratio of 83 % . ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular flow chart of the study showing the haemodynamic results in relation to the coronary sinus lead positions . ahr acute haemodynamic response , ant lat anterolateral , bas basal , cs coronary sinus , endo endocardial , lbbb left bundle branch block , lv left ventricular , pl posterolateral , pts patients , rv right ventricular when pacing endocardially , opposite the epicardial coronary sinus lead , we found that lvdp / dtmax did not differ significantly for any of the patients : lvdp / dtmax from the coronary sinus averaged 1046 292 mmhg / s vs. 1068 296 mmhg / s from the corresponding lv endocardial site ( p = 0.37 ) . we found that basal or mid - posterolateral segments had the highest ahr of the tested endocardial sites in all 14 patients who showed an ahr 15 % rise in lvdp / dtmax compared with baseline . we also found that in 12 out of the 14 patients ( 85 % ) with an ahr 15 % , the lv endocardial site with the longest time interval between the onset of the qrs complex and lv sensing ( q - lv interval ) resulted in the best haemodynamic response . the average q - lv / qrs width ratio , which is q - lv expressed as a percentage of qrs width , was 90 % . table 3haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax 15 % from endocardial pacingpts.ahr pl - best ( % ) ahr pl - basal ( % ) ahr pl - mid ( % ) ahr lv apical ( % ) ahr lv septal ( % ) qrs width ( ms)max q - lv interval ( ms)location longest q - lvpt.0124.224.22.71.72.1135128pl - baspt.0331.631.625.84.826.1174165pl - baspt.0424.818.724.814.219.6196205pl - midpt.0520.120.118.018.111.8165163pl - midpt.0851.051.046.022.029.2154138pl - baspt.1219.013.519.017.613.7198176pl - midpt.1316.55.316.68.89.6152128pl - midpt.1566.066.050.231.347.3200181pl - midpt.1625.825.814.1 0.612.2175147pl - baspt.2019.719.716.78.916.3156124pl - midpt.2134.928.034.916.96.9174132pl - midpt.2234.334.320.315.614.4175155pl - baspt.2331.026.231.026.718.6160138pl - midpt.2424.122.924.117.211.5210192pl - mid30.2 13.627.7 15.224.6 12.614.8 9.117.1 11.2173 22155 26 ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . haemodynamic results for all 4 endocardial locations and maximum q - lv interval for 14 patients that showed a rise in lvdp / dtmax 15 % from endocardial pacing ahr acute haemodynamic response expressed as the percentage rise in lvdp / dt from baseline , pl - best best result from either basal posterolateral ( pl - bas ) or mid - posterolateral ( pl - mid ) region , max q - lv longest interval measured between onset of the qrs complex and intrinsic activation at the lv electrode . grey shaded area indicates 2 patient in whom the longest q - lv interval did not correspond with the best haemodynamic response . ten patients were considered possible candidates for lv endocardial pacing : 9 patients in whom a rise in lvdp / dtmax 15 % level was only obtained by endocardial pacing , and the patient that showed a substantial additional rise in ahr with lv pacing compared with coronary sinus pacing ( patient 15 ) . one patient with a positive ahr from endocardial pacing also improved significantly after changing stimulation from the distal to the proximal electrode with a more basal position , and became a clinical responder after optimisation of the atrioventricular and interventricular interval . first , five clinical non - responders showed a clear ahr according to the accepted criteria ( even with the higher than usual cut - off value of 15 % increase in lvdp / dtmax to exclude borderline cases ) but failed to respond clinically . still , when a substantial further increase in lvdp / dtmax from endocardial pacing at a posterolateral position compared with the apical coronary sinus lead ( 66 vs. 19.7 % ) was obtained in one patient , endocardial lead implantation resulted in a positive clinical response . the longest q - lv interval is a reliable indicator for the optimal electrode position in the individual patient . endocardial pacing opposite epicardial sites does not show acute haemodynamic improvement , so epicardial pacing at the optimal site may be a good alternative for endocardial lv lead placement .

the interplay of kinetic and thermodynamic factors in regulating water partitioning between the condensed and gas phases in atmospheric aerosol is a subject of ongoing investigation . an assumption is frequently made that the activation of aerosol to form cloud condensation nuclei at supersaturated relative humidities ( rhs ) , and the hygroscopic growth in particle size at subsaturated rhs , are both regulated purely by thermodynamic principles ; the partitioning of water ( and any other semivolatile components ) between the condensed and gas phases is estimated according to aerosol volume rather than surface area . recently , the existence of secondary organic aerosol in kinetically arrested glassy states has been identified , with implications for the partitioning of water between the gas and condensed phases . aerosol particles with a highly viscous bulk , characterized as a rubber or glassy state , and with low molecular diffusivity are likely to exist in a state of disequilibrium from the surrounding gas phase due to the slow rate for the bulk transport of water . interpreting the molecular mechanism for the condensation or evaporation of water to / from an aerosol particle requires an understanding of the gas phase transport to the particle surface , the transport across the surface boundary and transport into the particle bulk . further , it has been recommended that the mass accommodation coefficient ( m ) for describing the kinetics for crossing the surface boundary be separated into surface and bulk accommodation contributions to discriminate between the kinetics of the surface accommodation process and the transport between the surface and near - surface bulk . low molecular diffusivity into the particle bulk may hinder condensation or evaporation through a slowing of bulk accommodation . in describing the kinetics of transport across the surface boundary , we choose here to refer to the mass accommodation coefficient rather than either the surface or bulk accommodation coefficients as we will discuss measurements that have reported this value for the uptake of water vapor at a liquid water surface . for this system , the transport between the surface and the bulk liquid is likely fast enough to justify the use of a single coefficient for the overall absorption process ( see , e.g. , shiraiwa et al . ) . the mass accommodation coefficient will also be used interchangeably with the evaporation coefficient , depending on the process under study ; the values for these two coefficients are assumed to be equivalent by microscopic reversibility . as a further consideration , molecular transport between the condensed and gas phases leads to the deposition or loss of heat from a droplet during condensation and evaporation , respectively , and the transport of heat must be understood if the molecular mass flux is to be rationalized . typically , for a particle of finite volume , energy is dissipated from the particle into the gas phase during condensation , and the heat flux between the droplet and the gas is dependent on the efficiency with which gas molecules colliding with the surface are able to transfer energy on collision with the condensed phase . this efficiency is quantified by a thermal accommodation coefficient ( t ) , the probability that an outgoing molecule , having scattered from the surface , is in thermal equilibrium with the surface . in this manuscript , we assume the thermal accommodation coefficient for gas phase molecules colliding with an aqueous solution to be unity , in agreement with experimental data found in the water growth literature ( see , e.g. , refs ( 18 ) , ( 20 ) , and ( 22 ) ) . it should be recognized that a value of unity is not observed for a wide range of other systems , with , for example , studies of the brownian motion of liquid oil droplets implying thermal accommodation coefficients on the order of 0.9 . however , for the purposes of this study we will not explore values of t other than unity . measurements of the mass accommodation coefficient of gas phase water at a liquid water surface have a long and contentious history with values spanning a range from 0.001 to 1 . indeed , even over the past decade the mass accommodation coefficient has been measured by a number of different approaches and reported values have spanned a range from 0.05 to 1.0 , albeit converging to the upper end of the earlier measurements . further , the influence of surface composition ( e.g. , the presence of a surface active organic component ) on mass accommodation coefficients remains ambiguous . confidence in the exact value of m is crucial , as it is a key determining factor in estimating the activated fraction of aerosol and the cloud droplet number in cloud parcel models , influencing the maximum supersaturation achieved in clouds . indeed , the uncertainty in this value could be a significant contributor to the uncertainty in the radiative forcing estimated for the indirect effect of aerosols on climate . at a limiting value for m of 1.0 , the gas phase diffusion of water vapor to the droplet surface and the removal of heat limit the rate of condensational growth , and cloud droplet growth is insensitive to the surface accommodation kinetics . if the value of the mass accommodation coefficient were to be below 0.1 , condensational growth would be limited by surface kinetics leading to an increased value for the water vapor saturation in clouds , with the consequence that a larger fraction of the aerosol distribution would be activated . in short , a large mass accommodation coefficient leads to a lower cloud droplet number concentration but larger droplets on average ; a small mass accommodation coefficient leads to a larger cloud droplet number concentration but smaller droplets on average . clouds containing a large number of smaller droplets are known to have a higher reflectivity and persist for longer in the atmosphere , increasing their radiative forcing effect . measurements of water condensation / evaporation at a range of pressures , water saturations ( relative humidities , rh ) , and temperatures are required to fully resolve the kinetics of the mass accommodation process from the limitations to the rate imposed by gas diffusion and to ensure consistency across a broad range of environmental conditions . to interpret these measurements , calculations of the mass and heat transfer between the gas and condensed phases are needed . the mass flux of water to or from a droplet during condensation or evaporation , respectively , is dependent on the concentration gradient of water in the gas phase . to calculate the mass flux , constraining the value of m from condensation / evaporation data requires knowledge of the pressure , temperature , and compositional dependence of these key thermophysical parameters and transport coefficients , such as the gas phase diffusion coefficients and thermal conductivities of the components . here , we assess the influence of the uncertainties associated with both these transport coefficients and the experimental environmental conditions , particularly the saturation / rh , in interpreting measurements of evaporation or condensation of water . in section ii we briefly review a semianalytic framework for calculating the molecular flux during condensation / evaporation and the time dependence in droplet size , an approach that can be used to study the condensation / evaporation of spherical liquid droplets . in section iii we assess the uncertainties associated with the key thermophysical properties required to predict evaporative or condensational fluxes , specifically the diffusion coefficients and gas phase thermal conductivities , the saturation vapor pressure of water and the enthalpy of vaporization of water . finally , in section iv we consider the impact of these uncertainties in interpreting measurements made with a range of experimental techniques that have been used to determine the mass accommodation coefficient of water . the techniques considered here include droplet growth measurements in a cloud expansion chamber , cloud condensation nuclei activation measurements in a flow tube instrument , condensation measurements with optical tweezers , and evaporation measurements with an electrodynamic balance . although there are differences between these approaches , the same semianalytic framework treating spherical liquid droplets can be used to simulate all of them . in addition , we identify the key quantities that determine the uncertainty with which the mass accommodation coefficient can be estimated from each of these approaches . the condensation ( evaporation ) rate to ( from ) a noninteracting liquid droplet suspended in a gaseous medium can be represented by applying the basic theories of mass and heat transport for a spherically symmetric system ( see , e.g. , refs ( 1 ) , ( 16 ) , ( 19 ) , and ( 5760 ) ) . the appropriate theory is chosen on the basis of the nondimensional size of the droplet , using the knusden number kn which is defined as the ratio of the mean free path of the gaseous medium and the radius of the studied droplet . if kn 1 ( continuum regime ) , the mass and heat transfer can be modeled with continuum theories such as diffusion and thermal conduction , whereas at kn 1 ( kinetic regime ) , kinetic gas theory can be used to model the collisions between the particle and the surrounding gas phase molecules . at kn values close to unity ( transition regime ) , the standard approach is to match the continuum and kinetic theories yielding a framework that applies over the whole kn space and reproduces the continuum and kinetic theories as the kn values approach 0 and infinity , respectively ( see , e.g. , refs ( 1 ) and ( 19 ) and references therein ) . have presented a semianalytic approach for predicting the rate of mass transfer between a liquid droplet and the surrounding gas phase , which can be applied in situations where the temperature gradients in the system are sufficiently small for the droplet temperature to remain approximately constant over each time step . the mass flux ( i , kg s ) of the condensing vapor ( in our case water ) on a droplet of radius r ( m ) can be written as1where mv is the molecular mass ( kg ) , dim is the binary diffusion coefficient of the vapor in the surrounding gas mixture ( m s ) , l is the enthalpy of vaporization of the vapor ( j kg ) , k is the thermal conductivity of the gas mixture ( w m k ) , pe(t ) is the equilibrium pressure of the vapor ( pa ) at the temperature of the gas phase far removed from the droplet , t , and r is the ideal gas constant ( j k mol ) . the degree of saturation of the vapor at infinite distance is given by s , and sr is the corresponding value at the droplet surface . the initial saturation values at t = 0 s when the droplet is in thermal equilibrium with the gas can be written as23where pr(t ) is the vapor pressure at the droplet surface including the kelvin correction and p(t ) is the partial pressure of the vapor at infinite distance . these are referenced to the equilibrium pressure of the vapor at the temperature of the gas phase above its pure liquid phase . implicit to eq 1 is an accounting for the influence of the change in droplet surface temperature on the mass flux to or from the droplet . more specifically , including the second term in the denominator of eq 1 accounts for the suppression of the growth rate due to elevation of the surface temperature by the latent heat produced during condensation , or conversely , the reduction in the evaporation rate due to depression of the surface temperature during evaporation . equation 1 holds for quasistationary droplet growth / evaporation by ordinary diffusion , where the droplet surface is in thermodynamic equilibrium with the gas layer just adjacent to it . the term a in eq 1 accounts for the influence of convective stefan flow on the flux:4where ptot is the total gas pressure ( pa ) . if the role of stefan flow is neglected ( a 1 ) and the second term in the denominator of eq 1 is ignored , the equation reduces to the conventional expression for isothermal mass transfer including the gas - diffusional correction ( see , e.g. , ref ( 60 ) ) . the transitional correction factors for mass and heat transfer accounting for the different kn regimes are given in eq 1 by m and t , respectively . use the fuchs sutugin transitional correction factors , which can be expressed in the form5where kni and i refer to the knudsen number for either mass ( i = m ) or heat ( i = t ) transfer , and the mass or thermal accommodation coefficients , respectively . knm is defined as the ratio of the mean free path of water molecules to the particle radius . we take the definition of knt reported by wagner as the ratio of the mean free path of the components responsible for heat transfer ( bath gas and water ) to the particle radius . the effective mean free paths for the mass transfer ( m ) and heat transfer ( t ) are calculated from the appropriate transport coefficients as67cv is the specific heat capacity of the gas at constant volume ( j k kg ) , is the mass concentration of the gas ( kg m ) , and c is the average mean speed of the gas molecules ( m s ) . two approximations are made in the derivation of eq 1 and the significance of the regimes under which these approximations fail must be noted . the first approximation determines the scaling of the vapor pressure at the droplet surface by the temperature change incurred due to the latent heat generated / removed during condensation / evaporation , eqs 19 and 22 of ref ( 16 ) . in short , the approximation made is8where ta is the temperature of the droplet surface . if the vapor is water , the difference between the exact and approximate values for this exponential is larger than 5% when the temperature difference exceeds 6 k at 300 k. we consider that eq 1 must be used with care if the difference between the temperatures of the droplet and gas exceeds this value . for the simulations presented later in this manuscript , the largest difference in temperature between the gas and particle is 5 k. the validity of using this treatment for examining sensitivities to the thermophysical parameters can therefore be assumed . the second approximation provides a simplification of the continuum regime mass flux expression , eqs 18 and 24 in ref ( 16 ) , leading to a mass flux that is linearly dependent on the vapor pressure . in short , the approximation made is9with a similar expression for the partial pressure of the vapor at infinite distance , p. given that the mass flux is directly proportional to the logarithmic quantity , when the partial pressure of the vapor is equal to the partial pressure of the buffer gas ( i.e. , air or nitrogen ) , the mass flux will be underestimated by 10% . at a temperature of 293 k and 100% rh , these conditions exist at a total gas pressure of 4.6 kpa when the gas phase is composed of 2.3 kpa of water and 2.3 kpa of air or nitrogen . when the total gas pressure is 10 kpa , the error in the approximation falls to less than 2% . thus , caution must be exercised when this semianalytical framework is used for modeling evaporation / condensation at low pressure when the partial pressure of the vapor constitutes more than 50% of the total pressure . equation 1 can be used to model the time evolution of a condensation or evaporation event by iterative propagation of time . for instance , in the expansion chamber work , knowledge of the initial temperature and supersaturation , total gas pressure , and particle size is required . for optical tweezers measurements , the initial droplet radius , the initial saturation ratio of water at the droplet surface and the final saturation ratio once the droplet has equilibrated with the gas phase , the temperature and the total gas pressure must be known . the key thermophysical parameters on which the semianalytic model relies are the vapor diffusion coefficient of water in the gas mixture , the enthalpy of vaporization of water from the solution and the thermal conductivity of the gas mixture . after each time step , the mass lost / gained from the droplet must be used to estimate the droplet radius for the beginning of the next time step from knowledge of the density of the droplet . a survey of mass accommodation studies published in the literature to date highlights the fact that there has been inconsistency in the parametrizations used for the thermophysical properties required for the analysis of kinetic measurements . in the following section we report the results of a literature survey of the diffusion coefficient , d , and thermal conductivity , k , for gas mixtures of air / water and nitrogen / water , with the aim of determining not only the most appropriate parametrizations to use but also their associated levels of uncertainty . any uncertainties in the values of d and k will propagate through the analysis to give limitations on the precision with which m and t can be determined . we also briefly review the accuracies of values of the saturation vapor pressure of water , density of solution , and the enthalpy of vaporization from water and aqueous solutions . for mass transfer of water to occur between a particle and a surrounding gas phase , water vapor molecules must diffuse to or from the droplet surface . the rate of diffusion is related to the magnitude of the gradient driving it , the concentration gradient , via a constant of proportionality known as the diffusion coefficient ( see eq 1 ) . the diffusion coefficient for water vapor in gaseous nitrogen or air is pressure dependent , increasing with a decrease in pressure and leading to an increased rate of diffusion to or from the particle surface . the diffusion coefficient of the vapor is dependent on the composition of the gas mixture with , for example , different values for diffusion in air and nitrogen . blanc s law relates the diffusion coefficient of a vapor i in a gas mixture , dim , to its diffusion coefficient in each of the pure components making up the mixture , dij , using the mole fraction , xj , of each component as a weighting.10 blanc s law is applicable in cases where the species i exists as a trace component in the mixture . some evaporation / condensation measurements have been carried out in air , some in nitrogen , and some in another gas such as helium , with measurements carried out in both humidified and unhumidified gas flows . we consider only the cases of air and nitrogen as a bath gas in the section below . the full details of the literature review are presented in the supporting information and only the conclusions will be presented here . eleven different parametrizations for calculating the diffusion coefficient of water in air , d(h2o air ) , as a function of temperature and pressure were found in the literature . detailed information on the origin and any stated uncertainty in each study is provided in the supporting information . both experimentally determined and theoretically predicted parametrizations were considered , as well as those that combined an element of both . for a temperature of 298.15 k , values of d(h2o air ) at 1 atm total pressure predicted by the different literature parametrizations were found to span the range 2.60 10 to 2.14 10 m s , a decrease of 17.6% . it is worth noting that numerous values for the interaction parameters and for air and water can be found in the literature , leading to differences in the diffusion coefficients calculated depending on which of the published values are used . following a critical examination of the different parametrizations , that of massman was deemed to be the most reliable as it arises from a locally weighted polynomial regression ( loess ) fit to 58 experimental d(h2o air ) values from a total of 27 different experiments . this was the largest body of solely water - in - air diffusion values considered in any of the parametrizations . all data were corrected to 1 atm pressure assuming an inverse pressure dependence and spanned the temperature range 273.15 to 373.15 k. the use of a loess fit allowed the identification and removal of anomalous data points from the fit . no attempt was made by the author to correct the body of literature data for compositional effects , the assumption being that each experiment was likely to possess more than one source of variability . the massman parametrization for calculating the diffusion coefficient of water in air ( cm s ) at the desired temperature ( t / k ) and pressure ( p / atm ) is given by11where p0 = 1 atm and t0 = 273.15 k. the value of d0 , equivalent to the diffusion coefficient at 273.15 k and 1 atm pressure , was found by the loess fit to be 0.2178 cm s. the value of was constrained as 1.81 on the basis of previous work . the absolute uncertainty in the massman fit for the diffusion coefficient of water in air is reported as 7% , representing the maximum percentage difference between the experimental data and the results of the loess fit . in the same paper , massman reports a parametrization for calculating the diffusion coefficient of water in nitrogen , which takes the same form as eq 11 . the value of d0 is marginally different from that for water in air , with a value of 0.2190 cm s. the absolute uncertainty associated with this parametrization is stated as 6% . for measurements performed at a range of total gas pressures but constant relative humidity , the relative proportions of water vapor and the gas components vary . in the extreme limit , when the total pressure equals the partial pressure of water , twelve different parametrizations have been reported for the self - diffusion coefficient for water - in - water vapor d(h2o h2o ) . h2o ) at 1 atm total pressure were found to span the range 2.59 10 to 3.29 10 m s , a decrease of 87% . nine of the studies gave diffusion coefficients at 298.15 k grouped within the range 1.90 10 to 1.46 10 m s , and these are considered to be the most accurate . these values can be compared with , for example , the reported values of d(h2o air ) at 1 atm total pressure which span the range 2.60 10 to 2.14 10 m s. thus , the diffusion coefficient for water in humid air / nitrogen can be expected to vary between the values in the dilute limit in dry air / nitrogen and the value for self - diffusion as the relative proportions of the gas phase constituents varies . recognizing that blanc s law is not strictly applicable to the case of calculating the diffusion coefficient for a mixture in which the trace vapor component is dominant and self - diffusion must be included , we choose to neglect explicitly this dependence on composition in our model calculations . it has been reported previously that such an assumption incurs at most an error of 5% in the diffusion coefficient . instead , we have limited the pressure range over which experimental measurements are simulated and recommend that when interpreting the sensitivity analyses in section iv for measurements at the lowest pressure ( 10 kpa total pressure , consisting of 3.1 kpa of water vapor at 100% rh and 298 k ) , the reader should consider that there exists a greater uncertainty in the binary diffusion coefficient at low pressure than at higher pressure . we have chosen not to be more explicit in treating the diffusion constant of the mixture here as the validity of the semianalytical treatment itself is questionable when the partial pressure of the vapor dominates the partial pressure of the gas . under such cirucmstances , a pressure gradient is established that can also be considered to drive mass flux . we shall return to a discussion of the conceptual problems in interpreting measurements at low pressure in our conclusions but limit the sensitivity analysis in section iv to a consideration of experimental regimes in which both the accuracy of the binary diffusion constant and the validity of eq 1 can be assumed . the conduction of heat to or from the droplet within the gas phase is critical in determining the time dependence of the surface temperature of the droplet and the steady state wet - bulb temperature , and thus plays an important role in governing the mass flux . it is therefore necessary to know the thermal conductivities for water vapor , air , nitrogen , and any other gases used in condensation / evaporation measurements . in addition , it is important to determine how to combine them to calculate the thermal conductivity of a mixture with specified composition . six different parametrizations were found in the literature for the thermal conductivity of air as a function of temperature and pressure . the origin of each of the parametrizations is given in the supporting information and only a brief overview is included here . the different parametrizations were found to match each other closely , with a decrease in the thermal conductivity of air of only 2.4% between the highest and lowest values calculated at a temperature of 298.15 k and a pressure of 1 atm . following an examination of the different parametrizations , the most reliable was judged to be a study originating from the university of idaho and the national institute of standards and technology ( nist ) by lemmon and jacobsen , currently used as the reference for the crc handbook . in this work , the authors performed a critical review of the literature and created a database of robust experimental values published over the temperature range 602500 k and pressure range 0.001101 mpa . these values were then fitted with a complex theoretical framework consisting of dilute gas , residual fluid , and critical enhancement terms to allow the thermal conductivity of air to be calculated accurately over a wide range of pressures and temperatures . for the purposes of the present study we need enskog theory with a collision integral fitted to experimental data and is suitable for measurements at atmospheric pressure and below . as the calculations even for this term are quite involved , we provide eq 12 , a quadratic fit to thermal conductivity values calculated using the dilute gas term over the temperature range 270300 k.12the uncertainty in the thermal conductivity of air calculated using this equation is 2% over the considered temperature range . similarly , the most reliable treatment for the thermal conductivity of nitrogen is also taken from the work of lemmon and jacobsen . as before , only the dilute term from the parametrization needs to be considered . a quadratic fit to the data calculated using this term over the temperature range 270 to 300 k takes the form13the uncertainty in the values calculated using this equation is 2% . six different parametrizations were found in the literature for the thermal conductivity of water vapor as a function of temperature and pressure . the origin of each of the parametrizations is given in detail in the supporting information and only a brief overview is included here . the different parametrizations were found to span the range 0.0191 to 0.0156 w m k at 298.15 k , a decrease of 18.3% . five of the parametrizations were found to give thermal conductivities closely grouped together , with the sixth predicting considerably lower values than the others . the parametrization considered to be the most reliable following the literature survey is that of sengers and watson , which originates from the nist and is based on a fit to experimental data . it is also endorsed by the international association for the properties of water and steam . using the dilute term of the parametrization only ( suitable for atmospheric pressure and below ) , a quadratic fit to values calculated over the temperature range 270 to 300 k gives14the uncertainty in the values calculated using this equation is 2% over the considered temperature range . seven different parametrizations for determining the thermal conductivity of a mixture were found in the literature , with two of these explicitly used for calculating the thermal conductivity of humid air . this is an empirical relationship based on kinetic theory and is shown in eq 15 for a binary mixture , requiring knowledge of the thermal conductivities of the pure components , ki , their mole fractions , xi , and the values of the parameters a12 and a21 , where 1 and 2 are the species of interest.15 the difference in the mixture thermal conductivities predicted by each of the parametrizations arises from the different methods used to calculate the parameters a12 and a21 . these are variously related to pure component viscosities , critical constants , molecular masses , normal boiling points , and the reduced temperature . the values of kmix predicted using each of the parametrizations were compared with experimental data from touloukian et al . , consisting of tabulated values for the thermal conductivity of a mixture of steam and air at 353.2 k with varying mole fractions of air . where viscosities were required to determine a12 and a21 , the viscosity of water was taken from work by huber et al . ( associated uncertainty 2% ) and the viscosities of air and nitrogen from lemmon and jacobsen ( associated uncertainties 1% and 0.5% , respectively ) . these studies were deemed the most appropriate following a survey of the literature , details of which are provided in the supporting information . it was found that for the seven different parametrizations for kmix tested , none reproduced the magnitude of the experimental data well and only three were able to reproduce the curvature seen in the thermal conductivity with increasing mole fraction of air ( figure s7 in the supporting information ) . of these , the parametrization of lindsay and bromley was chosen for use in this study , as it was reported in the original paper to reproduce 85 mixture thermal conductivities for 16 gas pairs from the literature with an average deviation of 1.9% ( 1% for water in air ) . saul and wagner and wagner and pruss have provided a parametrization for the temperature dependence of the saturation vapor pressure of water with an associated uncertainty reported to be 0.025% , consistent with the values reported by haar et al . uncertainties in the saturation vapor pressure lead to uncertainties in the mass flux calculated from eq 1 during condensation or evaporation . the uncertainties in the experimental saturation value are generally considerably larger than this small uncertainty in the saturation vapor pressure and so the uncertainty in this latter quantity can be largely neglected . however , the parametrization for the saturation vapor pressure used by winkler et al . provided by wukalowitsch systematically underestimates the vapor pressure by 0.2% ( > 5 pa ) in the temperature range of interest . the consequences of this will be discussed in section iv . for the sensitivity analyses presented in section iv , measurements on a range of mixed component aerosols are reviewed , including aqueous ammonium sulfate and sodium chloride aerosol , and pure water aerosol growing in supersaturated conditions . the enthalpy for vaporization of water from pure liquid water is reported as 43.98 kj mol at 298 k. values for the latent heat for ten aqueous solutions of sodium and ammonium salts at the saturation concentration have been reported by apelblat et al . these values span the range from 43.37 to 45.00 kj mol with the value for sodium chloride being 44.24 kj mol . given that most of the measurements assessed in section iv are made on aerosol in the dilute limit , the enthalpy for vaporization of pure water is assumed , consistent with the value used in most of the literature ( e.g. , see refs ( 18 ) and ( 48 ) ) . however , given that some of the aerosol evaporation / condensation measurements will pass through states with significant salt concentrations and may also be somewhat susceptible to the slight dependence of the enthalpy of vaporization on temperature , it is not unreasonable to consider the sensitivity of the different techniques to an uncertainty in the enthalpy of vaporization of 0.75 kj mol or 1.7% . this magnitude of error could be incurred by simply ignoring the compositional and temperature dependence of the enthalpy of vaporization . the density of solution is required to convert the change in particle mass calculated from the flux eq 1 to a change in droplet radius . for many of the sensitivity studies that will be performed the temperature dependence of the density of pure water is known accurately and a parametrization that is valid over the temperature range 0150 c has been provided by popiel and wojtkowiak . the estimated uncertainty in density calculated from this parametrization is between 0.002% and 0.004% . use of a parametrization provided by winkler et al . incurs a systematic underestimate of density by 0.03% at 290 k rising to an overestimate by 0.08% at 305 k. these differences are negligible for the experiments described by winkler et al . however , we must also consider the density of salt solutions for some of the experiments discussed here . for example , at 293 k the density of aqueous sodium chloride at a concentration of 0.5 m ( a water activity of 0.984 ) is 1002.5 kg m compared with the value for water of 998.2 kg m , a 0.3% difference . this rises to 2% at a water activity of 0.9 , with a density of 1018 kg m and a concentration of 2.44 m. a 2% error in density can lead to an error in diameter of 0.7% , which is usually considerably lower than the other uncertainties in any condensation / evaporation measurement . to achieve a similar level of error through an incorrect assignment of droplet temperature the kinetics of water condensation and evaporation in aerosol have been studied using a wide range of experimental techniques . measurements have been made on single particles in an electrodynamic balance and in optical tweezers ; on droplet trains with particles of close to monodisperse size and on liquid jets ; and on ensembles of growing particles in an expansion chamber , a continuous flow streamwise thermal gradient ccn chamber ( cfstgc ) , and the leipzig aerosol cloud interaction simulator under conditions of supersaturated growth . we do not intend to be comprehensive in examining all of these approaches in the analysis presented here . however , we will examine the sensitivities to uncertainties in the key thermophysical parameters and environmental conditions for measurements made in an expansion chamber , a cfstgc , optical tweezers , and an edb . we will also identify the predominant uncertainties that are likely to limit the accuracy of each technique for retrieving a value of the mass accommodation / evaporation coefficient . in each case , we consider the dynamics as occurring on single isolated particles , providing a baseline analysis of sensitivities that ignores the complexity that may arise from interparticle couplings . it is clear that including the possibility of interparticle couplings can only lead to an increase in the uncertainties in reported values . the values for , and the uncertainties associated with , the diffusion coefficients , thermal conductivities , saturation pressure , and enthalpy of vaporization were taken as recommended in section iii . condensational growth rates on 9 nm diameter silver seed particles have been studied in an expansion chamber during adiabatic expansion over a temperature range from 250 to 290 k and with water vapor supersaturations ranging from 1.3 to 1.5 . one additional measurement was made on 80 nm diameter diethylhexyl sebacate particles at a supersaturation of 1.02 . the pressure of the surrounding nitrogen atmosphere was in the range 10100 kpa . the growth in droplet size was monitored by the constant angle mie scattering technique with a laser of wavelength 632.8 nm and at a scattering angle of 15. extrema ( maxima and minima ) in the light scattering amplitudes were taken from the measurements and compared with theoretical calculations , identifying the time at which the droplets achieved a particular size . although not providing a continuous record of particle size , such an approach does allow the time at which the droplets reach a certain size to be identified with considerable accuracy . the uncertainty in droplet size arising from an uncertainty in refractive index ( due to a change in droplet temperature ) is < 0.1% ( < 2 nm for the upper size limit in particle radius of 2 m reported ) . uncertainties in the growth times were estimated to be 1 ms but were commonly reported as 2 ms in the published data sets , derived from the extent of the time frame early on in the expansion during which nucleation was assumed to be constrained . uncertainties in the temperature and pressure of the chamber were reported as 0.05 k and 0.2 kpa , respectively , and uncertainties in the saturation as 1% and 3% ( 0.01 and 0.03 as a fraction of saturation of 1 ) at pressures of 100 and 20 kpa , respectively . at the lowest pressures , droplet growth was found to be controlled by heat flux from the particle ; with increasing pressure , the gas phase diffusivity decreases and both the heat and mass flux were recognized as important in controlling the growth rate . equal sensitivity to the mass and thermal accommodation coefficients was established at a pressure of around 90 kpa . thus , low pressure measurements of the thermal accommodation coefficient were used to constrain the model and retrieve the mass accommodation coefficient from higher pressure measurements . simulations of the time - dependent droplet growth following activation of 9 nm diameter seed particles have been performed using values for the thermophysical parameters and environmental conditions selected to both maximize and minimize the apparent growth rate , based on the uncertainties in these quantities discussed above ( diffusion constant , thermal conductivity , enthalpy of vaporization , and saturation value ) . for a chosen value of m between 1.0 and 0.1 , the upper and lower limits of the size from the upper and lower limits of the growth rate can be used to define an envelope for the time - dependent droplet growth , as shown in figure 1 . the time frame for the simulations is typical of that studied in the measurements ( experimental data reported between 10 and 80 ms ) and incorporates the uncertainty in t = 0 s. uncertainties in the transport coefficients and the saturation value dominate the breadth of the envelope . as expected , use of an inaccurate treatment of the saturation vapor pressure for water discussed in section iii.c is insignificant . further , the accuracies associated with the pressure , temperature , temperature dependence of the density of water and the enthalpy of vaporization are sufficiently high that these do not contribute to the breadth of the envelope . although it is clear that the time - dependent trends for m values below 0.5 are clearly resolved , above this value the envelopes in uncertainty overlap and it is not possible to discriminate unambiguously between values of m . this is true for both the higher pressure and lower pressure measurements simulated here . sensitivity of the time dependence of droplet growth in an expansion chamber type measurement to the mass accommodation coefficient ( gray , m = 1 ; red , m = 0.5 ; blue , m = 0.2 ; green , m = 0.1 ) . the upper and lower limits of each envelope come from the uncertainties in the thermophysical parameters and the saturation , as described in the text . two pressures are considered : ( a ) 98.4 kpa and ( b ) 18 kpa . the sensitivity of the retrieved value of m to the uncertainties in the transport coefficients ( 0 to + 10% error ) and saturation value ( 0 to + 4% ) are presented in figure 2 ; these ranges in uncertainty are chosen to cover the expected ranges discussed earlier . the solid color bars indicate the range in uncertainty that must be considered for the various parameters . only errors that lead to a systematic overestimation of m are considered ; the sensitivity to negative errors in these values are not considered as these would only lead to the retrievals of values of m that are significantly larger than 1 . measurements made by the expansion chamber approach are insensitive to the other thermophysical parameters described in the previous paragraph and these are not shown . we have found that the value of m estimated from the data is also insensitive to the initial size of the particle within the spread of sizes reported as the size distribution for the seed particles . in figure 2 , the change in m that is required to counter a particular value of the uncertainty in the thermophysical property or environmental condition is reported , such that the change in the droplet size over the experimental time window of 1080 ms remains less than 20 nm . thus , setting a tolerance of 20 nm corresponds to an uncertainty in time of 1 ms or of 12% in size over the time frame during which radii are experimentally determined . in the absence of a quantitative value for this accuracy in the literature , we have deliberately chosen to assess the measurements based on a conservative estimate of the uncertainty in the size ; the uncertainty may be larger than this but is unlikely to be smaller given the breadth in size for the extrema in the light scattering intensity . if the uncertainty in the size at any time is greater than this , the spread of values of m with which the data are consistent would be larger than shown in figure 2 . sensitivity of the value of the mass accommodation coefficient retrieved from the time - dependent data from expansion chamber measurements at 98.4 kpa to uncertainties in diffusion coefficient ( black ) , thermal conductivity ( red ) , and saturation ( blue ) . the upper and the lower limits for each reflect mass accommodation coefficients that give rise to time - dependent growths in size that are consistent with the experimental measurements within the upper and lower limits on the size and time accuracy described in the text . the accepted range of the uncertainties for the diffusion coefficient , thermal conductivity and saturation is shown at the bottom by the solid bars ( same colors as above ) . figure 2 should be interpreted in the following way . for zero error in a thermophysical parameter or the saturation value , the experimentally measured time - dependent growth is consistent with m values in the range 0.621.3 when the set tolerances are satisfied in theoretically reproducing the measured time dependence in size . for a + 5% error in either the diffusion coefficient for water - in - nitrogen or the thermal conductivity of nitrogen , m values in the range 0.541.2 would provide a satisfactory fit to the experimental data within the criteria defined above . the similarity in the sensitivities to these two constants comes from the comparability in the heat and mass flux in limiting the condensational growth at pressures of around 100 kpa . for an error of + 1% in saturation , m values in the range 0.530.95 would provide a satisfactory fit to the data . put simply , these sensitivities again suggest that although it is possible to discriminate between values of 0.2 and 0.5 for the m from these measurements consistent with figure 1 , discriminating between 0.5 and 1.0 is not possible given the uncertainties in the key thermophysical and environmental parameters . in the treatment of the experimental data provided by winkler et al . , the parametrization used for the thermal conductivity of water vapor is not in agreement with the formalism determined in this work as being the most reliable as a result of the literature survey . at 298.15 k , the thermal conductivity calculated using the winkler parametrization is 15.5% lower than the value given using the best fit parametrization ( refer to supporting information figure s6 ) . however , given the insensitivity of the expansion chamber measurements to this value , no error in analysis has been incurred . as noted in section iii.c , the parametrization used for estimating the saturation vapor pressure of water also led to systematically low values by 0.25% when compared with the best available treatment . this level of uncertainty leads to an accumulated error in size due to the change in mass flux of only 0.1% after 100 ms , an error of < 2 nm , or equivalent to the error arising from uncertainties in refractive index . this is clearly much less than the influence of uncertainties in the quantities assessed in figure 2 . beyond the uncertainties in the transport coefficients , it is clear that the largest sensitivity in the retrieved value of the m arises in the value of the saturation , a value that is extremely hard to measure . although quoted uncertainties for this value range from 1% to 3% , a more accurate assessment of this uncertainty would be desirable . during the condensational growth , the temperature at the surface of droplets in the expansion has been shown to vary by less than 0.2 k in the first 200 ms , even if the number concentration of growing droplets is large . the temperature of the gas phase has been reported to vary by as much as 0.5 k , depending on droplet concentration . the effect of these two uncertainties on the retrieved value of m , arising as it does from interparticle couplings , has not been considered here although it should perhaps be better quantified . in these simulations , we have convolved the uncertainty in the exact time at which condensational growth starts ( t = 0 s ) with the other uncertainties . again , if the uncertainty in this time could be reduced , the sensitivity to m would be improved . the droplet radii recorded at times longer than 20 ms are only sensitive to m through the very rapid growth that occurs at early time ( < 10 ms ) and direct measurements of the sizes are typically not available at such an early time . from this analysis , we expect that exact values of m can not be resolved if the value is > 0.5 , on the basis of the uncertainties in the thermophysical properties , time , and supersaturation . thus , it can be concluded that these measurements are consistent with a value of m that is larger than 0.5 . , who suggested the value of the mass accommodation coefficient must be greater than 0.4 . the activation kinetics of inorganic salt particles have been studied with instruments such as the continuous flow streamwise thermal gradient ccn chamber ( cfstgc ) and the leipzig aerosol cloud interaction simulator ( lacis ) . typical inorganic salts studied include ammonium sulfate and sodium chloride , and quasi - monodisperse dry particle sizes of 50100 nm are typically selected by a dma . the principle of operation originates from the more rapid diffusion of water vapor mass than heat . aerosol is passed through a flow tube with wetted walls and a temperature gradient is imposed along the length of the tube . a radial supersaturation profile is established , which is a maximum at the center - line of the flow and which varies along the length of the tube . typical supersaturations span the range from 0 to 2.5% and 0 to 1.4% in the lacis and cfstgc instruments , respectively . the supersaturation achieves a maximum part way along the flow tube and then declines , consistent with the particles reaching a maximum size and then evaporating before their size is determined at the end of the tube . droplets typically grow as large as 10 m in diameter , and their size is determined by light scattering using , for example , an optical particle counter . growth times can be as long as 1.5 and 12 s in the lacis and cfstgc instruments , respectively . for cfstgc measurements , uncertainties in diameter can be typically 10% for particles 1 m in size , improving to 5% for particles 10 m in size . uncertainties in supersaturation are typically 0.025% for supersaturations in the range 0.21% . for lacis measurements , uncertainties in diameter are reported to be similar to cgstgc measurements and uncertainties in supersaturation are less clearly defined . calculations of the diameter of a droplet at a growth time of 10 s have been performed under conditions of constant supersaturation , with values for the thermophysical parameters and exact supersaturation selected to maximize or minimize the size achieved . the different instruments show characteristic variations in supersaturation and temperature along the flow length that we have not sought to reproduce here ; the sensitivities to the uncertainties in the thermophysical parameters and environmental conditions will not depend on the exact trajectory in rh taken by the aerosol . instead , we have focused on estimating the influence of uncertainties in the thermophysical parameters and the calibration of the supersaturation at a fixed temperature of 300 k for ammonium sulfate particles initially 90 nm in diameter . m has been varied between 1.0 and 0.1 and , for each value , the upper and lower limits of the predicted size have been used to define an upper and lower limit on an uncertainty envelope , as shown in figure 3 . although it is clearly possible to discriminate between values of m of 1.0 and 0.1 in these measurements , within the uncertainties of the measurements and model predictions , the uncertainty envelopes overlap considerably even for m values of 0.2 and 0.5 . sensitivity of growth size at 10 s in a continuous flow streamwise thermal gradient ccn chamber type measurement to the mass accommodation coefficient ( gray , m = 1 ; red , m = 0.5 ; blue , m = 0.2 ; green , m = 0.1 ) . the upper and lower limits of each envelope come from the uncertainties in the thermophysical parameters and the saturation , as described in the text . the envelopes at a saturation of 1% are taken to marginally different values to help indicate the range of values to be expected for each value of m . the sensitivity of m to the uncertainties in thermophysical parameters ( 0 to + 10% error ) and saturation are presented in figure 4a ; the ranges in uncertainty are chosen to reflect the expected ranges discussed earlier . the solid color bars indicate the range in uncertainty that must be considered for the various parameters . measurements made by this approach are insensitive to the self - diffusion coefficient of water - in - water vapor , to the thermal conductivity of water vapor and the initial droplet size , and these are not shown in figure 4 . similarly , sensitivities to uncertainties in solution density , refractive index when the droplets are sized , and enthalpy of vaporization are considerably smaller than those shown . the change in m that is required to counter the uncertainty in the thermophysical property or environmental condition is reported , such that the change in the droplet size at the measurement time of 10 s remains within 5% of the baseline size for the case without any errors in the thermophysical parameters or environmental conditions . an upper error on the size of + 5% is not considered , as this would lead to a retrieved value of m that would be considerably larger than 1 . sensitivity of the value of the mass accommodation coefficient retrieved from the growth size in cfstgc measurements to uncertainties in diffusion coefficient ( black ) , thermal conductivity ( red ) , and saturation ( blue ) . note the log scale for m . the accepted range of the uncertainties for the diffusion coefficient , thermal conductivity , and saturation is shown at the bottom by the solid bars ( same colors as above ) ( a ) sensitivities assuming an acceptable tolerance on the uncertainty in radius of 5% . ( b ) sensitivities assuming an acceptable tolerance on the uncertainty in radius of 2% . figure 4a should be read as follows . for zero error in the diffusion coefficient , thermal conductivity , and saturation , the minimum value of the mass accommodation coefficient that would be consistent with the measurements within the error on the size determination would be 0.27 . values considerably larger than 1 would also be consistent , although these are not considered here . these simulations are for a supersaturation of 0.3% ; higher values of the supersaturation lead to larger changes in the droplet size , and an even lower value of m is found to be consistent with the growth measurement . these simulations reflect the lack of discrimination apparent in the previous figure , even for values of m of 0.2 and 0.5 . if the accuracy in the size determination / spread in the final size distribution could be improved to be better than 2% , the ability to resolve between values of m would be improved and this is shown in figure 4b . however , the strong sensitivity to the saturation and to a lesser extent the thermal conductivity remains . in summary , measurements of the kinetics of ccn activation can allow discrimination between values of m if less than 0.2 . however , for values larger than this we suggest that the growth kinetics for all values of m are within the uncertainties of the thermophysical parameters and saturation value . this is indeed broadly consistent with previous assessments of the lacis and cfstgc techniques . once again , the importance of an accurate measurement of the saturation is crucial and with these techniques the value varies significantly during the trajectory taken by the aerosol through the instrument . from this analysis , we expect an upper limit for m that can be resolved by this technique of 0.25 . given the uncertainties in the thermophysical properties , size , and saturation , we suggest that the differences in the condensation kinetics for values larger than this can not be resolved . thus , these measurements can be stated as reporting a value for m that is larger than 0.25 , consistent with the values reported by the expansion chamber measurements . as for the expansion chamber work , the experimental times and droplet sizes at which measurements are made provide only an indirect signature of the value of m : the sizes at the time of measurement are governed by the early time dependence in the growth kinetics for particles 1 m in diameter during which m directly has an impact . we have recently reported the details of a new method for investigating condensation and evaporation from a water droplet surface with a resolution approaching a molecular layer . a solution droplet is initially captured by a single beam gradient force optical trap ( optical tweezers ) and rapidly equilibrates with the surrounding gas phase environment , achieving a steady size at which the vapor pressure is equal to the surrounding partial pressure of water . instantaneous perturbations to the droplet temperature of a few millikelvin are initiated by changing the extent of optical heating . the droplet size must then respond , with evaporation or condensation leading to a change in the solute concentration until the droplet vapor pressure is once again restored to balance the surrounding rh . measurements were made at rhs higher than 95% for droplets in a size range of between 3.5 and 5 m with each evaporation or condensation event leading to a size change of < 10 nm on a time scale of < 5 s. to attempt to resolve the influence of surface processes on the condensational / evaporation kinetics from limitations imposed by gas diffusion , measurements were made over a range in pressure from 4 to 100 kpa . the recorded time dependence of droplet size was fitted to a single exponential , representing the kinetics by a rate constant for the mass transfer with a typical error of 5% . uncertainties in the droplet size are 1 nm , temperatures are < 0.2 k , and times 50 ms . the largest uncertainty is expected to be the water activity , a factor that led to problems in initially interpreting our kinetic data . the water activity can be retrieved by fitting the refractive index of the droplet from the cavity enhanced raman fingerprint with a typical uncertainty of 0.06% in the refractive index . at an rh of 98.5% , typical of the measurements , this corresponds to a salt solution concentration of 20 4 g l , an uncertainty in rh of 0.2% . initially , we consider the sensitivity of the rate constant measured by the technique to the value of m , the gas phase saturation and the uncertainties in the thermophysical parameters at two pressures , 100 and 20 kpa . given the failing of a key assumption in the derivation of eq 1 under conditions of low pressure , uncertainties in the values in the transport coefficients , and ambiguities in the conceptual framework discussed below , a total pressure below 10 kpa is not considered . indeed , at the lowest pressures it is anticipated that the condensation / evaporation kinetics will be determined by the efficiency of heat transport rather than mass transfer . the rate constants are calculated for a typical condensation event with the droplet radius changing from 4000 to 4006 nm at an rh of 98.86% and temperature of 293 k. only the sensitivities to the thermophysical parameters that influence the rate constant significantly are shown at each pressure . at high pressure , figure 5a , the rate constant is insensitive to the self - diffusion coefficient and thermal conductivity of water vapor . the uncertainties in the diffusion coefficient of water - in - air and the thermal conductivity of air are such that the sensitivity of the rate constant to m is insufficiently pronounced to allow values larger than 0.4 to be resolved , as indicated by the dotted lines that designate the lowest rate constant that could be measured with the uncertainty in the transport coefficients . the solid color bars indicate the range in uncertainty that must be considered for the various parameters . at a pressure of 20 kpa , figure 5b , the rate constant for condensational growth is insensitive to all of the thermophysical parameters except for the thermal conductivity of air . the figure indicates that the optical tweezers technique would only be able to discriminate between values of m smaller than 0.4 due to the uncertainty in the thermal conductivity . sensitivity of rate constant for condensational growth measured in optical tweezers studies to the uncertainties in diffusion coefficient ( black ) , thermal conductivity of air ( red ) , and saturation ( blue ) . the sensitivity of the rate constant to the mass accommodation coefficient is also shown ( purple ) . the accepted range of the uncertainties for the diffusion coefficients and thermal conductivities is shown at the bottom by the solid bars ( same colors as above ) . the gray shaded box indicates the level of uncertainty in the measured rate constants . once the current uncertainty associated with the rate constant is considered ( 5% ) , an uncertainty indicated by the extent of the gray box in figure 5 , measurements of the rate constant are only expected to be outside this error if the value of m falls below 0.2 , with a marginal improvement at intermediate pressures . thus , we expect an upper limit for m that can be resolved by this technique of 0.2 . above this value , we can only expect to be able to conclude than m is greater than 0.2 . the sensitivities of the rate constants to the uncertainty in the saturation value are also shown in figure 5 . it is clear that the current accuracy with which the salt concentration and rh can be determined , 0.06% in refractive index and 0.2% in rh , is inadequate for us to determine the mass accommodation coefficient . a reduction in the uncertainty associated with the rh to 0.05% is possible from an improvement in the resolution of the resonant mode wavelengths . if this level of accuracy is achieved , examples of the pressure dependence of the condensational rate constant with the associated uncertainties in the thermophysical parameters and rh are shown in figure 6 for values of m between 1.0 and 0.05 . although these simulations clearly indicate that it will be possible to determine values of the mass accommodation coefficient of less than 0.15 , accurately determining the value of the mass accommodation coefficient when above 0.15 will not be possible within the uncertainties associated with the measurements and the thermophysical parameters . it will therefore only be possible to confirm if the value of m is larger than 0.15 with the optical tweezers technique . sensitivity of condensational growth rate measured by optical tweezers technique to the mass accommodation coefficient ( gray , m = 1 ; red , m = 0.5 ; blue , m = 0.2 ; green , m = 0.1 ; purple , m = 0.05 ) . the upper and lower limits of each envelope come from the uncertainties in the thermophysical parameters and the saturation , as described in the text . the envelopes at a pressure of 100 kpa are taken to marginally different values to help indicate the range of values to be expected for each value of m . the evaporation of water from aqueous droplets levitated in an electrodynamic balance ( edb ) has been studied by a number of research groups , including davis and co - workers , jakubczyk and co - workers , and reid and co - workers . a charged droplet , initially 1020 m radius , is generated by a droplet - on - demand generator with an induction electrode or by electrospray . the charged droplet is then captured within an edb , either based on a hyperboloidal electrodes design or having concentric cylindrical electrodes operating at atmospheric pressure . measurements are performed either in a chamber flushed through with gas or in a direct gas jet , and either in dry nitrogen / air or in humidified nitrogen / air . in the latter case , measurements of rh near saturation can not be performed with sufficient accuracy to perform kinetic analysis and the rh must instead be retrieved from the kinetic measurements directly , along with a value for the evaporation coefficient . typically , when evaporating into dry nitrogen / air , the droplet evaporates completely ( or becomes too small to be retained within the trap ) over a time scale of 2 s. evaporation into humidified nitrogen is considerably slower and has been studied for > 20 s with the radius decreasing from 10 to < 5 m . in all cases , the evolving size of the droplet is measured from elastic light scattering , either from a resonance spectrum or from recording the time - dependent phase function . typically , the accuracy of a single size estimate is 15 nm , once local minima in the fitting of the phase function are excluded from the fitting process . evaporation measurements have been performed for droplets containing inorganic salts and organic components , and at a range of temperatures . suppression of surface temperature due to evaporative cooling must be considered and , thus , the droplet temperature is marginally lower than that of the surrounding gas phase . reported that a correction factor is required that becomes significant as the freezing point of water is approached to account for thermal effusion , but the correction factor approaches 1 as the temperature at the droplet surface increases above 280 k. we consider specifically the evaporation of water droplets , initially 9.8 m in radius , into a humidified atmosphere of air , as these measurements have been used to estimate the evaporation coefficient . sensitivities of the time dependence of the evaporation to a specified error in the diffusion coefficient of water - in - air , the thermal conductivity of air , and the saturation have been investigated . the base case simulation is chosen , which uses the best estimates of all the thermophysical parameters and a saturation value of 0.9762 , typical of the reported experiments . to examine the sensitivities to errors in the transport coefficients or saturation , the evaporation coefficient was varied to compensate for the error in the chosen property such that the time dependence of size remained within the reported error of the experiments for all evaporation times up to 16 s. only positive errors in the transport coefficients are shown , as these lead to a reduction in the recovered value of the evaporation coefficient , which is reported as being unity by zientara et al . the uncertainties in the lower limit for the evaporation coefficient derived from this sensitivity analysis , indicated by the error bars in figure 7 , come from keeping the size within a 15 nm tolerance on the size for the base simulation . it is clear from figure 7 that the rather small uncertainties associated with the key transport coefficients can lead to quite significant systematic errors in the evaporation coefficient estimated from the time dependence of the droplet size . ignoring these uncertainties can lead to an estimation of the evaporation coefficient that is significantly below its true value : if values of the transport coefficients are used that are larger than the values used in the analysis previously , but still within the uncertainties , the value of the evaporation coefficient retrieved would be larger than previously reported . by comparison , for the expansion chamber measurements discussed in section iv.a , the retrieved value for the mass accommodation coefficient is much less sensitive to the uncertainties in the transport coefficients than the edb measurements . further , it can be seen from figure 7 that the retrieved value of the evaporation coefficient from the edb technique is strongly dependent on the saturation value inferred in the measurement . sensitivity of the retrieved value of the mass accommodation coefficient from measurements of the time - dependent evaporation of droplets trapped by an edb to uncertainties in diffusion coefficient ( black ) , thermal conductivity ( red ) , and saturation ( blue ) . the accepted range of the uncertainties for the diffusion coefficient and thermal conductivity is shown at the bottom by the solid bars ( same colors as above ) . two temperatures are considered : ( a ) 283 k and ( b ) 273 k. in figure 8 we show the simulated time dependencies of a water droplet during evaporation for a range of values of the evaporation coefficient and including the uncertainties in the values of the diffusion coefficient for water in air and for the thermal conductivity , but neglecting the uncertainty in the saturation value at this stage . it is clear that given the uncertainties in these transport coefficients , it is not possible to resolve between different values of the evaporation coefficient from these measurements if the mass accommodation coefficient is 0.1 or larger . it may be possible , if the saturation value is known accurately , to resolve the difference between a value of 0.1 and 0.05 . however , one factor not considered in the assessment of the evaporation kinetics of this approach is the accuracy with which the start time for the evaporation event is known . we have found that this uncertainty can induce large errors in interpreting the evaporation kinetics . indeed , there is a flight time for the travel of the injected droplet into the edb trap that can be in excess of 100 ms . this error in start time is equivalent to a systematic error in the size of 30 nm at any stated time , already a larger contribution to the uncertainty in size than quoted by jakubczyk and co - workers . sensitivity of time dependence of droplet evaporation in an edb measurement to the mass accommodation coefficient ( gray , m = 1 ; red , m = 0.5 ; blue , m = 0.2 ; green , m = 0.1 ; purple , m = 0.05 ) . the upper and lower limits of each envelope come from the uncertainties in the thermophysical parameters , as described in the text . the envelopes at the long time limit are taken to marginally different values to help indicate the range of values to be expected for each value of m . in measurements that use the electrodynamic balance approach to determine the evaporation coefficient , it is common to use the time dependence over the first few seconds until the droplet decreases below 6 m radius to estimate the saturation value . it is assumed that this early time portion of the evaporation process is independent of the evaporation surface kinetics . however , on the basis of the uncertainties resulting from the transport coefficients , we suggest it is not possible to even estimate the saturation value from the early time data . it is possible to compensate for changes in the saturation and the evaporation coefficient such that the time dependence of droplet radius remains the same within the uncertainty associated with the measurement of the size . this is shown in figure 9a , which shows two almost identical time - resolved profiles of droplet size but for evaporation coefficients as different as 0.1 and 1.0 . the differences in size between the two at all times is < 15 nm , the stated uncertainty in the size measurement . thus , we suggest that it is not possible to independently retrieve values of the saturation and evaporation coefficient from this approach , and figure 9b shows the interdependence of the two values . given the sensitivity to the saturation value identified by the analysis of the optical tweezers measurements , this is not surprising . ( a ) two simulated time dependencies for the evaporation of water droplets : m = 1 and s = 0.9762 , black line ; m = 0.1 and s = 0.9744 , red dashed line . ( b ) interdependence of the retrieved values of the mass accommodation coefficient and the saturation . a low value of the saturation with a low value of m is equivalent to a high value of the saturation with a high value of m , within the uncertainty in the size measurements . we have examined the impact of uncertainties in key transport coefficients , thermophysical parameters , and experimental conditions , such as saturation , on the ability of different measurement techniques to determine the value of the mass accommodation coefficient , m , for water condensing on or evaporating from a water surface . the key thermophysical parameters that must be used in retrieving values of m from experimental measurements are the diffusion coefficient and thermal conductivity of the gas phase surrounding the droplet . to determine the most reliable parametrizations to use for the diffusion coefficients of water in air / nitrogen and the thermal conductivities of air , nitrogen , and water vapor , a literature search has been undertaken and the results critically analyzed . the measurement techniques assessed are the expansion chamber , continuous flow streamwise thermal gradient ccn chamber and leipzig aerosol cloud interaction simulator , optical tweezers , and the electrodynamic balance . using the same semianalytic framework to model heat and mass transfer to / from aerosol particles in all the studied systems , two types of simulations the first highlights the ability of each technique to resolve between different values of m given the magnitude of the uncertainties in the thermophysical and environmental parameters used in the model ; the second shows how the value of m retrieved by a technique would be altered by compensating for a given uncertainty in a thermophysical or environmental parameter . in assessing each technique we have identified the observed quantities that could be better quantified to more tightly constrain the retrieved values of m and , given the limitations of current data , have recommended the limiting values of m that can be determined . in all cases , better quantification of the saturation value would lead to an improved constraint on the value of m and this is perhaps unsurprising . we suggest that the expansion chamber measurements are consistent with a value that is larger than 0.5 and the activation kinetics measurements with a value larger than 0.25 . measurements made by the former approach could be used to better constrain m if the induction time for the onset of heterogeneous nucleation was more accurately defined allowing a reduced uncertainty in growth time , and if measurements of evolving droplet size could be made at times earlier than 20 ms , typical of the current reported measurements . measurements of activation kinetics could be better used to constrain m if the droplet size was more accurately measured and if the errors in the saturation profile along the flow tubes were better quantified . in reconciling the values reported by these techniques , we consider that the value can be safely assumed to be larger than 0.5 for water adsorbing to a water surface , independently verifying assessments made by previous authors . notably , this limiting value is also consistent with the jet and droplet evaporation work of saykally and co - workers not evaluated in this study who report the evaporation coefficient to be larger than 0.5 . in contrast to these ensemble techniques , we consider that the single particle measurements performed so far have insufficient sensitivity to contribute to the debate on the value of m . the optical tweezers approach , although providing extremely accurate measurements of molecular fluxes , requires a considerable reduction in the uncertainty associated with the value of saturation . if this improvement is achieved , uncertainties in the thermophysical parameters will still conspire to limit the resolution of values of m if above 0.2 . the strengths of this particular technique are that near - isothermal growth kinetics near equilibrium can be accurately examined . this will allow measurements to be made on droplets coated in surface active organic films and will permit a direct comparison of the kinetics of condensation and evaporation . further , measurements can be made over a wide range of droplet compositions with varying subsaturated water activity , retaining approximately constant water activity in the droplet bulk throughout a growth / evaporation event . it is not clear that such measurements can be made by any other approach . the edb measurements performed by us and other workers should be considered to be the least reliable for retrieving values of m . the size regime within which measurements are performed requires that the thermophysical parameters be known with an accuracy that is not currently accessible . such a level of accuracy is required to allow an improved estimation of the saturation . currently , a unique retrieval of the saturation and the value of m from an evaporation profile is not possible . sequential measurements of mass transfer kinetics made for droplets of known or assumed m ( e.g. , pure water droplets ) followed by droplets with an unknown value of m ( e.g. , surfactant coated droplets ) could provide an extremely accurate method for comparatively assessing kinetics , something that it is difficult to achieve in ensemble measurements . however , we consider that values reported by this approach so far should be considered to be unreliable . a variety of other techniques have been used to estimate values of m . we have chosen not to assess this approach here as it has been extensively reviewed and discussed in the literature . it is also not straightforward to assess the technique by the semianalytic framework used here without resorting to complex fluid dynamics calculations , which we consider is beyond the remit of the current work . however , it should be recognized that there are large uncertainties in the diffusion coefficient of water in the low - pressure environment when water dominates the composition of the gas phase and also in the thermal conductivity of the mixture , and these will be particularly important to resolve when droplet train measurements at very low temperatures are interpreted . considering the problems associated with interpreting measurements at low pressure / high water mole fraction in the gas phase , two further problems immediately present themselves that challenge the current conceptual picture adopted in understanding mass and heat transfer during the condensation or evaporation of water . the first relates to our understanding of the process of water diffusion in the limit of very low inert gas concentrations . at the low pressure limit , considering in particular the case where the gas phase is composed entirely of water vapor , the concentration gradient that drives mass transport is equivalent to a pressure gradient , whereas eq 1 from kulmala et al . the accuracy of the correction for convective mass transport used in the semianalytical framework must therefore be better understood and the applicability of eq 1 at the limit of low - pressure condensation of water should be quantified . second , for all the cases considered in this manuscript the gross mass flux is considerably larger than the net mass flux , i.e. , the uptake coefficient 1 . at low pressure , as the mole fraction of water in the gas phase approaches 1 , the excess latent heat deposited in the droplet via condensation must still be carried away by desorbing molecules . if 1 , the mass and thermal accommodation coefficients could no longer be assumed to be independent . practically , this limit can only be achieved experimentally during condensation or evaporation in to a vacuum , or in molecular dynamics simulations . our results also highlight the fact that the simultaneous consideration of mass and heat transport is a prerequisite for a successful determination of accommodation coefficients from condensation / evaporation data .

we compare and contrast measurements of the mass accommodation coefficient of water on a water surface made using ensemble and single particle techniques under conditions of supersaturation and subsaturation , respectively . in particular , we consider measurements made using an expansion chamber , a continuous flow streamwise thermal gradient cloud condensation nuclei chamber , the leipzig aerosol cloud interaction simulator , aerosol optical tweezers , and electrodynamic balances . although this assessment is not intended to be comprehensive , these five techniques are complementary in their approach and give values that span the range from near 0.1 to 1.0 for the mass accommodation coefficient . we use the same semianalytical treatment to assess the sensitivities of the measurements made by the various techniques to thermophysical quantities ( diffusion constants , thermal conductivities , saturation pressure of water , latent heat , and solution density ) and experimental parameters ( saturation value and temperature ) . this represents the first effort to assess and compare measurements made by different techniques to attempt to reduce the uncertainty in the value of the mass accommodation coefficient . broadly , we show that the measurements are consistent within the uncertainties inherent to the thermophysical and experimental parameters and that the value of the mass accommodation coefficient should be considered to be larger than 0.5 . accurate control and measurement of the saturation ratio is shown to be critical for a successful investigation of the surface transport kinetics during condensation / evaporation . this invariably requires accurate knowledge of the partial pressure of water , the system temperature , the droplet curvature and the saturation pressure of water . further , the importance of including and quantifying the transport of heat in interpreting droplet measurements is highlighted ; the particular issues associated with interpreting measurements of condensation / evaporation rates with varying pressure are discussed , measurements that are important for resolving the relative importance of gas diffusional transport and surface kinetics .

Introduction The Semi Analytical Model of Condensation and Evaporation Assessment of the Uncertainties Associated with Key Thermophysical Quantities Sensitivities of Measurements of Evaporation and Condensation to Uncertainties in the Thermophysical Quantities and Environmental Conditions Conclusions

further , it has been recommended that the mass accommodation coefficient ( m ) for describing the kinetics for crossing the surface boundary be separated into surface and bulk accommodation contributions to discriminate between the kinetics of the surface accommodation process and the transport between the surface and near - surface bulk . in describing the kinetics of transport across the surface boundary , we choose here to refer to the mass accommodation coefficient rather than either the surface or bulk accommodation coefficients as we will discuss measurements that have reported this value for the uptake of water vapor at a liquid water surface . as a further consideration , molecular transport between the condensed and gas phases leads to the deposition or loss of heat from a droplet during condensation and evaporation , respectively , and the transport of heat must be understood if the molecular mass flux is to be rationalized . measurements of the mass accommodation coefficient of gas phase water at a liquid water surface have a long and contentious history with values spanning a range from 0.001 to 1 . at a limiting value for m of 1.0 , the gas phase diffusion of water vapor to the droplet surface and the removal of heat limit the rate of condensational growth , and cloud droplet growth is insensitive to the surface accommodation kinetics . if the value of the mass accommodation coefficient were to be below 0.1 , condensational growth would be limited by surface kinetics leading to an increased value for the water vapor saturation in clouds , with the consequence that a larger fraction of the aerosol distribution would be activated . measurements of water condensation / evaporation at a range of pressures , water saturations ( relative humidities , rh ) , and temperatures are required to fully resolve the kinetics of the mass accommodation process from the limitations to the rate imposed by gas diffusion and to ensure consistency across a broad range of environmental conditions . to calculate the mass flux , constraining the value of m from condensation / evaporation data requires knowledge of the pressure , temperature , and compositional dependence of these key thermophysical parameters and transport coefficients , such as the gas phase diffusion coefficients and thermal conductivities of the components . here , we assess the influence of the uncertainties associated with both these transport coefficients and the experimental environmental conditions , particularly the saturation / rh , in interpreting measurements of evaporation or condensation of water . in section iii we assess the uncertainties associated with the key thermophysical properties required to predict evaporative or condensational fluxes , specifically the diffusion coefficients and gas phase thermal conductivities , the saturation vapor pressure of water and the enthalpy of vaporization of water . finally , in section iv we consider the impact of these uncertainties in interpreting measurements made with a range of experimental techniques that have been used to determine the mass accommodation coefficient of water . the mass flux ( i , kg s ) of the condensing vapor ( in our case water ) on a droplet of radius r ( m ) can be written as1where mv is the molecular mass ( kg ) , dim is the binary diffusion coefficient of the vapor in the surrounding gas mixture ( m s ) , l is the enthalpy of vaporization of the vapor ( j kg ) , k is the thermal conductivity of the gas mixture ( w m k ) , pe(t ) is the equilibrium pressure of the vapor ( pa ) at the temperature of the gas phase far removed from the droplet , t , and r is the ideal gas constant ( j k mol ) . more specifically , including the second term in the denominator of eq 1 accounts for the suppression of the growth rate due to elevation of the surface temperature by the latent heat produced during condensation , or conversely , the reduction in the evaporation rate due to depression of the surface temperature during evaporation . the first approximation determines the scaling of the vapor pressure at the droplet surface by the temperature change incurred due to the latent heat generated / removed during condensation / evaporation , eqs 19 and 22 of ref ( 16 ) . in short , the approximation made is9with a similar expression for the partial pressure of the vapor at infinite distance , p. given that the mass flux is directly proportional to the logarithmic quantity , when the partial pressure of the vapor is equal to the partial pressure of the buffer gas ( i.e. the massman parametrization for calculating the diffusion coefficient of water in air ( cm s ) at the desired temperature ( t / k ) and pressure ( p / atm ) is given by11where p0 = 1 atm and t0 = 273.15 k. the value of d0 , equivalent to the diffusion coefficient at 273.15 k and 1 atm pressure , was found by the loess fit to be 0.2178 cm s. the value of was constrained as 1.81 on the basis of previous work . these values can be compared with , for example , the reported values of d(h2o air ) at 1 atm total pressure which span the range 2.60 10 to 2.14 10 m s. thus , the diffusion coefficient for water in humid air / nitrogen can be expected to vary between the values in the dilute limit in dry air / nitrogen and the value for self - diffusion as the relative proportions of the gas phase constituents varies . the conduction of heat to or from the droplet within the gas phase is critical in determining the time dependence of the surface temperature of the droplet and the steady state wet - bulb temperature , and thus plays an important role in governing the mass flux . this is an empirical relationship based on kinetic theory and is shown in eq 15 for a binary mixture , requiring knowledge of the thermal conductivities of the pure components , ki , their mole fractions , xi , and the values of the parameters a12 and a21 , where 1 and 2 are the species of interest.15 the difference in the mixture thermal conductivities predicted by each of the parametrizations arises from the different methods used to calculate the parameters a12 and a21 . however , given that some of the aerosol evaporation / condensation measurements will pass through states with significant salt concentrations and may also be somewhat susceptible to the slight dependence of the enthalpy of vaporization on temperature , it is not unreasonable to consider the sensitivity of the different techniques to an uncertainty in the enthalpy of vaporization of 0.75 kj mol or 1.7% . measurements have been made on single particles in an electrodynamic balance and in optical tweezers ; on droplet trains with particles of close to monodisperse size and on liquid jets ; and on ensembles of growing particles in an expansion chamber , a continuous flow streamwise thermal gradient ccn chamber ( cfstgc ) , and the leipzig aerosol cloud interaction simulator under conditions of supersaturated growth . however , we will examine the sensitivities to uncertainties in the key thermophysical parameters and environmental conditions for measurements made in an expansion chamber , a cfstgc , optical tweezers , and an edb . the values for , and the uncertainties associated with , the diffusion coefficients , thermal conductivities , saturation pressure , and enthalpy of vaporization were taken as recommended in section iii . simulations of the time - dependent droplet growth following activation of 9 nm diameter seed particles have been performed using values for the thermophysical parameters and environmental conditions selected to both maximize and minimize the apparent growth rate , based on the uncertainties in these quantities discussed above ( diffusion constant , thermal conductivity , enthalpy of vaporization , and saturation value ) . the time frame for the simulations is typical of that studied in the measurements ( experimental data reported between 10 and 80 ms ) and incorporates the uncertainty in t = 0 s. uncertainties in the transport coefficients and the saturation value dominate the breadth of the envelope . further , the accuracies associated with the pressure , temperature , temperature dependence of the density of water and the enthalpy of vaporization are sufficiently high that these do not contribute to the breadth of the envelope . sensitivity of the time dependence of droplet growth in an expansion chamber type measurement to the mass accommodation coefficient ( gray , m = 1 ; red , m = 0.5 ; blue , m = 0.2 ; green , m = 0.1 ) . the sensitivity of the retrieved value of m to the uncertainties in the transport coefficients ( 0 to + 10% error ) and saturation value ( 0 to + 4% ) are presented in figure 2 ; these ranges in uncertainty are chosen to cover the expected ranges discussed earlier . in figure 2 , the change in m that is required to counter a particular value of the uncertainty in the thermophysical property or environmental condition is reported , such that the change in the droplet size over the experimental time window of 1080 ms remains less than 20 nm . in the absence of a quantitative value for this accuracy in the literature , we have deliberately chosen to assess the measurements based on a conservative estimate of the uncertainty in the size ; the uncertainty may be larger than this but is unlikely to be smaller given the breadth in size for the extrema in the light scattering intensity . sensitivity of the value of the mass accommodation coefficient retrieved from the time - dependent data from expansion chamber measurements at 98.4 kpa to uncertainties in diffusion coefficient ( black ) , thermal conductivity ( red ) , and saturation ( blue ) . put simply , these sensitivities again suggest that although it is possible to discriminate between values of 0.2 and 0.5 for the m from these measurements consistent with figure 1 , discriminating between 0.5 and 1.0 is not possible given the uncertainties in the key thermophysical and environmental parameters . beyond the uncertainties in the transport coefficients , it is clear that the largest sensitivity in the retrieved value of the m arises in the value of the saturation , a value that is extremely hard to measure . from this analysis , we expect that exact values of m can not be resolved if the value is > 0.5 , on the basis of the uncertainties in the thermophysical properties , time , and supersaturation . the activation kinetics of inorganic salt particles have been studied with instruments such as the continuous flow streamwise thermal gradient ccn chamber ( cfstgc ) and the leipzig aerosol cloud interaction simulator ( lacis ) . the different instruments show characteristic variations in supersaturation and temperature along the flow length that we have not sought to reproduce here ; the sensitivities to the uncertainties in the thermophysical parameters and environmental conditions will not depend on the exact trajectory in rh taken by the aerosol . although it is clearly possible to discriminate between values of m of 1.0 and 0.1 in these measurements , within the uncertainties of the measurements and model predictions , the uncertainty envelopes overlap considerably even for m values of 0.2 and 0.5 . sensitivity of growth size at 10 s in a continuous flow streamwise thermal gradient ccn chamber type measurement to the mass accommodation coefficient ( gray , m = 1 ; red , m = 0.5 ; blue , m = 0.2 ; green , m = 0.1 ) . the change in m that is required to counter the uncertainty in the thermophysical property or environmental condition is reported , such that the change in the droplet size at the measurement time of 10 s remains within 5% of the baseline size for the case without any errors in the thermophysical parameters or environmental conditions . sensitivity of the value of the mass accommodation coefficient retrieved from the growth size in cfstgc measurements to uncertainties in diffusion coefficient ( black ) , thermal conductivity ( red ) , and saturation ( blue ) . the accepted range of the uncertainties for the diffusion coefficient , thermal conductivity , and saturation is shown at the bottom by the solid bars ( same colors as above ) ( a ) sensitivities assuming an acceptable tolerance on the uncertainty in radius of 5% . for zero error in the diffusion coefficient , thermal conductivity , and saturation , the minimum value of the mass accommodation coefficient that would be consistent with the measurements within the error on the size determination would be 0.27 . however , for values larger than this we suggest that the growth kinetics for all values of m are within the uncertainties of the thermophysical parameters and saturation value . once again , the importance of an accurate measurement of the saturation is crucial and with these techniques the value varies significantly during the trajectory taken by the aerosol through the instrument . as for the expansion chamber work , the experimental times and droplet sizes at which measurements are made provide only an indirect signature of the value of m : the sizes at the time of measurement are governed by the early time dependence in the growth kinetics for particles 1 m in diameter during which m directly has an impact . initially , we consider the sensitivity of the rate constant measured by the technique to the value of m , the gas phase saturation and the uncertainties in the thermophysical parameters at two pressures , 100 and 20 kpa . the uncertainties in the diffusion coefficient of water - in - air and the thermal conductivity of air are such that the sensitivity of the rate constant to m is insufficiently pronounced to allow values larger than 0.4 to be resolved , as indicated by the dotted lines that designate the lowest rate constant that could be measured with the uncertainty in the transport coefficients . once the current uncertainty associated with the rate constant is considered ( 5% ) , an uncertainty indicated by the extent of the gray box in figure 5 , measurements of the rate constant are only expected to be outside this error if the value of m falls below 0.2 , with a marginal improvement at intermediate pressures . the sensitivities of the rate constants to the uncertainty in the saturation value are also shown in figure 5 . although these simulations clearly indicate that it will be possible to determine values of the mass accommodation coefficient of less than 0.15 , accurately determining the value of the mass accommodation coefficient when above 0.15 will not be possible within the uncertainties associated with the measurements and the thermophysical parameters . sensitivities of the time dependence of the evaporation to a specified error in the diffusion coefficient of water - in - air , the thermal conductivity of air , and the saturation have been investigated . to examine the sensitivities to errors in the transport coefficients or saturation , the evaporation coefficient was varied to compensate for the error in the chosen property such that the time dependence of size remained within the reported error of the experiments for all evaporation times up to 16 s. only positive errors in the transport coefficients are shown , as these lead to a reduction in the recovered value of the evaporation coefficient , which is reported as being unity by zientara et al . ignoring these uncertainties can lead to an estimation of the evaporation coefficient that is significantly below its true value : if values of the transport coefficients are used that are larger than the values used in the analysis previously , but still within the uncertainties , the value of the evaporation coefficient retrieved would be larger than previously reported . by comparison , for the expansion chamber measurements discussed in section iv.a , the retrieved value for the mass accommodation coefficient is much less sensitive to the uncertainties in the transport coefficients than the edb measurements . sensitivity of the retrieved value of the mass accommodation coefficient from measurements of the time - dependent evaporation of droplets trapped by an edb to uncertainties in diffusion coefficient ( black ) , thermal conductivity ( red ) , and saturation ( blue ) . two temperatures are considered : ( a ) 283 k and ( b ) 273 k. in figure 8 we show the simulated time dependencies of a water droplet during evaporation for a range of values of the evaporation coefficient and including the uncertainties in the values of the diffusion coefficient for water in air and for the thermal conductivity , but neglecting the uncertainty in the saturation value at this stage . it is possible to compensate for changes in the saturation and the evaporation coefficient such that the time dependence of droplet radius remains the same within the uncertainty associated with the measurement of the size . we have examined the impact of uncertainties in key transport coefficients , thermophysical parameters , and experimental conditions , such as saturation , on the ability of different measurement techniques to determine the value of the mass accommodation coefficient , m , for water condensing on or evaporating from a water surface . the measurement techniques assessed are the expansion chamber , continuous flow streamwise thermal gradient ccn chamber and leipzig aerosol cloud interaction simulator , optical tweezers , and the electrodynamic balance . using the same semianalytic framework to model heat and mass transfer to / from aerosol particles in all the studied systems , two types of simulations the first highlights the ability of each technique to resolve between different values of m given the magnitude of the uncertainties in the thermophysical and environmental parameters used in the model ; the second shows how the value of m retrieved by a technique would be altered by compensating for a given uncertainty in a thermophysical or environmental parameter . we suggest that the expansion chamber measurements are consistent with a value that is larger than 0.5 and the activation kinetics measurements with a value larger than 0.25 . measurements made by the former approach could be used to better constrain m if the induction time for the onset of heterogeneous nucleation was more accurately defined allowing a reduced uncertainty in growth time , and if measurements of evolving droplet size could be made at times earlier than 20 ms , typical of the current reported measurements . in reconciling the values reported by these techniques , we consider that the value can be safely assumed to be larger than 0.5 for water adsorbing to a water surface , independently verifying assessments made by previous authors .

bronchiectasis is a suppurative airway disease that has been defined as an abnormal and permanent bronchial dilatation.1 although the exact pathogenesis of bronchiectasis is still not clear , it is believed to be due to the development of a vicious cycle of chronic inflammation and altered response to infection by compromised mucociliary clearance , leading to progressive airway destruction and distortion.2 furthermore , airway - wall thickening , chronic colonization of pseudomonas aeruginosa in the bronchial epithelium , excessive airway collapse during expiration , and bronchial hyperreactivity may contribute to deterioration of lung function in patients with bronchiectasis.36 one mainstay of bronchiectasis management is pharmacologic treatment.7,8 while there is accumulating evidence supporting the use of anti - inflammatory therapy ( eg , inhaled corticosteroids9,10 and macrolides1113 ) and antibiotics , data supporting bronchodilator use in patients with bronchiectasis are scarce and recommendations based mainly on expert opinion . the british thoracic society guideline for non - cystic fibrosis bronchiectasis suggests that bronchodilator use may be appropriate for patients with bronchiectasis who have reversible airflow limitation.7 however , data supporting this recommendation are from small - scale studies showing a significant reversibility of airflow limitation in response to short - acting bronchodilator use in patients with bronchiectasis.5,14 moreover , short - term bronchodilator response is a poor predictor of long - term treatment benefit for lung function in chronic obstructive pulmonary disease ( copd).15 on the other hand , the long - term effect of bronchodilator therapy on lung function has not been adequately investigated in patients with bronchiectasis.1618 therefore , the aims of our study were to explore the relationship between reversible airflow limitation , ie , positive bronchodilator response ( bdr ) at baseline , and lung - function improvement after long - term ( 312 months ) bronchodilator therapy in bronchiectasis patients with airflow limitation . the medical records of 206 patients aged 18 years or older at a university - based tertiary hospital were evaluated . all patients had bronchiectasis on chest computed tomography ( ct ) scan , and had performed pre- and postbronchodilator spirometry when they were clinically stable and repeated spirometry after 312 months of bronchodilator therapy between january 1995 and february 2015 . among these patients , we excluded those who had more than a 6-month interval between baseline spirometry and initiation of bronchodilator therapy ( n=34 ) and those without airflow limitation at baseline ( n=6 ) . a final total of 166 patients with concomitant bronchiectasis and airflow limitation were included ( figure 1 ) . the institutional review board of samsung medical center approved this study and waived the requirement for informed consent due to the retrospective nature of the study . spirometry was performed as recommended by the american thoracic society / european respiratory society using a vmax 22 ( sensormedics , yorba linda , ca , usa).19 the highest measured forced vital capacity ( fvc ) and forced expiratory volume in 1 second ( fev1 ) among three or more tests with acceptable curves were used . absolute values of fvc and fev1 were obtained , and the percentage of predicted values for fev1 and fvc were calculated from equations obtained in a representative south korean sample.20 airflow limitation was defined as prebronchodilator fev1/fvc < 70% . positive bdr at baseline was defined as postbronchodilator increase in fev1 and/or fvc of at least 12% and 200 ml from baseline values at 15 minutes after inhalation of 400 g of salbutamol.21 bronchiectasis was diagnosed when the following two key imaging findings were present on ct scan : 1 ) the internal diameter of the bronchus was larger than the accompanying vessels and 2 ) no bronchial tapering was present in the periphery of the lungs.22,23 all ct scans were reviewed by two pulmonologists ( hjj and jhk ) to confirm the diagnosis of bronchiectasis . in this study , long - term bronchodilator use was defined as administration of bronchodilator therapy for longer than 3 months . responders were defined as those whose fev1 values improved at least 12% and 200 ml from baseline fev1 following 312 months of bronchodilator therapy . patients who received long - term bronchodilator but did not meet these benchmarks were defined as poor responders . bronchodilators included inhaled long - acting 2-agonists ( labas ) and inhaled long - acting muscarinic antagonist ( lamas ) . inhaled corticosteroids ( ics ) exert an anti - inflammatory response rather than bronchodilation , but have beneficial effects on lung - function improvement;24 therefore , this category was counted as one of the inhalers . data are presented as medians and interquartile ranges ( iqrs ; first to third ) for continuous variables and as numbers and percentages for categorical variables . whitney u - test for continuous variables and pearson s test or fisher s exact test for categorical variables . to determine whether positive bdr at baseline was an independent factor for being a responder and to appreciate the influence of demographic , clinical , and treatment variables , a series of multiple logistic regression analyses were performed on variables with p<0.2 based on univariate results or clinically important : model 1 contained the demographic variables age ( continuous ) , sex , and body mass index ( bmi ) ( continuous ) ; model 2 additionally included pulmonary - related variables , which are generally considered important in pulmonary diseases ( smoking history [ non- vs ex- or current smokers ] and prebronchodilator fev1 < 50% predicted ) ; and finally model 3 additionally included the treatment variable and number of inhalers , as well as all of the aforementioned variables . the incremental values of each addition of variables in the three models were evaluated via test . next , we considered the stepwise selection of variables in a multiple logistic model to obtain a parsimonious prediction model containing only the relevant variables based on clinical and statistical significance . we used the hosmer lemeshow test to verify the goodness of fit for each model . all statistical tests were performed using spss version 22.0 ( ibm , armonk , ny , usa ) . the medical records of 206 patients aged 18 years or older at a university - based tertiary hospital were evaluated . all patients had bronchiectasis on chest computed tomography ( ct ) scan , and had performed pre- and postbronchodilator spirometry when they were clinically stable and repeated spirometry after 312 months of bronchodilator therapy between january 1995 and february 2015 . among these patients , we excluded those who had more than a 6-month interval between baseline spirometry and initiation of bronchodilator therapy ( n=34 ) and those without airflow limitation at baseline ( n=6 ) . a final total of 166 patients with concomitant bronchiectasis and airflow limitation were included ( figure 1 ) . the institutional review board of samsung medical center approved this study and waived the requirement for informed consent due to the retrospective nature of the study . spirometry was performed as recommended by the american thoracic society / european respiratory society using a vmax 22 ( sensormedics , yorba linda , ca , usa).19 the highest measured forced vital capacity ( fvc ) and forced expiratory volume in 1 second ( fev1 ) among three or more tests with acceptable curves were used . absolute values of fvc and fev1 were obtained , and the percentage of predicted values for fev1 and fvc were calculated from equations obtained in a representative south korean sample.20 airflow limitation was defined as prebronchodilator fev1/fvc < 70% . positive bdr at baseline was defined as postbronchodilator increase in fev1 and/or fvc of at least 12% and 200 ml from baseline values at 15 minutes after inhalation of 400 g of salbutamol.21 bronchiectasis was diagnosed when the following two key imaging findings were present on ct scan : 1 ) the internal diameter of the bronchus was larger than the accompanying vessels and 2 ) no bronchial tapering was present in the periphery of the lungs.22,23 all ct scans were reviewed by two pulmonologists ( hjj and jhk ) to confirm the diagnosis of bronchiectasis . in this study , long - term bronchodilator use was defined as administration of bronchodilator therapy for longer than 3 months . responders were defined as those whose fev1 values improved at least 12% and 200 ml from baseline fev1 following 312 months of bronchodilator therapy . patients who received long - term bronchodilator but did not meet these benchmarks were defined as poor responders . bronchodilators included inhaled long - acting 2-agonists ( labas ) and inhaled long - acting muscarinic antagonist ( lamas ) . inhaled corticosteroids ( ics ) exert an anti - inflammatory response rather than bronchodilation , but have beneficial effects on lung - function improvement;24 therefore , this category was counted as one of the inhalers . data are presented as medians and interquartile ranges ( iqrs ; first to third ) for continuous variables and as numbers and percentages for categorical variables . whitney u - test for continuous variables and pearson s test or fisher s exact test for categorical variables . to determine whether positive bdr at baseline was an independent factor for being a responder and to appreciate the influence of demographic , clinical , and treatment variables , a series of multiple logistic regression analyses were performed on variables with p<0.2 based on univariate results or clinically important : model 1 contained the demographic variables age ( continuous ) , sex , and body mass index ( bmi ) ( continuous ) ; model 2 additionally included pulmonary - related variables , which are generally considered important in pulmonary diseases ( smoking history [ non- vs ex- or current smokers ] and prebronchodilator fev1 < 50% predicted ) ; and finally model 3 additionally included the treatment variable and number of inhalers , as well as all of the aforementioned variables . the incremental values of each addition of variables in the three models were evaluated via test . next , we considered the stepwise selection of variables in a multiple logistic model to obtain a parsimonious prediction model containing only the relevant variables based on clinical and statistical significance . we used the hosmer lemeshow test to verify the goodness of fit for each model . all statistical tests were performed using spss version 22.0 ( ibm , armonk , ny , usa ) . participants comprised 113 men ( 68.1% ) and 53 women ( 31.9% ) with a median age of 64 years ( iqr 5670 years ) . the median bmi was 22.8 kg / m ( iqr 20.725.4 kg / m ) , and 93 patients ( 57.4% ) were current or ex - smokers . a total of 74 patients ( 44.6% ) had a history of pulmonary tuberculosis , and common coexisting pulmonary diseases included nontuberculous mycobacterial lung disease ( n=13 , 7.8% ) and chronic pulmonary aspergillosis ( n=5 , 3% ) . the most common extrapulmonary comorbidity was hypertension ( n=51 , 30.7% ) , followed by malignant disease ( n=23 , 13.9% ) , diabetes mellitus ( n=21 , 12.7% ) , chronic kidney disease ( n=7 , 4.2% ) , and cerebrovascular disease ( n=7 , 4.2% ) . the median fev1/fvc , fvc , and fev1 were 53.7% , 2.8 l ( 70.5% predicted ) , and 1.4 l ( 50% predicted ) , respectively . positive bdr at baseline was observed in 42 patients ( 25.3% ) . among the 166 patients , compared to poor responders , responders to treatment were more likely to be male ( 78.9% [ 45 of 57 ] vs 62.4% [ 68 of 109 ] , p=0.03 ) , current or ex - smokers ( 69.6% [ 39 of 56 ] vs 50.9% [ 54 of 106 ] , p=0.022 ) and have lower median fev1/fvc ( 50.5% [ iqr 43.5%58% ] vs 55% [ iqr 47%62.4% ] , p=0.011 ) and positive bdr at baseline ( 38.6% [ 22 of 57 ] vs 18.3% [ 20 of 109 ] , p=0.004 ) . there were no significant differences with respect to age , bmi , previous history of pulmonary tuberculosis , coexisting pulmonary or extrapulmonary comorbidities , or baseline pulmonary function tests , including fvc ( percentage predicted , liters ) and fev1 ( percentage predicted , liters ) between the responders and poor responders . as shown in table 2 , the median number of involved lobes was three ( iqr 25 ) . between responders and poor responders , there were no significant differences in the number of involved lobes or the locations of the involved lobes . a median of two inhalers ( iqr 12 ) were used , and there were no significant differences in the number of inhalers used between the responders and poor responders . there were no significant differences in ics use , laba ics , or lama ics between responders and poor responders . however , laba / lama ics was more frequently used by responders than poor responders ( 31.6% vs 16.5% , p=0.025 ) . the increase in fev1 was more significant in patients with positive bdr at baseline compared to those without positive bdr at baseline ( figure 2 ; median 210 ml [ iqr 130430 ml ] vs 130 ml [ iqr 10 to 250 ml ] , p=0.001 ) . in addition , patients with positive bdr at baseline were more likely to be responders to long - term bronchodilator therapy compared to those without positive bdr at baseline ( figure 3 ; 52.4% vs 28.2% , p=0.004 ) . however , the increase in fev1 following bronchodilator therapy was statistically significant in patients without positive bdr at baseline ( 130 ml [ iqr 10 to 250 ml ] , p<0.001 ) , as well as in those with positive bdr at baseline ( 210 ml [ iqr 130430 ml ] , p<0.001 ) ( figure 4 ) . the three sequential models were compared for their incremental importance to predicting responsiveness to bronchodilator therapy . compared to using only demographic variables ( age , sex , and bmi ) in model 1 , model 2 , which included pulmonary - related variables , including fev1<50% predicted and smoking history , was marginally better ( =0.47 , p=0.79 ) , while model 3 , including inhalers in addition to all previous variables , was significantly better in predicting the probability of responders ( =6.03 , p=0.049 ) . as shown in table 3 , positive bdr at baseline remained consistently a significant predictive factor for being a responder to long - term bronchodilator therapy , with the exception of model 3 , due to multiple insignificant variables making the model unstable . we then considered risk - factor modeling to predict responsiveness by removing irrelevant variables , and found that positive bdr at baseline was 2.3 times more ( confidence interval 1.0674.951 , p=0.034 ) significantly and independently associated with being a responder to long - term bronchodilator therapy than being a poor responder , in addition to the number of inhalers ( p=0.047 ) . the goodness of fit of the model was confirmed by the hosmer lemeshow test ( p>0.2 ) . participants comprised 113 men ( 68.1% ) and 53 women ( 31.9% ) with a median age of 64 years ( iqr 5670 years ) . the median bmi was 22.8 kg / m ( iqr 20.725.4 kg / m ) , and 93 patients ( 57.4% ) were current or ex - smokers . a total of 74 patients ( 44.6% ) had a history of pulmonary tuberculosis , and common coexisting pulmonary diseases included nontuberculous mycobacterial lung disease ( n=13 , 7.8% ) and chronic pulmonary aspergillosis ( n=5 , 3% ) . the most common extrapulmonary comorbidity was hypertension ( n=51 , 30.7% ) , followed by malignant disease ( n=23 , 13.9% ) , diabetes mellitus ( n=21 , 12.7% ) , chronic kidney disease ( n=7 , 4.2% ) , and cerebrovascular disease ( n=7 , 4.2% ) . the median fev1/fvc , fvc , and fev1 were 53.7% , 2.8 l ( 70.5% predicted ) , and 1.4 l ( 50% predicted ) , respectively . positive bdr at baseline was observed in 42 patients ( 25.3% ) . among the 166 patients , compared to poor responders , responders to treatment were more likely to be male ( 78.9% [ 45 of 57 ] vs 62.4% [ 68 of 109 ] , p=0.03 ) , current or ex - smokers ( 69.6% [ 39 of 56 ] vs 50.9% [ 54 of 106 ] , p=0.022 ) and have lower median fev1/fvc ( 50.5% [ iqr 43.5%58% ] vs 55% [ iqr 47%62.4% ] , p=0.011 ) and positive bdr at baseline ( 38.6% [ 22 of 57 ] vs 18.3% [ 20 of 109 ] , p=0.004 ) . there were no significant differences with respect to age , bmi , previous history of pulmonary tuberculosis , coexisting pulmonary or extrapulmonary comorbidities , or baseline pulmonary function tests , including fvc ( percentage predicted , liters ) and fev1 ( percentage predicted , liters ) between the responders and poor responders . as shown in table 2 , the median number of involved lobes was three ( iqr 25 ) . between responders and poor responders , there were no significant differences in the number of involved lobes or the locations of the involved lobes . a median of two inhalers ( iqr 12 ) were used , and there were no significant differences in the number of inhalers used between the responders and poor responders . there were no significant differences in ics use , laba ics , or lama ics between responders and poor responders . however , laba / lama ics was more frequently used by responders than poor responders ( 31.6% vs 16.5% , p=0.025 ) . the increase in fev1 was more significant in patients with positive bdr at baseline compared to those without positive bdr at baseline ( figure 2 ; median 210 ml [ iqr 130430 ml ] vs 130 ml [ iqr 10 to 250 ml ] , p=0.001 ) . in addition , patients with positive bdr at baseline were more likely to be responders to long - term bronchodilator therapy compared to those without positive bdr at baseline ( figure 3 ; 52.4% vs 28.2% , p=0.004 ) . however , the increase in fev1 following bronchodilator therapy was statistically significant in patients without positive bdr at baseline ( 130 ml [ iqr 10 to 250 ml ] , p<0.001 ) , as well as in those with positive bdr at baseline ( 210 ml [ iqr 130430 ml ] , p<0.001 ) ( figure 4 ) . the three sequential models were compared for their incremental importance to predicting responsiveness to bronchodilator therapy . compared to using only demographic variables ( age , sex , and bmi ) in model 1 , model 2 , which included pulmonary - related variables , including fev1<50% predicted and smoking history , was marginally better ( =0.47 , p=0.79 ) , while model 3 , including inhalers in addition to all previous variables , was significantly better in predicting the probability of responders ( =6.03 , p=0.049 ) . as shown in table 3 , positive bdr at baseline remained consistently a significant predictive factor for being a responder to long - term bronchodilator therapy , with the exception of model 3 , due to multiple insignificant variables making the model unstable . we then considered risk - factor modeling to predict responsiveness by removing irrelevant variables , and found that positive bdr at baseline was 2.3 times more ( confidence interval 1.0674.951 , p=0.034 ) significantly and independently associated with being a responder to long - term bronchodilator therapy than being a poor responder , in addition to the number of inhalers ( p=0.047 ) . the goodness of fit of the model was confirmed by the hosmer lemeshow test ( p>0.2 ) . in the present study of bronchiectasis patients with airflow limitation , we found that approximately one - third of patients were responders to long - term bronchodilator therapy , and positive bdr at baseline was significantly associated with an increase in fev1 following long - term bronchodilator therapy . however , the increase in fev1 was evident not only in patients with positive bdr at baseline but also in those without positive bdr at baseline . therefore , our findings suggest that patients with bronchiectasis who demonstrate poor bdr at baseline can benefit from long - term bronchodilator therapy . to the best of our knowledge , this is the first study to investigate the relationship between positive bdr at baseline and the long - term effects of bronchodilator therapy in patients with bronchiectasis . in the treatment of patients with bronchiectasis , bronchodilator use is recommended for patients with positive bdr at baseline.7 this recommendation is based on results from two previous studies that demonstrated significant bdr ( greater than 15% improvement in fev1 ) in a subset of patients with bronchiectasis after use of a short - acting bronchodilator.5,14 however , these studies evaluated bdr in fewer than 30 patients with bronchiectasis ( one study included 24 patients and the second included 16 patients ) , and did not confirm the long - term effect of bronchodilator therapy based on immediate bdr . in a relatively larger number of patients , our study confirmed and extended the previous findings , showing that about 25% of bronchiectasis patients with airflow limitation had positive bdr at baseline , and the presence of positive bdr at baseline was independently associated with improvement in lung function following long - term bronchodilator therapy in patients with bronchiectasis and airflow limitation . the predictive ability of airflow reversibility in a short - acting bronchodilator test on long - term improvements in lung function following bronchodilator therapy in patients with copd has been widely studied.15,25,26 a randomized controlled trial showed that copd patients with immediate bdr after the first dose of tiotropium showed a larger improvement in fev1 following 1 year of tiotropium use compared to copd patients without immediate bdr.15 other studies that evaluated the effects of salmeterol on copd patients have also shown that patients with positive bdr at baseline had larger improvements in lung function at 12 weeks and 1 year following the use of salmeterol compared with those without positive bdr at baseline.25,26 however , special attention should be given to patients without positive bdr at baseline . we observed that patients without positive bdr at baseline also exhibited significant improvement in fev1 following long - term bronchodilator therapy , although those with positive bdr at baseline had greater improvement in fev1 than those without positive bdr at baseline . this pattern has also been recognized in copd patients . despite the positive correlation between short - term response to bronchodilator at baseline and improvement in lung function following long - term bronchodilator therapy , several studies of copd patients have found that long - term bronchodilator therapy significantly improved lung function , dyspnea , and health status , irrespective of the presence or absence of positive bdr at baseline.15,2529 these findings led to the current guidelines that do not recommend use of reversibility testing with short - acting bronchodilator use to predict long - term response to treatment . therefore , our results suggest that long - term bronchodilator use needs to be extended to patients without positive bdr at baseline in patients with bronchiectasis and airflow limitation . however , a randomized clinical trial with a placebo group is needed to establish the efficacy of long - term bronchodilator use in patients with bronchiectasis . the coexistence of bronchial asthma in bronchiectasis patients has been reported,30,31 and a recent study has shown that the existence of bronchial asthma is an independent risk factor for bronchiectasis exacerbations.30 regarding lung function improvement following long - term bronchodilator therapy with ics , our study showed that coexisting bronchial asthma was not associated with being a responder in patients with bronchiectasis . our study included only bronchiectasis patients who had airflow limitation , which could have mitigated the effect of bronchodilator therapy with ics on coexisting bronchial asthma patients with bronchiectasis . further studies are necessary to investigate the effect of long - term bronchodilator therapy with ics on the rate of exacerbations in patients with both bronchial asthma and bronchiectasis . second , patients who had not undergone spirometry after 312 months following bronchodilator therapy were excluded , which may have led to selection bias . third , the rate of exacerbations , changes in symptoms , and health - related quality of life following long - term use of bronchodilators were not evaluated , which could represent additional long - term benefits to maintenance therapy with bronchodilators . however , the duration of 312 months was long enough to observe improvements in fev1 , and there was no significant difference in the median duration of bronchodilator use between responders and poor responders . finally , although we showed that bronchodilators are useful in improving lung function , the optimal inhalers for bronchiectasis patients could not be evaluated from our study . therefore , further studies are necessary to compare the effectiveness of each type of bronchodilator in patients with bronchiectasis . in summary , we conclude that positive bdr at baseline was an independent predictive factor for improvement in lung function after long - term bronchodilator therapy in patients with bronchiectasis and airflow limitation . we further suggest that clinicians should consider long - term bronchodilator therapy for patients with poor bdr at baseline , as it can exhibit beneficial response .

purposethe association between positive bronchodilator response ( bdr ) at baseline and the effect of long - term bronchodilator therapy has not been well elucidated in patients with bronchiectasis . the aims of our study were to explore the association between positive bdr at baseline and lung - function improvement following long - term ( 312 months ) bronchodilator therapy in bronchiectasis patients with airflow limitation.materials and methodsthe medical records of 166 patients with clinically stable bronchiectasis who underwent baseline pre- and postbronchodilator spirometry and repeated spirometry after 312 months of bronchodilator therapy were retrospectively reviewed . for analysis , patients were divided into two groups , responders and poor responders , based on achievement of at least 12% and 200 ml in forced expiratory volume in 1 second ( fev1 ) following bronchodilator therapy from baseline fev1.resultsa total of 57 patients ( 34.3% ) were responders . these patients were more likely to have positive bdr at baseline than poor responders ( 38.6% [ 22 of 57 ] vs 18.3% [ 20 of 109 ] , p=0.004 ) . this association persisted after adjustment for other confounding factors ( adjusted odds ratio 2.298 , p=0.034 ) . however , we found fev1 improved significantly following long - term bronchodilator therapy , even in patients without positive bdr at baseline ( change in fev1 130 ml , interquartile range 10 to 250 ml ; p<0.001).conclusionpositive bdr at baseline was independently associated with responsiveness to long - term bronchodilator therapy in bronchiectasis patients with airflow limitation . however , fev1 improvement was also evident in bronchiectasis patients without positive bdr at baseline , suggesting that these patients can benefit from long - term bronchodilator therapy .

Introduction Materials and methods Patients Pulmonary function test Definitions Statistical analysis Results Clinical characteristics of participants Comparison of CT findings and effect of inhaler therapy between responders and poor responders Association between positive BDR at baseline and being a responder following bronchodilator therapy Discussion Limitations Conclusion

the british thoracic society guideline for non - cystic fibrosis bronchiectasis suggests that bronchodilator use may be appropriate for patients with bronchiectasis who have reversible airflow limitation.7 however , data supporting this recommendation are from small - scale studies showing a significant reversibility of airflow limitation in response to short - acting bronchodilator use in patients with bronchiectasis.5,14 moreover , short - term bronchodilator response is a poor predictor of long - term treatment benefit for lung function in chronic obstructive pulmonary disease ( copd).15 on the other hand , the long - term effect of bronchodilator therapy on lung function has not been adequately investigated in patients with bronchiectasis.1618 therefore , the aims of our study were to explore the relationship between reversible airflow limitation , ie , positive bronchodilator response ( bdr ) at baseline , and lung - function improvement after long - term ( 312 months ) bronchodilator therapy in bronchiectasis patients with airflow limitation . all patients had bronchiectasis on chest computed tomography ( ct ) scan , and had performed pre- and postbronchodilator spirometry when they were clinically stable and repeated spirometry after 312 months of bronchodilator therapy between january 1995 and february 2015 . among these patients , we excluded those who had more than a 6-month interval between baseline spirometry and initiation of bronchodilator therapy ( n=34 ) and those without airflow limitation at baseline ( n=6 ) . a final total of 166 patients with concomitant bronchiectasis and airflow limitation were included ( figure 1 ) . spirometry was performed as recommended by the american thoracic society / european respiratory society using a vmax 22 ( sensormedics , yorba linda , ca , usa).19 the highest measured forced vital capacity ( fvc ) and forced expiratory volume in 1 second ( fev1 ) among three or more tests with acceptable curves were used . positive bdr at baseline was defined as postbronchodilator increase in fev1 and/or fvc of at least 12% and 200 ml from baseline values at 15 minutes after inhalation of 400 g of salbutamol.21 bronchiectasis was diagnosed when the following two key imaging findings were present on ct scan : 1 ) the internal diameter of the bronchus was larger than the accompanying vessels and 2 ) no bronchial tapering was present in the periphery of the lungs.22,23 all ct scans were reviewed by two pulmonologists ( hjj and jhk ) to confirm the diagnosis of bronchiectasis . in this study , long - term bronchodilator use was defined as administration of bronchodilator therapy for longer than 3 months . responders were defined as those whose fev1 values improved at least 12% and 200 ml from baseline fev1 following 312 months of bronchodilator therapy . patients who received long - term bronchodilator but did not meet these benchmarks were defined as poor responders . to determine whether positive bdr at baseline was an independent factor for being a responder and to appreciate the influence of demographic , clinical , and treatment variables , a series of multiple logistic regression analyses were performed on variables with p<0.2 based on univariate results or clinically important : model 1 contained the demographic variables age ( continuous ) , sex , and body mass index ( bmi ) ( continuous ) ; model 2 additionally included pulmonary - related variables , which are generally considered important in pulmonary diseases ( smoking history [ non- vs ex- or current smokers ] and prebronchodilator fev1 < 50% predicted ) ; and finally model 3 additionally included the treatment variable and number of inhalers , as well as all of the aforementioned variables . all patients had bronchiectasis on chest computed tomography ( ct ) scan , and had performed pre- and postbronchodilator spirometry when they were clinically stable and repeated spirometry after 312 months of bronchodilator therapy between january 1995 and february 2015 . among these patients , we excluded those who had more than a 6-month interval between baseline spirometry and initiation of bronchodilator therapy ( n=34 ) and those without airflow limitation at baseline ( n=6 ) . a final total of 166 patients with concomitant bronchiectasis and airflow limitation were included ( figure 1 ) . spirometry was performed as recommended by the american thoracic society / european respiratory society using a vmax 22 ( sensormedics , yorba linda , ca , usa).19 the highest measured forced vital capacity ( fvc ) and forced expiratory volume in 1 second ( fev1 ) among three or more tests with acceptable curves were used . positive bdr at baseline was defined as postbronchodilator increase in fev1 and/or fvc of at least 12% and 200 ml from baseline values at 15 minutes after inhalation of 400 g of salbutamol.21 bronchiectasis was diagnosed when the following two key imaging findings were present on ct scan : 1 ) the internal diameter of the bronchus was larger than the accompanying vessels and 2 ) no bronchial tapering was present in the periphery of the lungs.22,23 all ct scans were reviewed by two pulmonologists ( hjj and jhk ) to confirm the diagnosis of bronchiectasis . in this study , long - term bronchodilator use was defined as administration of bronchodilator therapy for longer than 3 months . responders were defined as those whose fev1 values improved at least 12% and 200 ml from baseline fev1 following 312 months of bronchodilator therapy . patients who received long - term bronchodilator but did not meet these benchmarks were defined as poor responders . to determine whether positive bdr at baseline was an independent factor for being a responder and to appreciate the influence of demographic , clinical , and treatment variables , a series of multiple logistic regression analyses were performed on variables with p<0.2 based on univariate results or clinically important : model 1 contained the demographic variables age ( continuous ) , sex , and body mass index ( bmi ) ( continuous ) ; model 2 additionally included pulmonary - related variables , which are generally considered important in pulmonary diseases ( smoking history [ non- vs ex- or current smokers ] and prebronchodilator fev1 < 50% predicted ) ; and finally model 3 additionally included the treatment variable and number of inhalers , as well as all of the aforementioned variables . a total of 74 patients ( 44.6% ) had a history of pulmonary tuberculosis , and common coexisting pulmonary diseases included nontuberculous mycobacterial lung disease ( n=13 , 7.8% ) and chronic pulmonary aspergillosis ( n=5 , 3% ) . positive bdr at baseline was observed in 42 patients ( 25.3% ) . among the 166 patients , compared to poor responders , responders to treatment were more likely to be male ( 78.9% [ 45 of 57 ] vs 62.4% [ 68 of 109 ] , p=0.03 ) , current or ex - smokers ( 69.6% [ 39 of 56 ] vs 50.9% [ 54 of 106 ] , p=0.022 ) and have lower median fev1/fvc ( 50.5% [ iqr 43.5%58% ] vs 55% [ iqr 47%62.4% ] , p=0.011 ) and positive bdr at baseline ( 38.6% [ 22 of 57 ] vs 18.3% [ 20 of 109 ] , p=0.004 ) . there were no significant differences with respect to age , bmi , previous history of pulmonary tuberculosis , coexisting pulmonary or extrapulmonary comorbidities , or baseline pulmonary function tests , including fvc ( percentage predicted , liters ) and fev1 ( percentage predicted , liters ) between the responders and poor responders . between responders and poor responders , there were no significant differences in the number of involved lobes or the locations of the involved lobes . a median of two inhalers ( iqr 12 ) were used , and there were no significant differences in the number of inhalers used between the responders and poor responders . there were no significant differences in ics use , laba ics , or lama ics between responders and poor responders . however , laba / lama ics was more frequently used by responders than poor responders ( 31.6% vs 16.5% , p=0.025 ) . the increase in fev1 was more significant in patients with positive bdr at baseline compared to those without positive bdr at baseline ( figure 2 ; median 210 ml [ iqr 130430 ml ] vs 130 ml [ iqr 10 to 250 ml ] , p=0.001 ) . in addition , patients with positive bdr at baseline were more likely to be responders to long - term bronchodilator therapy compared to those without positive bdr at baseline ( figure 3 ; 52.4% vs 28.2% , p=0.004 ) . however , the increase in fev1 following bronchodilator therapy was statistically significant in patients without positive bdr at baseline ( 130 ml [ iqr 10 to 250 ml ] , p<0.001 ) , as well as in those with positive bdr at baseline ( 210 ml [ iqr 130430 ml ] , p<0.001 ) ( figure 4 ) . as shown in table 3 , positive bdr at baseline remained consistently a significant predictive factor for being a responder to long - term bronchodilator therapy , with the exception of model 3 , due to multiple insignificant variables making the model unstable . we then considered risk - factor modeling to predict responsiveness by removing irrelevant variables , and found that positive bdr at baseline was 2.3 times more ( confidence interval 1.0674.951 , p=0.034 ) significantly and independently associated with being a responder to long - term bronchodilator therapy than being a poor responder , in addition to the number of inhalers ( p=0.047 ) . a total of 74 patients ( 44.6% ) had a history of pulmonary tuberculosis , and common coexisting pulmonary diseases included nontuberculous mycobacterial lung disease ( n=13 , 7.8% ) and chronic pulmonary aspergillosis ( n=5 , 3% ) . positive bdr at baseline was observed in 42 patients ( 25.3% ) . among the 166 patients , compared to poor responders , responders to treatment were more likely to be male ( 78.9% [ 45 of 57 ] vs 62.4% [ 68 of 109 ] , p=0.03 ) , current or ex - smokers ( 69.6% [ 39 of 56 ] vs 50.9% [ 54 of 106 ] , p=0.022 ) and have lower median fev1/fvc ( 50.5% [ iqr 43.5%58% ] vs 55% [ iqr 47%62.4% ] , p=0.011 ) and positive bdr at baseline ( 38.6% [ 22 of 57 ] vs 18.3% [ 20 of 109 ] , p=0.004 ) . there were no significant differences with respect to age , bmi , previous history of pulmonary tuberculosis , coexisting pulmonary or extrapulmonary comorbidities , or baseline pulmonary function tests , including fvc ( percentage predicted , liters ) and fev1 ( percentage predicted , liters ) between the responders and poor responders . between responders and poor responders , there were no significant differences in the number of involved lobes or the locations of the involved lobes . a median of two inhalers ( iqr 12 ) were used , and there were no significant differences in the number of inhalers used between the responders and poor responders . however , laba / lama ics was more frequently used by responders than poor responders ( 31.6% vs 16.5% , p=0.025 ) . the increase in fev1 was more significant in patients with positive bdr at baseline compared to those without positive bdr at baseline ( figure 2 ; median 210 ml [ iqr 130430 ml ] vs 130 ml [ iqr 10 to 250 ml ] , p=0.001 ) . in addition , patients with positive bdr at baseline were more likely to be responders to long - term bronchodilator therapy compared to those without positive bdr at baseline ( figure 3 ; 52.4% vs 28.2% , p=0.004 ) . however , the increase in fev1 following bronchodilator therapy was statistically significant in patients without positive bdr at baseline ( 130 ml [ iqr 10 to 250 ml ] , p<0.001 ) , as well as in those with positive bdr at baseline ( 210 ml [ iqr 130430 ml ] , p<0.001 ) ( figure 4 ) . as shown in table 3 , positive bdr at baseline remained consistently a significant predictive factor for being a responder to long - term bronchodilator therapy , with the exception of model 3 , due to multiple insignificant variables making the model unstable . we then considered risk - factor modeling to predict responsiveness by removing irrelevant variables , and found that positive bdr at baseline was 2.3 times more ( confidence interval 1.0674.951 , p=0.034 ) significantly and independently associated with being a responder to long - term bronchodilator therapy than being a poor responder , in addition to the number of inhalers ( p=0.047 ) . in the present study of bronchiectasis patients with airflow limitation , we found that approximately one - third of patients were responders to long - term bronchodilator therapy , and positive bdr at baseline was significantly associated with an increase in fev1 following long - term bronchodilator therapy . however , the increase in fev1 was evident not only in patients with positive bdr at baseline but also in those without positive bdr at baseline . therefore , our findings suggest that patients with bronchiectasis who demonstrate poor bdr at baseline can benefit from long - term bronchodilator therapy . to the best of our knowledge , this is the first study to investigate the relationship between positive bdr at baseline and the long - term effects of bronchodilator therapy in patients with bronchiectasis . in the treatment of patients with bronchiectasis , bronchodilator use is recommended for patients with positive bdr at baseline.7 this recommendation is based on results from two previous studies that demonstrated significant bdr ( greater than 15% improvement in fev1 ) in a subset of patients with bronchiectasis after use of a short - acting bronchodilator.5,14 however , these studies evaluated bdr in fewer than 30 patients with bronchiectasis ( one study included 24 patients and the second included 16 patients ) , and did not confirm the long - term effect of bronchodilator therapy based on immediate bdr . in a relatively larger number of patients , our study confirmed and extended the previous findings , showing that about 25% of bronchiectasis patients with airflow limitation had positive bdr at baseline , and the presence of positive bdr at baseline was independently associated with improvement in lung function following long - term bronchodilator therapy in patients with bronchiectasis and airflow limitation . the predictive ability of airflow reversibility in a short - acting bronchodilator test on long - term improvements in lung function following bronchodilator therapy in patients with copd has been widely studied.15,25,26 a randomized controlled trial showed that copd patients with immediate bdr after the first dose of tiotropium showed a larger improvement in fev1 following 1 year of tiotropium use compared to copd patients without immediate bdr.15 other studies that evaluated the effects of salmeterol on copd patients have also shown that patients with positive bdr at baseline had larger improvements in lung function at 12 weeks and 1 year following the use of salmeterol compared with those without positive bdr at baseline.25,26 however , special attention should be given to patients without positive bdr at baseline . we observed that patients without positive bdr at baseline also exhibited significant improvement in fev1 following long - term bronchodilator therapy , although those with positive bdr at baseline had greater improvement in fev1 than those without positive bdr at baseline . despite the positive correlation between short - term response to bronchodilator at baseline and improvement in lung function following long - term bronchodilator therapy , several studies of copd patients have found that long - term bronchodilator therapy significantly improved lung function , dyspnea , and health status , irrespective of the presence or absence of positive bdr at baseline.15,2529 these findings led to the current guidelines that do not recommend use of reversibility testing with short - acting bronchodilator use to predict long - term response to treatment . therefore , our results suggest that long - term bronchodilator use needs to be extended to patients without positive bdr at baseline in patients with bronchiectasis and airflow limitation . however , a randomized clinical trial with a placebo group is needed to establish the efficacy of long - term bronchodilator use in patients with bronchiectasis . the coexistence of bronchial asthma in bronchiectasis patients has been reported,30,31 and a recent study has shown that the existence of bronchial asthma is an independent risk factor for bronchiectasis exacerbations.30 regarding lung function improvement following long - term bronchodilator therapy with ics , our study showed that coexisting bronchial asthma was not associated with being a responder in patients with bronchiectasis . our study included only bronchiectasis patients who had airflow limitation , which could have mitigated the effect of bronchodilator therapy with ics on coexisting bronchial asthma patients with bronchiectasis . further studies are necessary to investigate the effect of long - term bronchodilator therapy with ics on the rate of exacerbations in patients with both bronchial asthma and bronchiectasis . second , patients who had not undergone spirometry after 312 months following bronchodilator therapy were excluded , which may have led to selection bias . third , the rate of exacerbations , changes in symptoms , and health - related quality of life following long - term use of bronchodilators were not evaluated , which could represent additional long - term benefits to maintenance therapy with bronchodilators . however , the duration of 312 months was long enough to observe improvements in fev1 , and there was no significant difference in the median duration of bronchodilator use between responders and poor responders . therefore , further studies are necessary to compare the effectiveness of each type of bronchodilator in patients with bronchiectasis . in summary , we conclude that positive bdr at baseline was an independent predictive factor for improvement in lung function after long - term bronchodilator therapy in patients with bronchiectasis and airflow limitation . we further suggest that clinicians should consider long - term bronchodilator therapy for patients with poor bdr at baseline , as it can exhibit beneficial response .

cyclic sulfamides and their analogues have been the subject of many organic and medicinal chemistry studies due to their interesting biological activities that include anti - hiv and serine protease [ 14 ] . in addition , some cyclic sulfamide derivatives have been reported as nonhydrolyzable peptidomimetics [ 5 , 6 ] , metalloprotease inhibitors , and constrained peptides [ 810 ] . the benzothiadiazepine ring system has been considered as cyclic sulfamides , and these derivatives have been the subject , especially in the field of medicinal chemistry , because many useful therapeutic agents contain this heterocyclic system . for example , the nevirapine analogs , the pyrrolo[1,2-b]benzothiadiazepine-1,1-dioxides ( pbtds ) and the pyrrolo[2,1-d]benzothiadiazepine-1,1-dioxides , were tested and reported as potential nonnucleosidic reverse transcriptase inhibitors . furthermore , the pyrrolo[1,2-b]benzothiadiazepine ( pbtd ) derivatives were also reported to exert potent anticancer activities . considering the diverse biological properties of this class of compounds and as part of continuous work on the synthesis of biologically active heterocycles , we herein report simple and efficient procedures for the synthesis of a new class of fused benzothiadiazepine derivatives ( a ) , ( b ) , ( c ) ( figure 1 ) . these derivatives include two thiadiazepine rings and macrocyclic molecules containing a sulfamide functionality ( n so2n ) , which were synthesised using previously described n , n - disubstituted symmetric sulfamides and n(boc ) , n(alkyl)sulfamide [ 1418 ] . in particular , we report the synthesis and spectroscopic properties of novel macrocyclic rings containing the sulfamide unit , which was incorporated by a direct reaction between m - dibromomethylbenzene derivatives and n , n-disubstituted symmetric sulfamide . this strategy provides a ready access to a broad range of products . beyond their pivotal role in the development of supramolecular chemistry [ 19 , 20 ] , this class of molecules has also served as the basis for designing various receptors of organic molecules . moreover , they have become useful building blocks for constructing nanoporous structures [ 22 , 23 ] . our earlier studies involved the synthesis of heterocyclic compounds containing sulfonyl groups [ 9 , 10 , 15 , 16 ] . chlorosulfonyl isocyanate ( csi ) and sulfuryl chloride ( so2cl2 ) have been shown to be versatile reagents in the synthesis of heterocyclic chemistry . several total syntheses of n , n-disubstituted symmetric sulfamides ( 1a - d ) have been reported in the literature including the original synthetic approaches [ 2427 ] . thus , the starting material , sulfuryl chloride , was treated with an excess of the corresponding amine in dichloromethane for 24 h ( scheme 1 ) , and this resulted in the formation of products 1a - d in moderate yields . the synthesis of the key intermediates n(boc ) , n-alkyl - sulfamide ( 2a - f ) and n-((boc)sulfamoylamino)carboxylates ( 2g - j ) was accomplished as shown in scheme 1 . the carbamylation of chlorosulfonyl isocyanate with tert - butyl alcohol at 0c in dichloromethane followed by in situ sulfamoylation with the corresponding amine , amino acid ester hydrochloride , or diamine in the presence of triethylamine ( tea ) gave the desired n(boc ) , n(alkyl)sulfamide ( 2a - b ) , n(boc ) , n-sufamoylamino acid esters ( 2g - j ) or bis - carboxylsulfamides ( 2c - f ) [ 28 , 29 ] . as outlined in scheme 2 , the n , n-disubstituted symmetric sulfamides ( 1a - d ) are a suitable starting material for the synthesis of an array of new benzocondensed scaffolds ( 4a - c ) in good yields 7579% . the starting materials , n , n-disubstituted symmetric sulfamides 1a - d , were condensed with 1,2,4,5-tetrakis(bromomethyl)benzene ( 0.5 equiv ) by refluxing in acetonitrile for 10 h in the presence of potassium carbonate ( k2co3 ) to afford fused benzo - di - thiadiazepines 4a - c . in the second route , after replacing n , n-disubstituted sulfamides by n(boc)sulfamides derivatives ( 2a - j ) under the same conditions , products 4d - f and 4d-f were formed . both isomers ( symmetric and asymmetric ) these yields seem to be strongly dependent upon the reaction conditions ( solvent , temperature , and steric effect ) . therefore , further optimization of the reaction conditions might improve the yield of these reactions . the prepared n(boc)-protected compounds ( 2a - j ) have traditionally been a starting point for the design of novel benzocondensed derivatives ( 3a - e ) by condensation with ,-dibromo - o - xylene in acetonitrile in the presence of potassium carbonate ( k2co3 ) ( scheme 2 ) . the presence of the tert - butoxycarbonyl ( boc ) group , which activates the sulfamide nitrogen nucleophilicity , was required for substitution . this protecting group was removed by trifluoroacetic acid to yield the unprotected fused cyclic sulfamides . these deprotected compounds were considered excellent starting materials for preparation of biomolecule analogues employing different types of reactions such as regioselective mitsunobu reaction ( dead , pph3 , thf at room temperature , 2 h ) [ 31 , 32 ] . the structures of fused compounds were confirmed by ir , mass spectrometry , and nmr ( h , c ) , and the results are presented in table 1 . the ir spectra of compounds 3a - e displayed the characteristic absorption bonds near 1370 for so2 , near 1140 cm for so2 and strong absorption in the vicinity of 1740 cm due to c = o stretching . if the substituent r is an ester group , there must also be an intense stretch in the carbonyl region of the spectrum near to 1750 cm . at ambient temperature , the h - nmr spectra of the benzothiadiazepines showed sharp signals near 1.40 ppm indicating the presence of boc group . the aromatic proton signals appear at 7 ppm as one multiplet of 4h for ( 3a - c ) and 9h for ( 3d - e ) . esi - ms spectra of the compounds 3a - e showed ion peaks due to [ m+na ] and [ m+2na ] . the structures of fused a , b , and c compounds were supported by analysis of the mass spectra esi - ms , which showed peaks respectively at m / z 509 , 567 , and 710 indicating molecular masses of ions [ m+na ] . as shown in table 1 , all the hnmr spectra showed one singlet peak near 7 ppm , which is a strong indication of the presence of aromatic protons . in the infrared data , all spectra showed bands near 1150 and 1350 cm due to so2 stretching . in the esi - ms spectra , all the prepared fused compounds 4d - f exhibit intense peaks corresponding to the molecular weight [ m+na ] . since the symmetric and asymmetric fused compounds have the same molecular weight , it was difficult to extract all the rich structural information from the mass spectra . in the ir spectra ( table 1 ) , there are peaks at about ( 11401170 ) and ( 13681390 ) cm , due to the sulfonyl group ( so2 ) stretching , and at about 17151732 cm , due to c = o stretching vibrations . for the compounds containing an ester group , ir spectra showed also bands near 1700 cm due to c = o stretching . the one difference in the h nmr spectra between the symmetric and asymmetric fused is the aromatic region . the h nmr spectra of symmetric fused 4d - f showed resonances attributed a two aromatic protons , which appeared as one singlet with a relative integration of 2 indicating the equivalency of the two hydrogens . however , for the asymmetric fused derivatives , the h - nmr spectra showed two different kinds of aromatic protons with relative integration of 1 : 1 . there are many strategies available for the synthesis of benzylic amide macrocycles that involve the reaction of an ester group with an amino group [ 3335 ] . in this work as shown in scheme 3 , the desired macrocyclic sulfamides 5 were synthesized in one step [ 2 + 2 ] condensation under high dilution conditions . a solution of 1-methoxy-4-tert - butyl-(2,6-dibromomethyl)benzene ( 1 equiv ) in 20 ml of acetonitrile and a solution of n , n-disubstituted symmetric sulfamide ( 1 equiv ) in 20 ml of ch3cn were added dropwise using two mechanically driven syringes over 5 h into solution of k2co3 ( 4.5 equiv ) in 130 ml of ch3cn under nitrogen with stirring at reflux for 24 h. the reaction mixture was subsequently cooled down , and the solvent was removed . dichloromethane was added to the obtained crude , and this solution was washed with 2 n hcl then with water and dried with magnesium sulfate . the solvent was evaporated to give the macrocycle 5a in 58% yield . in the macrocyclization reactions , it was critical to find suitable reaction conditions that maintain the correct condensation , while keeping the reactions fast enough to prevent buildup of reactive intermediates . the structure of macrocyclic compounds 5a was unambiguously confirmed by ir , mass spectrometry , and nmr ( h , c ) spectroscopy . the infrared spectrum showed characteristic bands at 1148 and 1361 cm , which were assigned to the sulfonyl group ( so2 ) . in addition , the 300 mhz h spectrum , measured on a sample dissolved in cdcl3 , showed a relative integration of 4 : 20 for the two sets of peaks at 6.78 and 7.307.45 ppm . in summary , we have successfully synthesized and characterized a new series of n - protected fused benzothiadiazepines , which offer good starting materials for the synthesis of new molecules with interesting biological activities . in the second part , we described an efficient method for the synthesis of new macrocycle with sulfamide moiety with potential diverse applications in supramolecular chemistry and as starting materials for further synthetic transformations . the synthetic example presented in this work is one of the simplest and most efficient macrocyclization reactions based on the technique of high - dilution conditions . the biological evaluation of the compounds synthesized in this work is currently being carried out . nmr spectra were acquired on commercial instruments ( bruker avance 300 mhz or bruker amx 400 mhz ) and chemical shifts ( ) are reported in parts per million downfield from internal me4si ( s = singlet , d = doublet , dd = double of doublet , t = triplet , q = quartet , m = multiplet ) . mass spectrometry data were obtained with an hp ms apparatus 5989a , at 70 ev for ei spectra and with methane as reagent gas for ci spectra . the esi - ms were obtained on mariner ( esi tof ) and api 365 ( esi 3q ) mass spectrometers with methanol as a spray solvent . melting points ( not corrected ) were determined using a reichert thermovar or electrothermal 9200 apparatus . all reactions were done in a 10 ml glass tube sealed with a teflon stopper unless stated otherwise . the reaction was carried out by dropwise addition of a solution of sulfuryl chloride ( 1 equiv ) in 20 ml of dichloromethane to a solution of the corresponding amine ( 46 equiv ) in 50 ml of ch2cl2 at 0c in darkness . the reaction mixture was warmed to room temperature ( rt ) , stirred for 24 h , and monitored by tlc ( sio2 ) . the crude was washed by hcl ( 2 n , 2 20 ml ) water ( 2 30 ml ) and dried over na2so4 . the solution was filtered and then concentrated under reduced pressure to leave yellow solid as the crude product . column chromatography ( ch2cl2 , meoh 95 : 5 ) afforded the n , n-dialkyl sulfamide . n , n-dipropylsulfamide ( 1a)this compound was prepared according to the general procedure a , using a solution of propylamine ( 6 equiv ) in ch2cl2 and so2cl2 ( 1 equiv ) in ch2cl2 . yield = 60% ( was obtained as a white solid ) ; rf = 0.45 [ sio2 , ch2cl2/meoh ( 95 : 5 ) ] ; mp 6465c ( described : 6263c ) . ir ( kbr , cm ) : 3280 ( nh ) , 1333 and 1150 ( so2 ) . h nmr ( cdcl3 ) : 0.95 ( t , j = 7.2 hz , 6h , ch3 ) , 1.57 ( sext , j = j = 7.1 hz , 4h , -ch2 ) , 2.99 ( q , 4h , -ch2 ) , 4.27 ( t broad , 2h , nh ) . c nmr ( cdcl3 ) : 11.26 ( -c ) , 22.89 ( -c ) , 44.95 ( -c ) . lrms ( ci ) : 181 [ m+h ] . this compound was prepared according to the general procedure a , using a solution of propylamine ( 6 equiv ) in ch2cl2 and so2cl2 ( 1 equiv ) in ch2cl2 . yield = 60% ( was obtained as a white solid ) ; rf = 0.45 [ sio2 , ch2cl2/meoh ( 95 : 5 ) ] ; mp 6465c ( described : 6263c ) . ir ( kbr , cm ) : 3280 ( nh ) , 1333 and 1150 ( so2 ) . h nmr ( cdcl3 ) : 0.95 ( t , j = 7.2 hz , 6h , ch3 ) , 1.57 ( sext , j = j = 7.1 hz , 4h , -ch2 ) , 2.99 ( q , 4h , -ch2 ) , 4.27 ( t broad , 2h , nh ) . c nmr ( cdcl3 ) : 11.26 ( -c ) , 22.89 ( -c ) , 44.95 ( -c ) . n , n-dibutylsulfamide ( 1b)yield = 58% ( was obtained as a white solid ) ; rf = 0.36 [ ( sio2 , ch2cl2 ) ) ] ; mp : 126127c ( described 126.5c ) . ir ( kbr , cm ) : 3281 ( nh ) , 1314 and 1145 ( so2 ) . h nmr ( cdcl3 ) : 4.33 ( t broad , 2h , nh ) , 3.04 ( m , 4h , -ch2 ) , 1.54 ( m , 4h , -ch2 ) , 1.38 ( m , 4h , -ch2 ) , 0.93 ( t , j = 7.1 hz , 6h , ch3 ) . c nmr ( cdcl3 ) : 43.2 ( -c ) , 31.7 ( -c ) , 20.11 ( -c ) , 13.88 ( ch3 ) . lrms ( ci ) : 209 [ m+h ] . yield = 58% ( was obtained as a white solid ) ; rf = 0.36 [ ( sio2 , ch2cl2 ) ) ] ; mp : 126127c ( described 126.5c ) . ir ( kbr , cm ) : 3281 ( nh ) , 1314 and 1145 ( so2 ) . h nmr ( cdcl3 ) : 4.33 ( t broad , 2h , nh ) , 3.04 ( m , 4h , -ch2 ) , 1.54 ( m , 4h , -ch2 ) , 1.38 ( m , 4h , -ch2 ) , 0.93 ( t , j = 7.1 hz , 6h , ch3 ) . c nmr ( cdcl3 ) : 43.2 ( -c ) , 31.7 ( -c ) , 20.11 ( -c ) , 13.88 ( ch3 ) . lrms ( ci ) : 209 [ m+h ] . n , n-di(2methoxyethyl)sulfamide ( 1c)yield = 61% ( was obtained as a viscous oil ) ; rf = 0.36 [ sio2 , ch2cl2/meoh ( 95 : 5 ) ] ; ir ( kbr , cm ) : 3279 ( nh ) , 1316 and 1147 ( so2 ) . h nmr ( cdcl3 ) : 3.22 ( q , 4h , -ch2 ) , 3.36 ( s , 6h , ch3 ) , 3.52 ( m , 4h , -ch2 ) , 5.28 ( t , 2h , nh ) , c nmr ( cdcl3 ) : 42.67 ( -c ) , 58.57 ( ch3 ) , 70.93 ( -c ) . lrms ( ci ) : 213 [ m+h ] . yield = 61% ( was obtained as a viscous oil ) ; rf = 0.36 [ sio2 , ch2cl2/meoh ( 95 : 5 ) ] ; ir ( kbr , cm ) : 3279 ( nh ) , 1316 and 1147 ( so2 ) . h nmr ( cdcl3 ) : 3.22 ( q , 4h , -ch2 ) , 3.36 ( s , 6h , ch3 ) , 3.52 ( m , 4h , -ch2 ) , 5.28 ( t , 2h , nh ) , c nmr ( cdcl3 ) : 42.67 ( -c ) , 58.57 ( ch3 ) , 70.93 ( -c ) . n , n-dibenzylsulfamide ( 1d)yield = 59% ( was obtained as a white solid ) ; rf = 0.37 ( sio2 , ch2cl2 ) ; mp : 182184c ( described 180182c ) . ir ( kbr , cm ) : 3270 ( nh ) , 3034 ( ch - ar ) , 1350 and 1143 ( so2 ) . h nmr ( cdcl3 ) : 4.17 ( d , 4h , ch2 ) , 4.37 ( t broad , 2h , nh ) , 7.287.34 ( m , 10h , ar - h ) . lrms ( ci ) : 277 [ m+h ] , 199 , 91 . yield = 59% ( was obtained as a white solid ) ; rf = 0.37 ( sio2 , ch2cl2 ) ; mp : 182184c ( described 180182c ) . ir ( kbr , cm ) : 3270 ( nh ) , 3034 ( ch - ar ) , 1350 and 1143 ( so2 ) . h nmr ( cdcl3 ) : 4.17 ( d , 4h , ch2 ) , 4.37 ( t broad , 2h , nh ) , 7.287.34 ( m , 10h , ar - h ) . to a stirred solution of csi ( 1 equiv , 10 mmol , 1.4153 g ) in 20 ml of anhydrous dichloromethane at 0c was added a solution of tert - butyl alcohol ( 1 equiv , 10 mmol , 0.7412 g ) in 20 ml of dried ch2cl2 . after being stirred for 30 min , the resulting solution of n(boc)-sulfamoyl chloride and triethylamine ( tea ) in 20 ml dichloromethane was added dropwise to a solution of amine ( 1 equiv ) or ( diamine 0.5 equiv ) in 20 ml of ch2cl2 . the resulting reaction solution was allowed to warm up to rt over 3 h. the reaction mixture was diluted with dichloromethane and washed with 0.1 n hcl and brine . the organic layer was dried ( na2so4 ) and concentrated in vacuo to give the crude product . n , n-bis(tert - butoxycarbonylsulfamoyl)-1,2-diaminoethane ( 2c)this compound was prepared according to the general procedure b using a solution of 1,2-ethan diamine ( 0.5 equiv , 5 mmol , 0.6010 g ) in ch2cl2 . yield = 70% ( was obtained as a white solid ) ; rf = 0.20 [ sio2 , ch2cl2-meoh 95 : 5 ] ; ir(kbr , cm ) : 3286 and 3311(nh ) ; 1709 ( c = o ) ; 1346 and 1141 ( so2 ) . h nmr ( dmso - d6 ) : 10.90 ( s , 2h , nhboc ) ; 7.65 ( s , 2h , nh ) ; 2.98 ( s , 4h , ch2 ) ; 1.43 ( s , 18h , tbu ) . hrms esi : m / z : 441 [ m+na ] . this compound was prepared according to the general procedure b using a solution of 1,2-ethan diamine ( 0.5 equiv , 5 mmol , 0.6010 g ) in ch2cl2 . yield = 70% ( was obtained as a white solid ) ; rf = 0.20 [ sio2 , ch2cl2-meoh 95 : 5 ] ; ir(kbr , cm ) : 3286 and 3311(nh ) ; 1709 ( c = h nmr ( dmso - d6 ) : 10.90 ( s , 2h , nhboc ) ; 7.65 ( s , 2h , nh ) ; 2.98 ( s , 4h , ch2 ) ; 1.43 ( s , 18h , tbu ) . n , n-bis(tert - butoxycarbonylsulfamoyl)-1,3-diaminopropane ( 2d)this compound was prepared according to the general procedure b using a solution of 1,3-propandiamine ( 0.5 equiv , 5 mmol , 0.3706 g ) in dichloromethane yield = 70% ( was obtained as a white solid ) ; rf = 0.25 [ sio2 , ch2cl2-meoh 95 : 5 ] ; mp : 175177c . ir ( kbr , cm ) : 3266 and 3212(nh ) ; 1697 ( c = o ) ; 1348 and 1138 ( so2 ) . h nmr ( dmso - d6 ) : 10.79 ( s , 2h , nhboc ) ; 7.52 ( t , 2h , nh ) ; 2.89 ( q , 4h , ch2-n ) ; 1.63 ( m , 2h , ch2 ) ; 1.42 ( s , 18h , tbu ) . hrms . esi : m / z : 455 [ m+na ] , 887 [ m*2+na ] . this compound was prepared according to the general procedure b using a solution of 1,3-propandiamine ( 0.5 equiv , 5 mmol , 0.3706 g ) in dichloromethane yield = 70% ( was obtained as a white solid ) ; rf = 0.25 [ sio2 , ch2cl2-meoh 95 : 5 ] ; mp : 175177c . ir ( kbr , cm ) : 3266 and 3212(nh ) ; 1697 ( c = o ) ; 1348 and 1138 ( so2 ) . h nmr ( dmso - d6 ) : 10.79 ( s , 2h , nhboc ) ; 7.52 ( t , 2h , nh ) ; 2.89 ( q , 4h , ch2-n ) ; 1.63 ( m , 2h , ch2 ) ; 1.42 ( s , 18h , tbu ) . esi : m / z : 455 [ m+na ] , 887 [ m*2+na ] . compound ( 2e)this compound was prepared according to the general procedure b using a solution of l - cystine methyl ester dihydrochloride ( 0.5 equiv , 5 mmol , 1.7064 g ) in ch2cl2 and triethylamine ( 2 equiv , 20 mmol , 2.0238 g ) . yield = 71% ( was obtained as a white solid ) ; rf = 0.37 [ sio2 , ch2cl2-meoh 95 : 5 ] ; ir ( kbr , cm ) : 3289 and 3240(nh ) ; 1709 and 1748 ( c = o ) ; 1364 and 1139 ( so2 ) . h nmr ( dmso - d6 ) : 10.98 ( s , 2h , nhboc ) ; 8.40 ( d , 2h , nh ) ; 4.22 ( q , 2h , ch ) ; 3.66 ( s , 6h , ch3 ) ; 3.00 ( d , 4h , ch2 ) ; 1.41 ( s , 18h , tbu ) . hrms esi : m / z : 659 [ m+na ] , 1275 [ m*2+na ] . this compound was prepared according to the general procedure b using a solution of l - cystine methyl ester dihydrochloride ( 0.5 equiv , 5 mmol , 1.7064 g ) in ch2cl2 and triethylamine ( 2 equiv , 20 mmol , 2.0238 g ) . yield = 71% ( was obtained as a white solid ) ; rf = 0.37 [ sio2 , ch2cl2-meoh 95 : 5 ] ; ir ( kbr , cm ) : 3289 and 3240(nh ) ; 1709 and 1748 ( c = o ) ; 1364 and 1139 ( so2 ) . h nmr ( dmso - d6 ) : 10.98 ( s , 2h , nhboc ) ; 8.40 ( d , 2h , nh ) ; 4.22 ( q , 2h , ch ) ; 3.66 ( s , 6h , ch3 ) ; 3.00 ( d , 4h , ch2 ) ; 1.41 ( s , 18h , tbu ) . hrms esi : m / z : 659 [ m+na ] , 1275 [ m*2+na ] . dimethyl-5,5-oxybis(2-(n-(tert - butoxycarbonyl)sulfamoylamino)pentanoate ) ( 2f)this compound was prepared according to the general procedure b using a solution of 1,3-propan diamine ( 0.5 equiv , 5 mmol , 0.661 g ) . yield = 73% ( was obtained as a white solid ) ; rf = 0.49 [ sio2 , ch2cl2-meoh 95 : 5 ] ; mp : 132134c . ir ( kbr , cm ) : 3296 ( nh ) ; 1713 and 1738 ( c = o ) ; 1371 and 1143 ( so2 ) . h nmr ( cdcl3 ) : 7.52 ( s , 2h , nhboc ) ; 5.79 ( t , 2h , nh ) ; 3.57 ( t , 4h , ch2o ) ; 3.20 ( q , 4h , ch2n ) ; 1.84 ( m , 4h , ch2 ) ; 1.49 ( s , 18h , tbu ) . hrms esi : m / z : 514 [ m+na ] , 1004 [ m*2+na ] . this compound was prepared according to the general procedure b using a solution of 1,3-propan diamine ( 0.5 equiv , 5 mmol , 0.661 g ) . yield = 73% ( was obtained as a white solid ) ; rf = 0.49 [ sio2 , ch2cl2-meoh 95 : 5 ] ; mp : 132134c . ir ( kbr , cm ) : 3296 ( nh ) ; 1713 and 1738 ( c = o ) ; 1371 and 1143 ( so2 ) . h nmr ( cdcl3 ) : 7.52 ( s , 2h , nhboc ) ; 5.79 ( t , 2h , nh ) ; 3.57 ( t , 4h , ch2o ) ; 3.20 ( q , 4h , ch2n ) ; 1.84 ( m , 4h , ch2 ) ; 1.49 ( s , 18h , tbu ) . hrms esi : m / z : 514 [ m+na ] , 1004 [ m*2+na ] . to a suspension of the amino acid ester hydrochloride ( 1 equiv , 10 mmol ) was added triethylamine ( 1 equiv , 10 mmol , 1.0119 g ) in 20 ml of dichloromethane . simultaneously the tert - butyl chlorosulfonyl carbamate was prepared by addition of tert - butyl alcohol ( 1 equiv , 10 mmol , 0.7412 g ) in 20 ml of ch2cl2 to an ice - cooled solution containing csi ( 1 equiv , 10 mmol , 1.4153 g ) in 20 ml of dichloromethane . the obtained reagent solution was slowly added to the solution of amino acid ester hydrochloride in 30 ml of dichloromethane at the same time as of et3n ( 1 equiv , 10 mmol , 1.0119 g ) . the mixture was then diluted with ch2cl2 ( 100 ml ) and washed with 2 portions of 1 m hcl and water . the solution was then dried with na2so4 and concentrated in vacuum to give the crude product . recrystallization from ch2cl2 at low temperature or chromatography on silica gel ( eluent : ch2cl2/meoh , 9 : 1 ) afforded the pure carboxyl sulfamide . the synthesis of the compounds , starting from csi , tert - butyl alcohol and methyl esters of amino acids ( l - alanine 2j and l - phenylalanine 2 h ) has been previously reported [ 9 , 10 ] . ethyl [ n,(n-tert - butyloxycarbonyl)-sulfamoyl]-acetate ( 2 g ) this compound was prepared according to the general procedure c using a solution of glycine ethyl ester hydrochloride ( 1 equiv , 10 mmol , 1.396 g ) . yield = 72% ( was obtained as a white solid ) ; rf = 0.60 [ sio2 , ch2cl2-meoh 9 : 1 ] ; mp 122123c . ir ( kbr , cm ) : 1352 and 1126 ( so2 ) , 1735 and 1675 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.5 ( s , 9h , tbu ) , 1.30 ( t , 3h , ch3 ) , 3.96 ( d , 2h , n - ch2 ) , 4.24 ( q , 2h , ch2 ) , 5.63 ( t , 1h , nh - ch2 ) , 7.25 ( s , 1h , nhboc ) . this compound was prepared according to the general procedure c using a solution of glycine ethyl ester hydrochloride ( 1 equiv , 10 mmol , 1.396 g ) . yield = 72% ( was obtained as a white solid ) ; rf = 0.60 [ sio2 , ch2cl2-meoh 9 : 1 ] ; mp 122123c . ir ( kbr , cm ) : 1352 and 1126 ( so2 ) , 1735 and 1675 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.5 ( s , 9h , tbu ) , 1.30 ( t , 3h , ch3 ) , 3.96 ( d , 2h , n - ch2 ) , 4.24 ( q , 2h , ch2 ) , 5.63 ( t , 1h , nh - ch2 ) , 7.25 ( s , 1h , nhboc ) methyl [ n,(n-tert - butyloxycarbonyl)-sulfamoyl]-2-phenyglycynate ( 2i)this compound was prepared according to the general procedure c using a solution of phenyl glycine methyl ester hydrochloride ( 1 equiv , 10 mmol , 2.0165 g ) . yield = 76% ( was obtained as a white solid ) ; rf = 0.70 [ sio2 , ch2cl2-meoh 9 : 1 ] ; mp 144146c . ir ( kbr , cm ) : 1362 and 1141 ( so2 ) , 17351712 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.44 ( s , 9h , tbu ) , 3.74 ( s , 3h , o - ch3 ) , 5.27 ( d , 1h , ch ) , 6.27 ( d , 1h , nh ) , 7.36 ( s , 5h , ph ) , 7.44 ( s , 1h , nhboc ) . this compound was prepared according to the general procedure c using a solution of phenyl glycine methyl ester hydrochloride ( 1 equiv , 10 mmol , 2.0165 g ) . yield = 76% ( was obtained as a white solid ) ; rf = 0.70 [ sio2 , ch2cl2-meoh 9 : 1 ] ; mp 144146c . ir ( kbr , cm ) : 1362 and 1141 ( so2 ) , 17351712 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.44 ( s , 9h , tbu ) , 3.74 ( s , 3h , o - ch3 ) , 5.27 ( d , 1h , ch ) , 6.27 ( d , 1h , nh ) , 7.36 ( s , 5h , ph ) , 7.44 ( s , 1h , nhboc ) . to a stirring solution of [ n - tert - butyloxycarbonyl , n-alkyl]-sulfamide ( 1 equiv , 1 mmol ) in ch3cn ( 50 ml ) in a 100 ml round bottom flask was added k2co3 ( 2.5 equiv , 2.5 mmol , 0.34552 g ) and ,-dibromo - o - xylene ( 1 equiv , 1 mmol , 0.26396 g ) . the resulting mixture was stirred at reflux for 4 hours then cooled to room temperature . the residue was dissolved in ch2cl2 , washed with 2 portions of hcl ( 1 m ) and water and dried with na2so4 , and the solvent was removed under reduced pressure to give the crude oil . flash chromatography on silica gel ch2cl2 to furnish the pure fused cyclic sulfamide in 7085% yields the following . 1,5-dihydro-2-tert - butoxycarbonyl-4-benzylbenzo[d]thiadiazepine-3,3-dioxides ( 3a)this compound was prepared according to the general procedure d , using a solution of 2a ( 1 equiv , 1 mmol , 0.28635 g ) . yield = 71% ( was obtained as a viscous oil ) ; rf = 0.36 [ sio2 , ch2cl2 ] . ir ( kbr , cm ) : 1389 and 1141 ( so2 ) , 1732 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.43 ( s , 9h , tbu ) , 4.14 ( s , 2h , ch2-n ) , 4.37 ( s , 2h , ch2-n - boc ) , 4.91 ( s , 2h , ch2-ph ) , 7.037.37 ( m , 9h , h - ar ) . this compound was prepared according to the general procedure d , using a solution of 2a ( 1 equiv , 1 mmol , 0.28635 g ) . yield = 71% ( was obtained as a viscous oil ) ; rf = 0.36 [ sio2 , ch2cl2 ] . ir ( kbr , cm ) : 1389 and 1141 ( so2 ) , 1732 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.43 ( s , 9h , tbu ) , 4.14 ( s , 2h , ch2-n ) , 4.37 ( s , 2h , ch2-n - boc ) , 4.91 ( s , 2h , ch2-ph ) , 7.037.37 ( m , 9h , h - ar ) . 1,5-dihydro-2-tert - butoxycarbonyl-4-(cyclohexyl)benzo[d]thiadiazepine-3,3-dioxides ( 3b)this compound was prepared according to the general procedure d , using a solution of 2b ( 1 equiv , 1 mmol , 0.27837 g ) . yield = 80% ( was obtained as a white solid ) ; rf = 0.42 [ sio2 , ch2cl2 ] ; mp 9698c . ir ( kbr , cm ) : 1376 and 1145 ( so2 ) , 1726 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.231.72 ( m , 10h , cyclohexyl ) ; 1.41 ( s , 9h , tbu ) , 3.94 ( m , 1h , ch ) , 4.58 ( s , 2h , ch2 ) , 4.88 ( s , 2h , ch2 ) , 7.207.28 ( m , 4h , h - ar ) . this compound was prepared according to the general procedure d , using a solution of 2b ( 1 equiv , 1 mmol , 0.27837 g ) . yield = 80% ( was obtained as a white solid ) ; rf = 0.42 [ sio2 , ch2cl2 ] ; mp 9698c . ir ( kbr , cm ) : 1376 and 1145 ( so2 ) , 1726 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.231.72 ( m , 10h , cyclohexyl ) ; 1.41 ( s , 9h , tbu ) , 3.94 ( m , 1h , ch ) , 4.58 ( s , 2h , ch2 ) , 4.88 ( s , 2h , ch2 ) , 7.207.28 ( m , 4h , h - ar ) . 1,5-dihydro-2-tert - butoxycarbonyl-4-((ethoxycarbonyl)methyl)benzo[d]thiadiazepine-3,3-dioxides ( 3c)this compound was prepared according to the general procedure d , using a solution of 2 g ( 1 equiv , 1 mmol , 0.28231 g ) . yield = 79% ( was obtained as a white solid ) ; rf = 0.20 [ sio2 , ch2cl2 ] ; mp 9192c . ir ( kbr , cm ) : 1382 and 1134 ( so2 ) , 1729 and 1748 2(c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.28 ( t , 3h , ch3 ) , 1.41 ( s , 9h , tbu ) , 3.77 ( s , 2h , ch2co ) , 4.23 ( q , 2h , ch2-o ) , 4.72 ( s , 2h , ch2-n ) , 4.88 ( s , 2h , ch2-n - boc ) , 7.207.35 ( m , 4h , h - ar ) . hrms esi : m / z : 407 [ m+na ] , 791[m*2+na ] . this compound was prepared according to the general procedure d , using a solution of 2 g ( 1 equiv , 1 mmol , 0.28231 g ) . yield = 79% ( was obtained as a white solid ) ; rf = 0.20 [ sio2 , ch2cl2 ] ; mp 9192c . ir ( kbr , cm ) : 1382 and 1134 ( so2 ) , 1729 and 1748 2(c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.28 ( t , 3h , ch3 ) , 1.41 ( s , 9h , tbu ) , 3.77 ( s , 2h , ch2co ) , 4.23 ( q , 2h , ch2-o ) , 4.72 ( s , 2h , ch2-n ) , 4.88 ( s , 2h , ch2-n - boc ) , 7.207.35 ( m , 4h , h - ar ) . hrms esi : m / z : 407 [ m+na ] , 791[m*2+na ] . 1,5-dihydro-2-tert - butoxycarbonyl-4-(benzyl(methoxycarbonyl)methyl)benzo[d]thiadiazepine-3,3-dioxides ( 3d)this compound was prepared according to the general procedure d , using a solution of 2 h ( 1 equiv , 1 mmol , 0.35841 g ) . yield = 76% ( was obtained as a viscous oil ) ; rf = 0.37 [ sio2 , ch2cl2 ] . ir ( kbr , cm ) : 1368 and 1142 ( so2 ) , 1735 and 1742 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.40 ( s , 9h , tbu ) , 3.25 ( s , 3h , ch3o ) , 5.01 ( t , 1h , ch ) , 4.76 ( d , 2h , ch2-ph ) , 4.73 ( s , 2h , ch2n boc ) , 4.65 ( s , 2h , ch2n ) , 7.087.28 ( m , 9h , ar - h ) . hrms esi : m / z : 483 [ m+na ] , 944[m*2+na ] . this compound was prepared according to the general procedure d , using a solution of 2 h ( 1 equiv , 1 mmol , 0.35841 g ) . yield = 76% ( was obtained as a viscous oil ) ; rf = 0.37 [ sio2 , ch2cl2 ] . ir ( kbr , cm ) : 1368 and 1142 ( so2 ) , 1735 and 1742 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.40 ( s , 9h , tbu ) , 3.25 ( s , 3h , ch3o ) , 5.01 ( t , 1h , ch ) , 4.76 ( d , 2h , ch2-ph ) , 4.73 ( s , 2h , ch2n boc ) , 4.65 ( s , 2h , ch2n ) , 7.087.28 ( m , 9h , ar - h ) . hrms esi : m / z : 483 [ m+na ] , 944[m*2+na ] . 1,5-dihydro-2-tert - butoxycarbonyl-4-(phenyl(methoxycarbonyl)methyl)benzo[d]thiadiazepine-3,3-dioxides ( 3e)this compound was prepared according to the general procedure d , using a solution of 2i ( 1 equiv , 1 mmol , 0.34438 g ) . yield = 66% ( was obtained as a white solid ) ; rf = 0.38 [ sio2 , ch2cl2 ] ; mp : 9092c . ir ( kbr , cm ) : 1314 and 1138 ( so2 ) , 1732 and 1708 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.43 ( s , 9h , tbu ) , 3.66 ( s , 3h , ch3-o ) , 5.96 ( s , 1h , ch ) , 6.987.32 ( m , 9h , h - ar ) , 4.384.80 ( dd , 2h , chahb - n ) , 4.855.05 ( dd , 2h , chahb - n - boc ) , 6.987.32 ( m , 9 h , h - ar ) . hrms esi : m / z : 469 [ m+na ] , 915[m*2+na ] . this compound was prepared according to the general procedure d , using a solution of 2i ( 1 equiv , 1 mmol , 0.34438 g ) . yield = 66% ( was obtained as a white solid ) ; rf = 0.38 [ sio2 , ch2cl2 ] ; mp : 9092c . ir ( kbr , cm ) : 1314 and 1138 ( so2 ) , 1732 and 1708 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.43 ( s , 9h , tbu ) , 3.66 ( s , 3h , ch3-o ) , 5.96 ( s , 1h , ch ) , 6.987.32 ( m , 9h , h - ar ) , 4.384.80 ( dd , 2h , chahb - n ) , 4.855.05 ( dd , 2h , chahb - n - boc ) , 6.987.32 ( m , 9 h , h - ar ) . an acetonitrile solution ( 50 ml ) containing n , n-disubstituted symmetric sulfamide ( 2 equiv , 2 mmol ) , 2,3,4,5-tetrakis(bromomethyl)benzene ( 1 equiv , 1 mmol , 0.44980 g ) , and anhydrous k2co3 ( 4.5 equiv , 4.5 mmol , 0.6219 g ) was heated at reflux for 10 hours . the reaction mixture was cooled to room temperature , filtered , and after removal of the solvent under reduced pressure a solid was obtained . the solid was redissolved in dichloromethane ( ch2cl2 ) , washed with 2 portions of hcl 1 m ( 2 20 ml ) followed with water ( 2 30 ml ) , and dried with na2so4 . the residue was purified by chromatography on silica gel using ch2cl2 to yield fused symmetric and asymmetric benzothiadiazepines compound ( 4a)this compound was prepared according to the general procedure e , using a solution of 1a ( 2 equiv , 2 mmol and 0.36054 g ) . yield = 76% ( was obtained as a white solid ) ; rf = 0.25 [ sio2 , ch2cl2 ] ; mp 264266c . ir ( kbr , cm ) : 1339 and 1148 ( so2 ) . h nmr ( 300 mhz , cdcl3 ) : 0.90 ( t , 12h , ch3 ) , 1.58 ( m , 8h , -ch2 ) , 2.91 ( t , 8h , -ch2 ) , 4.46 ( s , 8h , n - ch2-ar ) , 7.15 ( s , 2h , h - ar ) . c nmr ( cdcl3 ) : 11.02 ( -c ) , 21.08 ( -c ) , 48.90 ( -c ) , 50.03 ( ch2 ) , 132.50 and 136.85 ( car ) . lrms ( ci ) : 487 [ m+h ] ; hrms esi : m / z : 509 [ m+na ] . this compound was prepared according to the general procedure e , using a solution of 1a ( 2 equiv , 2 mmol and 0.36054 g ) . yield = 76% ( was obtained as a white solid ) ; rf = 0.25 [ sio2 , ch2cl2 ] ; mp 264266c . ir ( kbr , cm ) : 1339 and 1148 ( so2 ) . h nmr ( 300 mhz , cdcl3 ) : 0.90 ( t , 12h , ch3 ) , 1.58 ( m , 8h , -ch2 ) , 2.91 ( t , 8h , -ch2 ) , 4.46 ( s , 8h , n - ch2-ar ) , 7.15 ( s , 2h , h - ar ) . c nmr ( cdcl3 ) : 11.02 ( -c ) , 21.08 ( -c ) , 48.90 ( -c ) , 50.03 ( ch2 ) , 132.50 and 136.85 ( car ) . lrms ( ci ) : 487 [ m+h ] ; hrms esi : m / z : 509 [ m+na ] . compound ( 4b)this compound was prepared according to the general procedure e , using a solution of 1b ( 2 equiv , 2 mmol and 0.41664 g ) . yield = 79% ( was obtained as a white solid ) ; rf = 0.29 [ sio2 , ch2cl2 ] ; mp 141143c . ir ( kbr , cm ) : 1354 and 1149 ( so2 ) . h nmr ( 300 mhz , cdcl3 ) : 0.91 ( t , 12h , ch3 ) , 1.34 ( m , 8h , -ch2 ) , 1.52 ( m , 8h , -ch2 ) , 2.94 ( t , 8h , -ch2 ) , 4.46 ( s , 8h , n - ch2 ) , 7.16 ( s , 2h , h - ar ) . c nmr ( cdcl3 ) : 13.68 ( -c ) , 19.58 ( -c ) , 29.79 ( -c ) , 46.82 ( -c ) , 49.97 ( ch2 ) , 132.49 and136.85 ( car ) . lrms ( ci ) : 543 [ m+h ] ; hrms esi : m / z : 567 [ m+na ] . this compound was prepared according to the general procedure e , using a solution of 1b ( 2 equiv , 2 mmol and 0.41664 g ) . yield = 79% ( was obtained as a white solid ) ; rf = 0.29 [ sio2 , ch2cl2 ] ; mp 141143c . ir ( kbr , cm ) : 1354 and 1149 ( so2 ) . h nmr ( 300 mhz , cdcl3 ) : 0.91 ( t , 12h , ch3 ) , 1.34 ( m , 8h , -ch2 ) , 1.52 ( m , 8h , -ch2 ) , 2.94 ( t , 8h , -ch2 ) , 4.46 ( s , 8h , n - ch2 ) , 7.16 ( s , 2h , h - ar ) . c nmr ( cdcl3 ) : 13.68 ( -c ) , 19.58 ( -c ) , 29.79 ( -c ) , 46.82 ( -c ) , 49.97 ( ch2 ) , 132.49 and136.85 ( car ) . lrms ( ci ) : 543 [ m+h ] ; hrms esi : m / z : 567 [ m+na ] . compound ( 4c)this compound was prepared according to the general procedure e , using a solution of 1d ( 2 equiv , 2 mmol and 0.5527 g ) . yield = 75% ( was obtained as a white solid ) ; rf = 0.46 [ sio2 , ch2cl2 ] ; mp 313314c . ir ( kbr , cm ) : 1364 and 1157 ( so2 ) . h nmr ( 300 mhz , cdcl3 ) : 4.25 ( s , 8h , ch2-ph ) , 4.35 ( s , 8h , n - ch2 ) , 6.79 ( s , 2h , h - ar ) , 7.287.37 ( m , 20h , ph ) . this compound was prepared according to the general procedure e , using a solution of 1d ( 2 equiv , 2 mmol and 0.5527 g ) . yield = 75% ( was obtained as a white solid ) ; rf = 0.46 [ sio2 , ch2cl2 ] ; mp 313314c . ir ( kbr , cm ) : 1364 and 1157 ( so2 ) . h nmr ( 300 mhz , cdcl3 ) : 4.25 ( s , 8h , ch2-ph ) , 4.35 ( s , 8h , n - ch2 ) , 6.79 ( s , 2h , h - ar ) , 7.287.37 ( m , 20h , ph ) . hrms esi : m / z : 701 [ m+na ] . compound ( 4d)this compound was prepared according to the general procedure e , using a solution of 2a ( 2 equiv , 2 mmol , 0.5727 g ) . yield = 55% ( was obtained as a white solid ) ; rf = 0.22 [ sio2 , ch2cl2 ] ; mp 160162c . ir ( kbr , cm ) : 1392 and 1150 ( so2 ) , 1715 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.42 ( s , 18h , tbu ) , 4.05 ( s , 4h , ch2-n - bn ) , 4.39 ( s , 4h , ch2-n - boc ) , 4.90 ( s , 4h , ch2-ph ) , 7.13 ( s , 2h , h - ar ) , 7.367.44 ( m , 10h , ph ) . this compound was prepared according to the general procedure e , using a solution of 2a ( 2 equiv , 2 mmol , 0.5727 g ) . yield = 55% ( was obtained as a white solid ) ; rf = 0.22 [ sio2 , ch2cl2 ] ; mp 160162c . ir ( kbr , cm ) : 1392 and 1150 ( so2 ) , 1715 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.42 ( s , 18h , tbu ) , 4.05 ( s , 4h , ch2-n - bn ) , 4.39 ( s , 4h , ch2-n - boc ) , 4.90 ( s , 4h , ch2-ph ) , 7.13 ( s , 2h , h - ar ) , 7.367.44 ( m , 10h , ph ) . hrms esi : m / z : 721 [ m+na ] . compound ( 4d)yield = 15% ( was obtained as a white solid ) ; rf = 0.25 [ sio2 , ch2cl2 ] ; mp 159161c . ir ( kbr , cm ) : 1371 and 1155 ( so2 ) , 1715 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.45 ( s , 18h , tbu ) , 4.17 ( s , 4h , ch2-n - bn ) , 4.40 ( s , 4h , ch2-n - boc ) , 4.90 ( s , 4h , ch2-ph ) , 6.75 ( s,1h , h - ar ) , 7.27 ( s , 1h , h - ar ) , 7.367.44 ( m , 10h , ph ) . hrms esi : m / z : 721 [ m+na ] . yield = 15% ( was obtained as a white solid ) ; rf = 0.25 [ sio2 , ch2cl2 ] ; mp 159161c . ir ( kbr , cm ) : 1371 and 1155 ( so2 ) , 1715 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.45 ( s , 18h , tbu ) , 4.17 ( s , 4h , ch2-n - bn ) , 4.40 ( s , 4h , ch2-n - boc ) , 4.90 ( s , 4h , ch2-ph ) , 6.75 ( s,1h , h - ar ) , 7.27 ( s , 1h , h - ar ) , 7.367.44 ( m , 10h , ph ) . hrms esi : m / z : 721 [ m+na ] . compound ( 4f)this compound was prepared according to the general procedure e , using a solution of 2j ( 2 equiv , 2 mmol , 0.5646 g ) . yield = 37% ( was obtained as a white solid ) ; rf = 0.20 [ sio2 , ch2cl2 ] ; mp 169171c . ir ( kbr , cm ) : 1368 and 1144 ( so2 ) , 1732 and 1745 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.44 ( s , 18h , tbu ) , 1.34 ( d , 6h , ch3 ) , 3.53 ( s , 6h , o - ch3 ) , 4.65 ( q , 2h , ch ) , 4.86 ( s , 4h , n - ch2-ar ) , 5.00 ( s , 4h , ch2-n - boc ) , 7.13 ( s , 2h , h - ar ) . this compound was prepared according to the general procedure e , using a solution of 2j ( 2 equiv , 2 mmol , 0.5646 g ) . yield = 37% ( was obtained as a white solid ) ; rf = 0.20 [ sio2 , ch2cl2 ] ; mp 169171c . ir ( kbr , cm ) : 1368 and 1144 ( so2 ) , 1732 and 1745 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.44 ( s , 18h , tbu ) , 1.34 ( d , 6h , ch3 ) , 3.53 ( s , 6h , o - ch3 ) , 4.65 ( q , 2h , ch ) , 4.86 ( s , 4h , n - ch2-ar ) , 5.00 ( s , 4h , ch2-n - boc ) , 7.13 ( s , 2h , h - ar ) . compound ( 4f)yield = 34% ( was obtained as a white solid ) ; rf = 0.24 [ sio2 , ch2cl2 ] ; mp 168170c . ir ( kbr , cm ) : 1390 and 1144 ( so2 ) , 1732 and 1750 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.42 ( s , 18h , tbu ) , 1.30 ( d , 6h , ch3 ) , 3.47 ( s , 6h , o - ch3 ) , 4.63 ( q , 2h , ch ) , 4.84 ( s , 4h , n - ch2-ar ) , 4.96 ( s , 4h , ch2-n - boc ) , 7.00 ( s , 1h , h - ar ) , 7.24 ( s , 1h , h - ar ) . yield = 34% ( was obtained as a white solid ) ; rf = 0.24 [ sio2 , ch2cl2 ] ; mp 168170c . ir ( kbr , cm ) : 1390 and 1144 ( so2 ) , 1732 and 1750 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.42 ( s , 18h , tbu ) , 1.30 ( d , 6h , ch3 ) , 3.47 ( s , 6h , o - ch3 ) , 4.63 ( q , 2h , ch ) , 4.84 ( s , 4h , n - ch2-ar ) , 4.96 ( s , 4h , ch2-n - boc ) , 7.00 ( s , 1h , h - ar ) , 7.24 ( s , 1h , h - ar ) . compound ( 4e)this compound was prepared according to the general procedure e , using a solution of 2 g ( 2 equiv , 2 mmol , 0.5646 g ) . yield = 48% ( was obtained as a white solid ) ; rf = 0.18 [ sio2 , ch2cl2 ] ; mp 181183c . ir ( kbr , cm ) : 1380 and 1139 ( so2 ) , 1723 and 1751 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.40 ( s , 18h , tbu ) , 1.29 ( t , 6h , ch3 ) , 3.75 ( s , 4h , ch2co ) , 4.22 ( q , 4h , ch2ch3 ) , 4.73 ( s , 4h , ch2 ) , 4.86 ( s , 4h , ch2-n - boc ) , 7.21 ( s , 2h , h - ar ) . hrms esi : m / z : 713 [ m+na ] . this compound was prepared according to the general procedure e , using a solution of 2 g ( 2 equiv , 2 mmol , 0.5646 g ) . yield = 48% ( was obtained as a white solid ) ; rf = 0.18 [ sio2 , ch2cl2 ] ; mp 181183c . ir ( kbr , cm ) : 1380 and 1139 ( so2 ) , 1723 and 1751 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.40 ( s , 18h , tbu ) , 1.29 ( t , 6h , ch3 ) , 3.75 ( s , 4h , ch2co ) , 4.22 ( q , 4h , ch2ch3 ) , 4.73 ( s , 4h , ch2 ) , 4.86 ( s , 4h , ch2-n - boc ) , 7.21 ( s , 2h , h - ar ) . hrms esi : m / z : 713 [ m+na ] . compound ( 4e)yield = 24% ( was obtained as a white solid ) ; rf = 0.22 [ sio2 , ch2cl2 ] ; mp 179181c . ir ( kbr , cm ) : 1380 and 1139 ( so2 ) , 1723 and 1755 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.43 ( s , 18h : tbu ) , 1.28 ( t , 6h : ch3 ) , 3.77 ( s , 4h , ch2co ) , 4.20 ( q , 4h , ch2ch3 ) , 4.71 ( s , 4h , ch2 ) , 4.90 ( s , 4h , ch2-n - boc ) , 7.05 ( s , 1h , h - ar ) , 7.32 ( s , 1h , h - ar ) . yield = 24% ( was obtained as a white solid ) ; rf = 0.22 [ sio2 , ch2cl2 ] ; mp 179181c . ir ( kbr , cm ) : 1380 and 1139 ( so2 ) , 1723 and 1755 ( c = o ) . h nmr ( 300 mhz , cdcl3 ) : 1.43 ( s , 18h : tbu ) , 1.28 ( t , 6h : ch3 ) , 3.77 ( s , 4h , ch2co ) , 4.20 ( q , 4h , ch2ch3 ) , 4.71 ( s , 4h , ch2 ) , 4.90 ( s , 4h , ch2-n - boc ) , 7.05 ( s , 1h , h - ar ) , 7.32 ( s , 1h , h - ar ) . hrms esi : m / z : 713 [ m+na ] . a solution of 1-methoxy-4-tert - butyl-(2,6-dibromomethyl)benzene ( 1 equiv , 1 mmol , 0.350 g ) in 20 ml of anhydrous ch3cn and a solution of n , n - disubstituted sulfamide 1d ( 1 equiv , 1 mmol , 0.2763 g ) in ch3cn ( 20 ml ) were slowly added by syringe pump over several 5 hours at the same rate to a mixture of k2co3 ( 4.5 equiv , 4.5 mmol , 0.622 g ) and ch3cn ( 150 ml ) . after stirring at reflux for 24 h , the residue was dissolved in ch2cl2 , washed with hcl ( 0.1 n ) ( 2 20 ml ) water ( 2 30 ml ) , and dried with na2so4 and the solvent was evaporated under reduced pressure . the residue was purified by chromatography on silica gel using ch2cl2 to yield the pure macrocyclic sulfamide 5a . macrocyclic sulfamide ( 5a)yield = 58% ( was obtained as a white solid ) ; rf = 0.47 [ sio2 , ch2cl2 ] ; mp > 350c . ir ( kbr , cm ) : 1361 and 1148 ( so2 ) . h nmr ( 300 mhz , cdcl3 ) : 1.35 ( s , 18h , tbu ) , 3.10 ( s , 6h , ch3 ) , 3.8 ( br , 8h , n - ch2 ) , 4.85 ( br , 8h , ch2-ph ) , 6.78 ( s , 4h , h - ar ) , 7.307.45 ( m , 20h , ph ) . yield = 58% ( was obtained as a white solid ) ; rf = 0.47 [ sio2 , ch2cl2 ] ; mp > 350c . ir ( kbr , cm ) : 1361 and 1148 ( so2 ) . h nmr ( 300 mhz , cdcl3 ) : 1.35 ( s , 18h , tbu ) , 3.10 ( s , 6h , ch3 ) , 3.8 ( br , 8h , n - ch2 ) , 4.85 ( br , 8h , ch2-ph ) , 6.78 ( s , 4h , h - ar ) , 7.307.45 ( m , 20h , ph ) .

herein , we describe an efficient one - step synthesis of new fused benzothiadiazepine-1,1-dioxides and macrocyclic sulfamides . the synthesis of these compounds was achieved in moderate yields starting from previously described n , n-disubstituted symmetric sulfamides and n - tert - butoxycarbonyl , n-alkyl sulfamide . the chemical structures of all the new compounds reported in this work were confirmed by nmr , ir , and mass spectrometry . these compounds are beneficial building blocks that can be used in deriving new chemical entities that exert a wide spectrum of pharmacological activities .

1. Introduction 2. Results and Discussion 3. Synthesis of New Macrocyclic Sulfamides 4. Conclusion 5. Experimental Section

cyclic sulfamides and their analogues have been the subject of many organic and medicinal chemistry studies due to their interesting biological activities that include anti - hiv and serine protease [ 14 ] . in addition , some cyclic sulfamide derivatives have been reported as nonhydrolyzable peptidomimetics [ 5 , 6 ] , metalloprotease inhibitors , and constrained peptides [ 810 ] . the benzothiadiazepine ring system has been considered as cyclic sulfamides , and these derivatives have been the subject , especially in the field of medicinal chemistry , because many useful therapeutic agents contain this heterocyclic system . considering the diverse biological properties of this class of compounds and as part of continuous work on the synthesis of biologically active heterocycles , we herein report simple and efficient procedures for the synthesis of a new class of fused benzothiadiazepine derivatives ( a ) , ( b ) , ( c ) ( figure 1 ) . these derivatives include two thiadiazepine rings and macrocyclic molecules containing a sulfamide functionality ( n so2n ) , which were synthesised using previously described n , n - disubstituted symmetric sulfamides and n(boc ) , n(alkyl)sulfamide [ 1418 ] . in particular , we report the synthesis and spectroscopic properties of novel macrocyclic rings containing the sulfamide unit , which was incorporated by a direct reaction between m - dibromomethylbenzene derivatives and n , n-disubstituted symmetric sulfamide . moreover , they have become useful building blocks for constructing nanoporous structures [ 22 , 23 ] . our earlier studies involved the synthesis of heterocyclic compounds containing sulfonyl groups [ 9 , 10 , 15 , 16 ] . chlorosulfonyl isocyanate ( csi ) and sulfuryl chloride ( so2cl2 ) have been shown to be versatile reagents in the synthesis of heterocyclic chemistry . several total syntheses of n , n-disubstituted symmetric sulfamides ( 1a - d ) have been reported in the literature including the original synthetic approaches [ 2427 ] . thus , the starting material , sulfuryl chloride , was treated with an excess of the corresponding amine in dichloromethane for 24 h ( scheme 1 ) , and this resulted in the formation of products 1a - d in moderate yields . the synthesis of the key intermediates n(boc ) , n-alkyl - sulfamide ( 2a - f ) and n-((boc)sulfamoylamino)carboxylates ( 2g - j ) was accomplished as shown in scheme 1 . the carbamylation of chlorosulfonyl isocyanate with tert - butyl alcohol at 0c in dichloromethane followed by in situ sulfamoylation with the corresponding amine , amino acid ester hydrochloride , or diamine in the presence of triethylamine ( tea ) gave the desired n(boc ) , n(alkyl)sulfamide ( 2a - b ) , n(boc ) , n-sufamoylamino acid esters ( 2g - j ) or bis - carboxylsulfamides ( 2c - f ) [ 28 , 29 ] . as outlined in scheme 2 , the n , n-disubstituted symmetric sulfamides ( 1a - d ) are a suitable starting material for the synthesis of an array of new benzocondensed scaffolds ( 4a - c ) in good yields 7579% . the starting materials , n , n-disubstituted symmetric sulfamides 1a - d , were condensed with 1,2,4,5-tetrakis(bromomethyl)benzene ( 0.5 equiv ) by refluxing in acetonitrile for 10 h in the presence of potassium carbonate ( k2co3 ) to afford fused benzo - di - thiadiazepines 4a - c . in the second route , after replacing n , n-disubstituted sulfamides by n(boc)sulfamides derivatives ( 2a - j ) under the same conditions , products 4d - f and 4d-f were formed . both isomers ( symmetric and asymmetric ) these yields seem to be strongly dependent upon the reaction conditions ( solvent , temperature , and steric effect ) . therefore , further optimization of the reaction conditions might improve the yield of these reactions . the presence of the tert - butoxycarbonyl ( boc ) group , which activates the sulfamide nitrogen nucleophilicity , was required for substitution . the structures of fused compounds were confirmed by ir , mass spectrometry , and nmr ( h , c ) , and the results are presented in table 1 . the structures of fused a , b , and c compounds were supported by analysis of the mass spectra esi - ms , which showed peaks respectively at m / z 509 , 567 , and 710 indicating molecular masses of ions [ m+na ] . in the esi - ms spectra , all the prepared fused compounds 4d - f exhibit intense peaks corresponding to the molecular weight [ m+na ] . since the symmetric and asymmetric fused compounds have the same molecular weight , it was difficult to extract all the rich structural information from the mass spectra . in the ir spectra ( table 1 ) , there are peaks at about ( 11401170 ) and ( 13681390 ) cm , due to the sulfonyl group ( so2 ) stretching , and at about 17151732 cm , due to c = o stretching vibrations . for the compounds containing an ester group , ir spectra showed also bands near 1700 cm due to c = o stretching . there are many strategies available for the synthesis of benzylic amide macrocycles that involve the reaction of an ester group with an amino group [ 3335 ] . in this work as shown in scheme 3 , the desired macrocyclic sulfamides 5 were synthesized in one step [ 2 + 2 ] condensation under high dilution conditions . a solution of 1-methoxy-4-tert - butyl-(2,6-dibromomethyl)benzene ( 1 equiv ) in 20 ml of acetonitrile and a solution of n , n-disubstituted symmetric sulfamide ( 1 equiv ) in 20 ml of ch3cn were added dropwise using two mechanically driven syringes over 5 h into solution of k2co3 ( 4.5 equiv ) in 130 ml of ch3cn under nitrogen with stirring at reflux for 24 h. the reaction mixture was subsequently cooled down , and the solvent was removed . dichloromethane was added to the obtained crude , and this solution was washed with 2 n hcl then with water and dried with magnesium sulfate . the structure of macrocyclic compounds 5a was unambiguously confirmed by ir , mass spectrometry , and nmr ( h , c ) spectroscopy . in summary , we have successfully synthesized and characterized a new series of n - protected fused benzothiadiazepines , which offer good starting materials for the synthesis of new molecules with interesting biological activities . in the second part , we described an efficient method for the synthesis of new macrocycle with sulfamide moiety with potential diverse applications in supramolecular chemistry and as starting materials for further synthetic transformations . the synthetic example presented in this work is one of the simplest and most efficient macrocyclization reactions based on the technique of high - dilution conditions . the biological evaluation of the compounds synthesized in this work is currently being carried out . nmr spectra were acquired on commercial instruments ( bruker avance 300 mhz or bruker amx 400 mhz ) and chemical shifts ( ) are reported in parts per million downfield from internal me4si ( s = singlet , d = doublet , dd = double of doublet , t = triplet , q = quartet , m = multiplet ) . the reaction mixture was warmed to room temperature ( rt ) , stirred for 24 h , and monitored by tlc ( sio2 ) . the crude was washed by hcl ( 2 n , 2 20 ml ) water ( 2 30 ml ) and dried over na2so4 . column chromatography ( ch2cl2 , meoh 95 : 5 ) afforded the n , n-dialkyl sulfamide . n , n-dipropylsulfamide ( 1a)this compound was prepared according to the general procedure a , using a solution of propylamine ( 6 equiv ) in ch2cl2 and so2cl2 ( 1 equiv ) in ch2cl2 . n , n-dibutylsulfamide ( 1b)yield = 58% ( was obtained as a white solid ) ; rf = 0.36 [ ( sio2 , ch2cl2 ) ) ] ; mp : 126127c ( described 126.5c ) . n , n-di(2methoxyethyl)sulfamide ( 1c)yield = 61% ( was obtained as a viscous oil ) ; rf = 0.36 [ sio2 , ch2cl2/meoh ( 95 : 5 ) ] ; ir ( kbr , cm ) : 3279 ( nh ) , 1316 and 1147 ( so2 ) . n , n-dibenzylsulfamide ( 1d)yield = 59% ( was obtained as a white solid ) ; rf = 0.37 ( sio2 , ch2cl2 ) ; mp : 182184c ( described 180182c ) . to a stirred solution of csi ( 1 equiv , 10 mmol , 1.4153 g ) in 20 ml of anhydrous dichloromethane at 0c was added a solution of tert - butyl alcohol ( 1 equiv , 10 mmol , 0.7412 g ) in 20 ml of dried ch2cl2 . n , n-bis(tert - butoxycarbonylsulfamoyl)-1,2-diaminoethane ( 2c)this compound was prepared according to the general procedure b using a solution of 1,2-ethan diamine ( 0.5 equiv , 5 mmol , 0.6010 g ) in ch2cl2 . simultaneously the tert - butyl chlorosulfonyl carbamate was prepared by addition of tert - butyl alcohol ( 1 equiv , 10 mmol , 0.7412 g ) in 20 ml of ch2cl2 to an ice - cooled solution containing csi ( 1 equiv , 10 mmol , 1.4153 g ) in 20 ml of dichloromethane . the synthesis of the compounds , starting from csi , tert - butyl alcohol and methyl esters of amino acids ( l - alanine 2j and l - phenylalanine 2 h ) has been previously reported [ 9 , 10 ] . h nmr ( 300 mhz , cdcl3 ) : 1.5 ( s , 9h , tbu ) , 1.30 ( t , 3h , ch3 ) , 3.96 ( d , 2h , n - ch2 ) , 4.24 ( q , 2h , ch2 ) , 5.63 ( t , 1h , nh - ch2 ) , 7.25 ( s , 1h , nhboc ) . to a stirring solution of [ n - tert - butyloxycarbonyl , n-alkyl]-sulfamide ( 1 equiv , 1 mmol ) in ch3cn ( 50 ml ) in a 100 ml round bottom flask was added k2co3 ( 2.5 equiv , 2.5 mmol , 0.34552 g ) and ,-dibromo - o - xylene ( 1 equiv , 1 mmol , 0.26396 g ) . the residue was dissolved in ch2cl2 , washed with 2 portions of hcl ( 1 m ) and water and dried with na2so4 , and the solvent was removed under reduced pressure to give the crude oil . h nmr ( 300 mhz , cdcl3 ) : 1.43 ( s , 9h , tbu ) , 3.66 ( s , 3h , ch3-o ) , 5.96 ( s , 1h , ch ) , 6.987.32 ( m , 9h , h - ar ) , 4.384.80 ( dd , 2h , chahb - n ) , 4.855.05 ( dd , 2h , chahb - n - boc ) , 6.987.32 ( m , 9 h , h - ar ) . an acetonitrile solution ( 50 ml ) containing n , n-disubstituted symmetric sulfamide ( 2 equiv , 2 mmol ) , 2,3,4,5-tetrakis(bromomethyl)benzene ( 1 equiv , 1 mmol , 0.44980 g ) , and anhydrous k2co3 ( 4.5 equiv , 4.5 mmol , 0.6219 g ) was heated at reflux for 10 hours . the reaction mixture was cooled to room temperature , filtered , and after removal of the solvent under reduced pressure a solid was obtained . the solid was redissolved in dichloromethane ( ch2cl2 ) , washed with 2 portions of hcl 1 m ( 2 20 ml ) followed with water ( 2 30 ml ) , and dried with na2so4 . h nmr ( 300 mhz , cdcl3 ) : 0.90 ( t , 12h , ch3 ) , 1.58 ( m , 8h , -ch2 ) , 2.91 ( t , 8h , -ch2 ) , 4.46 ( s , 8h , n - ch2-ar ) , 7.15 ( s , 2h , h - ar ) . h nmr ( 300 mhz , cdcl3 ) : 0.91 ( t , 12h , ch3 ) , 1.34 ( m , 8h , -ch2 ) , 1.52 ( m , 8h , -ch2 ) , 2.94 ( t , 8h , -ch2 ) , 4.46 ( s , 8h , n - ch2 ) , 7.16 ( s , 2h , h - ar ) . h nmr ( 300 mhz , cdcl3 ) : 0.91 ( t , 12h , ch3 ) , 1.34 ( m , 8h , -ch2 ) , 1.52 ( m , 8h , -ch2 ) , 2.94 ( t , 8h , -ch2 ) , 4.46 ( s , 8h , n - ch2 ) , 7.16 ( s , 2h , h - ar ) . h nmr ( 300 mhz , cdcl3 ) : 4.25 ( s , 8h , ch2-ph ) , 4.35 ( s , 8h , n - ch2 ) , 6.79 ( s , 2h , h - ar ) , 7.287.37 ( m , 20h , ph ) . h nmr ( 300 mhz , cdcl3 ) : 4.25 ( s , 8h , ch2-ph ) , 4.35 ( s , 8h , n - ch2 ) , 6.79 ( s , 2h , h - ar ) , 7.287.37 ( m , 20h , ph ) . h nmr ( 300 mhz , cdcl3 ) : 1.44 ( s , 18h , tbu ) , 1.34 ( d , 6h , ch3 ) , 3.53 ( s , 6h , o - ch3 ) , 4.65 ( q , 2h , ch ) , 4.86 ( s , 4h , n - ch2-ar ) , 5.00 ( s , 4h , ch2-n - boc ) , 7.13 ( s , 2h , h - ar ) . h nmr ( 300 mhz , cdcl3 ) : 1.42 ( s , 18h , tbu ) , 1.30 ( d , 6h , ch3 ) , 3.47 ( s , 6h , o - ch3 ) , 4.63 ( q , 2h , ch ) , 4.84 ( s , 4h , n - ch2-ar ) , 4.96 ( s , 4h , ch2-n - boc ) , 7.00 ( s , 1h , h - ar ) , 7.24 ( s , 1h , h - ar ) . h nmr ( 300 mhz , cdcl3 ) : 1.42 ( s , 18h , tbu ) , 1.30 ( d , 6h , ch3 ) , 3.47 ( s , 6h , o - ch3 ) , 4.63 ( q , 2h , ch ) , 4.84 ( s , 4h , n - ch2-ar ) , 4.96 ( s , 4h , ch2-n - boc ) , 7.00 ( s , 1h , h - ar ) , 7.24 ( s , 1h , h - ar ) . a solution of 1-methoxy-4-tert - butyl-(2,6-dibromomethyl)benzene ( 1 equiv , 1 mmol , 0.350 g ) in 20 ml of anhydrous ch3cn and a solution of n , n - disubstituted sulfamide 1d ( 1 equiv , 1 mmol , 0.2763 g ) in ch3cn ( 20 ml ) were slowly added by syringe pump over several 5 hours at the same rate to a mixture of k2co3 ( 4.5 equiv , 4.5 mmol , 0.622 g ) and ch3cn ( 150 ml ) . h nmr ( 300 mhz , cdcl3 ) : 1.35 ( s , 18h , tbu ) , 3.10 ( s , 6h , ch3 ) , 3.8 ( br , 8h , n - ch2 ) , 4.85 ( br , 8h , ch2-ph ) , 6.78 ( s , 4h , h - ar ) , 7.307.45 ( m , 20h , ph ) . h nmr ( 300 mhz , cdcl3 ) : 1.35 ( s , 18h , tbu ) , 3.10 ( s , 6h , ch3 ) , 3.8 ( br , 8h , n - ch2 ) , 4.85 ( br , 8h , ch2-ph ) , 6.78 ( s , 4h , h - ar ) , 7.307.45 ( m , 20h , ph ) .

glaucoma is a complex , multifactorial , polygenetic disease that is the leading cause of irreversible blindness worldwide . trends indicate that by 2020 , as many as 80 million people will have glaucoma worldwide , with as many as 11 million being blinded . the various subtypes of glaucoma share the common clinical pathologies of retinal ganglion cell ( rgc ) damage , optic nerve axonal destruction , and visual field defects . several risk factors are known for glaucoma [ 5 - 7 ] , and elevated intraocular pressure ( iop ) is the major risk factor linked to the development and progression of primary open angle glaucoma ( poag ) [ 3,5,8 - 10 ] . the elevation in iop can be traced back to several causative factors , including faulty fluid dynamics of the aqueous humor within the anterior segment ocular structures . currently , elevated iop is the only clinical manifestation of glaucoma that can be treated . pigment dispersion glaucoma ( pg)a form of secondary open angle glaucoma is the second most common form of glaucoma in young adults . in this disease , pigment - containing particles slough off the posterior iris and block the drainage of aqueous humor through the trabecular meshwork . in most cases , the resultant iop increase leads to glaucoma , yet in some cases , the iop increase does not , indicating that additional factors , including differentially expressed genes and/or genetic heterogeneity , may influence disease progression . dba/2j ( d2 ) mice have served as the leading model for pg for more than two decades [ 12 - 16 ] . it has been well documented that the pigmentary dispersion syndrome in d2 mice is due to recessive mutations in the gpnmb and tyrp1b genes [ 12,16 - 22 ] with other pigmentation genes modulating the phenotype . in addition to iris stromal atrophy and pigment dispersion , d2 mice develop elevated iop , rgc loss , optic nerve damage , and optic disc cup excavation that is age - related and progressive , yet highly variable in severity with some mice having relatively normal pressure and optic nerves . recombinant inbred ( ri ) strains of mice are a valuable resource for identifying genetic sources of variation in disease risk and progression , in this case , the various molecular and clinical phenotypes associated with glaucoma onset and severity . the largest panel of these strains the bxd family consists of approximately 150 inbred strains generated from crosses between the wild - type normotensive b6 strain and the pg - prone d2s train . the bxd strains have been used extensively over the past two decades in genetic and genomic studies of the eye and the primary visual system [ 21,22,25 - 28 ] . in the present study , we determine whether an increase in the severity of iris transillumination defects ( tids ) or mutations in tyrp1 and gpnmb modulate iop levels within the bxd family . the current study was motivated by initial observations that several bxd strains carrying wild - type alleles of both tyrp1 and gpnmb ( wt / wt ) had elevated iop , while others had low iop . this range of variation in iop values suggested that mutations in tyrp1 and gpnmb are neither necessary nor sufficient to cause iop elevation . mice were handled in accordance with the guide for the care and use of laboratory animals . studies involving all mice were approved the animal care and use review board of the aalac - accredited university of tennessee health science center and followed the arvo statement for the use of animals in ophthalmic and vision research in addition to the guidelines for laboratory animal experiments ( institute of laboratory animal resources , public health service policy on humane care and use of laboratory animals ) . lights were maintained on a 12 h:12 h light - dark lighting cycle with light onset occurring at 6 am . a total of 3,856 mice were used in this study and were distributed as follows : 3,548 mice from 73 bxd strains ( 268 for microarray studies and 3280 for phenotyping studies ) , 226 mice from parental strains ( 16 for microarray studies and 210 for phenotyping studies ) , and 82 mice from f1 crosses ( eight for microarray studies and 74 for phenotyping studies ) . the bxd24 strain was excluded from this study because this strain harbors a spontaneous mutation in cep290 , which causes severe retinal degeneration . the sex was relatively well balanced with 1,965 females/1,599 males used in the phenotyping studies and 146 females/146 males used in the microarray studies . given the known time course of glaucoma in the d2 parent , the bxd strains were classified by age and grouped into five categories : 12 months ( immature and without disease ) , 35 months ( possible early onset preclinical symptoms ) , 69 months ( early - stage to mid - stage disease ) , 1013 months ( advanced glaucoma ) , and older than 13 months ( final disease outcome ) . briefly , the bxd genotype file was created using classical microsatellite marker maps and high - density single nucleotide polymorphism ( snp ) genotypes generated using affymetrix and mega mouse universal genotyping arrays ( megamuga ) . this genotype file includes all markers , both snps and microsatellites , with unique strain distribution patterns ( sdps ) , as well as pairs of markers for the sdps represented by two or more markers . the iop of both eyes of all mice were measured using an induction - impact tonometer ( tonolab , colonial medical supply , franconia , nh ) between 9:00 am and 6:00 pm during the light cycle . mice were lightly anesthetized with ketamine and xylazine ( 25 mg / kg and 5 mg / kg ; butler schein , dublin , oh ) and iop measurements were taken as previously described . to determine whether iop has a diurnal rhythm , iop was measured in both eyes of the b6 mice every 2 h from 8 am until 6 pm ( i.e. , 8 am , 10 am , 12 pm , 2 pm , 4 pm and 6 pm ) for five consecutive days ( n=5 mice ) . these data are presented as meansem and were analyzed using one - way anovas followed by tukey s hsd test for differences between means ( analystsoftstatplus : mac software , version 5.8.3.8 ; walnut , ca ) . p<0.05 was considered significant . to determine whether iop was influenced by sidedness , we measured and recorded individually the iop values of the left and right eyes of all mice . previous reports have shown sex differences in measurements of iop in various mice strains . to assess the possibility of such differences in the bxd mice , we analyzed the iop of each sex separately within each age group . to determine whether the iop values of the left and right eyes of the mice differed among mice of each age group or if iop was influenced by sex of the mouse , two - tailed student s t - tests were performed on data within each age group ( analystsoftstatplus : mac software , version 5.8.3.8 ; walnut , ca ) . the grading of the tid phenotype is described in detail in our previously published paper . briefly , the iris phenotype of each mouse was photodocumented using a slit - lamp biomicroscope using narrow and wide beam illumination . a grade from 0 ( lowest ) to 4 ( highest ) the iop values ( mmhg ) and the tid grades were plotted in separate panels along with their respective heritability values across the five age groups . heritability of both the iop and the tid phenotypes were calculated using the formula : h2 = 0.5vg/(0.5vg + ve ) , which accounts for genetic and environmental variance . the factor of 0.5 was included to adjust for the twofold increase in additive genetic variance among the inbred strains relative to the outbred populations . because the mean iop was highest in the 6- to 9-month - old cohort , in subsequent analyses we focused our attention on this age group . to determine the effect of the mutations in tyrp1 and gpnmb on the iop values and tid scores , we stratified the bxd strains based on their genotype . using genenetwork , we plotted a bar graph ( by rank ) for the iop and organized the tid graph to match the strain order of the iop graph to enable comparisons . the strains were further represented using different colors based on their genotypes at tyrp1/gpnmb ( wt / wt = green , wt / mut = orange , mut / wt = blue , mut / mut = red , and the parents = black ) . we also plotted the iop and tid values for the 6- to 9-month - old cohort and evaluated whether the iop and tid phenotypes segregate based on the bxd genotypes for the genes tyrp1 and gpnmb . simple interval mapping using 5,000 permutation tests was performed [ 21 - 23,30 ] to identify quantitative trait loci ( qtls ) that modulate iop ( record i d 16339 , n=71 strains ) and tid ( record i d 12324 , n=73 strains ) in mice aged 6 - 9 months . logarithm of odds ( lod ) values can be obtained by dividing the likelihood ratio statistic ( lrs ) by 4.61 . the iop simple interval map was examined for the presence of significant or suggestive expression qtls ( eqtls ) at the locations of tyrp1 ( chr4 at ~80.5 mb ) and gpnmb ( chr6 at ~49 mb ) , as well as any other eqtls . mice were handled in accordance with the guide for the care and use of laboratory animals . studies involving all mice were approved the animal care and use review board of the aalac - accredited university of tennessee health science center and followed the arvo statement for the use of animals in ophthalmic and vision research in addition to the guidelines for laboratory animal experiments ( institute of laboratory animal resources , public health service policy on humane care and use of laboratory animals ) . lights were maintained on a 12 h:12 h light - dark lighting cycle with light onset occurring at 6 am . a total of 3,856 mice were used in this study and were distributed as follows : 3,548 mice from 73 bxd strains ( 268 for microarray studies and 3280 for phenotyping studies ) , 226 mice from parental strains ( 16 for microarray studies and 210 for phenotyping studies ) , and 82 mice from f1 crosses ( eight for microarray studies and 74 for phenotyping studies ) . the bxd24 strain was excluded from this study because this strain harbors a spontaneous mutation in cep290 , which causes severe retinal degeneration . the sex was relatively well balanced with 1,965 females/1,599 males used in the phenotyping studies and 146 females/146 males used in the microarray studies . given the known time course of glaucoma in the d2 parent , the bxd strains were classified by age and grouped into five categories : 12 months ( immature and without disease ) , 35 months ( possible early onset preclinical symptoms ) , 69 months ( early - stage to mid - stage disease ) , 1013 months ( advanced glaucoma ) , and older than 13 months ( final disease outcome ) . briefly , the bxd genotype file was created using classical microsatellite marker maps and high - density single nucleotide polymorphism ( snp ) genotypes generated using affymetrix and mega mouse universal genotyping arrays ( megamuga ) . this genotype file includes all markers , both snps and microsatellites , with unique strain distribution patterns ( sdps ) , as well as pairs of markers for the sdps represented by two or more markers . the iop of both eyes of all mice were measured using an induction - impact tonometer ( tonolab , colonial medical supply , franconia , nh ) between 9:00 am and 6:00 pm during the light cycle . mice were lightly anesthetized with ketamine and xylazine ( 25 mg / kg and 5 mg / kg ; butler schein , dublin , oh ) and iop measurements were taken as previously described . to determine whether iop has a diurnal rhythm , iop was measured in both eyes of the b6 mice every 2 h from 8 am until 6 pm ( i.e. , 8 am , 10 am , 12 pm , 2 pm , 4 pm and 6 pm ) for five consecutive days ( n=5 mice ) . these data are presented as meansem and were analyzed using one - way anovas followed by tukey s hsd test for differences between means ( analystsoftstatplus : mac software , version 5.8.3.8 ; walnut , ca ) . p<0.05 was considered significant . to determine whether iop was influenced by sidedness , we measured and recorded individually the iop values of the left and right eyes of all mice . previous reports have shown sex differences in measurements of iop in various mice strains . to assess the possibility of such differences in the bxd mice , we analyzed the iop of each sex separately within each age group . to determine whether the iop values of the left and right eyes of the mice differed among mice of each age group or if iop was influenced by sex of the mouse , two - tailed student s t - tests were performed on data within each age group ( analystsoftstatplus : mac software , version 5.8.3.8 ; walnut , ca ) . the grading of the tid phenotype is described in detail in our previously published paper . briefly , the iris phenotype of each mouse was photodocumented using a slit - lamp biomicroscope using narrow and wide beam illumination . a grade from 0 ( lowest ) to 4 ( highest ) the iop values ( mmhg ) and the tid grades were plotted in separate panels along with their respective heritability values across the five age groups . heritability of both the iop and the tid phenotypes were calculated using the formula : h2 = 0.5vg/(0.5vg + ve ) , which accounts for genetic and environmental variance . the factor of 0.5 was included to adjust for the twofold increase in additive genetic variance among the inbred strains relative to the outbred populations . because the mean iop was highest in the 6- to 9-month - old cohort , in subsequent analyses we focused our attention on this age group . to determine the effect of the mutations in tyrp1 and gpnmb on the iop values and tid scores , we stratified the bxd strains based on their genotype . using genenetwork , we plotted a bar graph ( by rank ) for the iop and organized the tid graph to match the strain order of the iop graph to enable comparisons . the strains were further represented using different colors based on their genotypes at tyrp1/gpnmb ( wt / wt = green , wt / mut = orange , mut / wt = blue , mut / mut = red , and the parents = black ) . we also plotted the iop and tid values for the 6- to 9-month - old cohort and evaluated whether the iop and tid phenotypes segregate based on the bxd genotypes for the genes tyrp1 and gpnmb . simple interval mapping using 5,000 permutation tests was performed [ 21 - 23,30 ] to identify quantitative trait loci ( qtls ) that modulate iop ( record i d 16339 , n=71 strains ) and tid ( record i d 12324 , n=73 strains ) in mice aged 6 - 9 months . logarithm of odds ( lod ) values can be obtained by dividing the likelihood ratio statistic ( lrs ) by 4.61 . the iop simple interval map was examined for the presence of significant or suggestive expression qtls ( eqtls ) at the locations of tyrp1 ( chr4 at ~80.5 mb ) and gpnmb ( chr6 at ~49 mb ) , as well as any other eqtls . to determine whether the time of the day has any influence on iop measurements , we selected b6 mice as the representative strain and measured iop at six different time points during the light cycle . the iop values of the b6 mice ranged between 15.10.3 and 15.50.2 mmhg in each age group and did not differ significantly throughout the day ( figure 1a , p>0.05 ) . other groups have shown a distinct circadian fluctuation of iop over 24 h that included a dark period ; however , the present study measured iop only during normal working hours ( 8 am to 6 pm ) . during that time interval , we detected no statistically significant change in iop , although there was a trend toward a reduction in iop during the middle to the latter part of the light cycle , in agreement with published reports . it is also possible that the use of anesthesia blunted larger iop differences that may have been present , as has been described previously . nonetheless , the study measurements detected no statistically significant iop variation from 8 am to 6 pm , and therefore , we pooled the iop data from the different age groups in each bxd strain into a single data set that was stratified only by age . a : the intraocular pressure ( iop ) values in the b6 mice ranged between 15.10.3 and 15.50.2 mmhg and did not differ significantly between 8 am and 6 pm ( p>0.05 ; n = 5 mice at each time of day ) . b : the elevated iop phenotype in the bxd mice is independent of sidedness . within each age cohort , no significant difference was observed between the right and left eyes ( p>0.05 ; the n values , superimposed on each bar of the graph , represent the number of mice ) . c : the elevated iop phenotype in bxd mice is independent of gender . within each age group , no significant differences were observed between female and male mice ( p>0.05 ; the n values , superimposed on each bar of the graph , represent the number of mice ) . within each age cohort , no significant difference was observed between the right and left eyes in each age group ( figure 1b , p>0.05 ) . likewise , no significant differences were observed between females and males of both eyes within each age group ( figure 1c , p>0.05 ) . we have also shown in our previous work that the tid phenotype showed no significant differences between females and males or between right and left eyes . although there may be individual strains of bxd mice in which the iop segregates with the genotypes at tyrp1 and/or gpnmb , on average , there is no association in this genetic reference panel . although some investigations using d2 mice have demonstrated that there may be sex differences in basal iop , other studies from the same group have illustrated that the data to support a universal discrepancy in iop based upon sex is inconsistent , especially when working with genetically diverse mice . consistent with this conclusion , we did not observe a disparity between sexes regarding iop or tid values in the bxd mice , thus allowing us to average the phenotypic data for each age group . if iop and tid are linked by shared genetic determinants , we would expect a similar age of peak expression in both phenotypes . to test this hypothesis , we plotted the iop and the tid across the five age cohorts in separate panels along with the respective heritability values . the mean iop values ranged between 14.310.36 ( the > 13-month - old cohort ) and 16.4560.28 mmhg ( the 6- to 9-month - old cohort ) across the bxd mice ( figure 2a ) . iop increased progressively with age until 69 months , followed by a reduction in iop with age ( figure 2a ) , which is likely due to ciliary body atrophy , as well as the changes in the plasticity and viscoelastic properties of the mouse eye . a : intraocular pressure ( iop ) values increased with age until the 6- to 9-month - old cohort and then progressively decreased in the older cohorts . the mean iop values ranged between 14.140.42 ( the > 13-month - old cohort ) and 16.480.29 mmhg ( the 6- to 9-month - old cohort ) across the bxd mice . heritability of the iop phenotype ranged between 28.07% and 39.87% , with the highest values obtained in the oldest cohort . the average tid grade ranged between 0.710.10 ( the 1- to 2-month - old cohort ) and 1.290.15 ( the > 13-month - old cohort ) . the heritability of the tid phenotype ranged between 88.59% and 97.22% , with the highest values obtained in the 10- to 13-month - old cohort . in previously published work , we demonstrated that the tid phenotype develops in a progressive fashion within this bxd family . we measured a progressive increase in the tid among the five age cohorts , in which the average tid grade within the bxd mice ranged from 0.710.10 ( the 1- to 2-month - old cohort ) to 1.290.15 ( the > 13-month - old cohort ; figure 2b ) . the tid average grade of the d2 and b6 parents was significantly different at all ages . in contrast , the d2 parents had defects that averaged a grade of 2.020.21 as early as 12 months of age and increased to a maximum of 3.450.13 in the mice older than 13 months of age . figure 2 also highlights the heritability of both phenotypes . as seen in figure 2a , heritability of the iop phenotype ranged between 28.07% and 39.87% , with the highest values obtained in the oldest cohort . the tid phenotype , however ( figure 2b ) , ranged between 88.59% and 97.22% , with the highest values obtained in the 10- to 13-month - old cohort . the lower heritability values of the iop phenotype compared to the tid phenotype also likely explain the more complex polygenetic nature of iop as a phenotype compared to the tid phenotype . the lower values also indicate the extent of the influence of genetic and environmental factors on the phenotypic variations in iop and tid across the bxd mice population used in these studies . interestingly , in the bxd mice , the tid grade increased with age similar to what has been observed by various groups for the d2 iris disease , highlighting the importance of the mutations in tyrp1 and gpnmb for the existence of iris disease in the bxd family . we have previously demonstrated that mutations in tyrp1 and gpnmb alter the functional categories of the transcripts with which the mutations are correlated . specifically , the wild - type alleles of both genes are correlated with melanin synthesis . in contrast , the mutant alleles of both genes lose many of those associations and correlate with transcripts that have little or no functional relationship to pigmentation , including glycoprotein metabolism and amino acid phosphorylation . these functional categories have no apparent influence on iop , and thus , it can be postulated that the genetic regulatory networks of iop and tids are composed of distinct groups of gene products that are active in functions within the eye . to test for possible genetic correlations between the iop and tid phenotypes , we examined the influence of mutations in tyrp1 and gpnmb on the iop values and tid grades in the 6- to 9-month - old cohort . after the iop and tid bar graphs were stratified and color - coded based on the genotypes at tyrp1 or gpnmb , important differences were observed ( figure 3a , b ) . the bxd strains carrying wild - type alleles of tyrp1 and gpnmb ( wt / wt ) had iops that varied greatly with some as high as 22.52.7 mmhg in some strains ( e.g. , bxd66 ) , while others had iop values as low as 12.01.1 mmhg ( e.g. , bxd42 ) . if iop were influenced only by the tyrp1 and gpnmb genotype , we would expect to see higher iop values in strains that have mutations in both tyrp1 and gpnmb ( mut / mut ) . however , the iop of the bxd strains that are mut / mut did not show such a trend . specifically , the bxd98 mice had iop of 12.170.55 mmhg , while the bxd95 mice had higher iop of 19.941.4 mmhg . the lack of association between iop elevation and increased tid is also evident in the low correlation values noted between these two traits ( r=0.173 at 6 - 9 months ) . a : bxd strains carrying wild - type alleles of tyrp1 and gpnmb ( wt / wt ) had intraocular pressure ( iop ) values that ranged between 10 and 21 mmhg ( green bars ) . mice with mutant alleles of both genes ( red bars ) had a similar range of iops . b : when plotted in the same rank order as iop , the transillumination defect ( tid ) values had no pattern in ranking . at 6 to 9 months , the only bxd strains that had tid grades above 0 were those that had mutations in tyrp1 . those lacking mutations in either gene had little to no tid , despite having elevated iops in some cases . the n values range from 4 to 13 mice per bxd strain . in contrast , as we reported previously , tids are dependent upon mutations in tyrp1 and , to a more limited degree , gpnmb . high tid values were observed consistently in the bxd strains carrying mutant alleles of both tyrp1 and gpnmb ( mut / mut ) , as opposed to what was seen in the iop values of these strains ( contrast figure 3a , b ) . similarly , we observed high tid values for many bxd strains carrying mutant versions of tyrp1 and wild - type versions of gpnmb ( mut / wt ) in the 6- to 9-month - old mice , yet the iop values did not follow a similar correlation . for example , the bxd21 mice had a high tid value of 2.00 , yet the iop was 13.50 mmhg . based on these findings , we conclude that the genetic modulators of these two phenotypes are likely separate and distinct . these findings also lend strong support to our hypotheses that neither tid nor tyrp1/gpnmb correlate with iop . moreover , this finding also suggests that other genetic modulators or factors may could be responsible for the variation in iop across all bxd strains that may not have a direct role in modulation of tid . to further understand the relationship between iop and tids , we plotted iop versus tids for each strain in the 6- to 9-month - old cohort and color - coded the strains by genotype ( figure 4 ) . some strains that are wild - type at tyrp1 and gpnmb developed high iop ( about 20 mmhg ) but showed negligible tids . in contrast , some strains that are mutant at tyrp1 and gpnmb had low iop values ( about 12 mmhg ) and high tid grades . however , consistently elevated tid grades were observed only in the bxd strains that harbored the mutant alleles of tyrp1 and gpnmb . moreover , in strains that are mut / wt , we observed an inverse relationship , in which higher tid values were present in the strains that had lower iop values . these results suggest that tids do not influence iop directly , and likely the main genetic modulators tyrp1 and gpnmb have no direct roles in modulating iop in the bxd strains . the transillumination defect ( tid ) does not influence intraocular pressure ( iop ) in bxd murine strains when they are stratified based on the tyrp1/gpnmb genotype . some strains that are wild - type at tyrp1 and gpnmb ( green symbols ) developed high iop but showed negligible tid . however , some that are mutant at tyrp1 and gpnmb ( red symbols ) had low iop values and high grades of tid , thus demonstrating that tid does not directly influence iop . the n values ranged from 4 to 13 mice per bxd strain . to further assess the possible relationship of iop with tids , we performed simple interval mapping for iop and tid for all bxd mice in the 6- to 9-month - old cohort . the interval maps for tids and iop were distinct , and each contained unique peaks . importantly , the iop map does not show a statistically suggestive or statistically significant peak at the chromosomal locations of tyrp1 ( figure 5 , left vertical box ) or gpnmb ( figure 5 , right vertical box ) , suggesting that these genes do not directly modulate iop in bxd mice ( figure 5a ) . in contrast , the tids had a large qtl at the location of tyrp1 in the 6- to 9-month - old bxd strains ( figure 5b ) . we had previously reported that tids also map to the location of gpnmb in the older than 13-month - old cohort of bxd mice . moreover , we have demonstrated conclusively that tyrp1 and gpnmb influence the tid phenotype in a significant manner . tid and iop interval maps are unique and contain non - overlapping peaks . a : intraocular pressure ( iop ) does not show statistically suggestive or statistically significant peaks at the locations of tyrp1 ( left vertical box ) or gpnmb ( right vertical box ) , suggesting that these genes do not modulate iop in bxd mice . b : in contrast , the transillumination defect ( tid ) mapped with a high likelihood ratio statistic ( lrs ) score to tyrp1 in the 6- to 9-month - old cohort of the bxd strains . the role of tyrp1 and gpnmb in tids is well established in the d2 and bxd strains of mice . based on the premise that mutations in tyrp1 and gpnmb that are present in d2 mice are responsible for the development of glaucoma , anderson and colleagues introduced the d2-derived tyrp1b and gpnmb mutations on the widely used b6 background to generate several lines of b6 double - congenic mice . these strains were used in genetic epistasis experiments and compared with d2 regarding clinical indices of iris disease , iop elevation , and optic nerve damage . the researchers discovered that similar to d2 mice , b6 double - congenic mice develop a severe depigmentation iris disease leading to massive pigment dispersion . to their surprise , in comparison to d2 , the b6 double - congenic mice were much less likely to develop increased iop in response to this pigment dispersing disease . these results indicated that initiation ( iris disease ) and progression ( high iop ) of glaucomatous phenotypes in d2 mice are genetically separable and that additional genetic factors relevant to glaucoma remain to be characterized . these data also suggest that additional important allelic variants specific to the b6 strain may mitigate elevations in iop . the identification of susceptibility and resistance loci and alleles in the mouse has tremendous potential to enhance our understanding of glaucoma and may help define corresponding loci and genes in human populations . we evaluated the relationship of tids and iop and further studied the influence of tyrp1 and gpnmb on these traits in the bxd murine panel . we conclude that the elevated iop phenotype in bxd mice is independent of sidedness and sex . we also observed that high iop and the most severe tid phenotypes differed in the age of peak expression . the tyrp1 and gpnmb gene expression levels correlated with the tid phenotype but not with iop . remarkably , the severity of tids did not correlate with iop in any age group . we conclude that the presence of mutations in tyrp1 and gpnmb are a prerequisite for tids but not for elevated iop in the bxd family of mice . to determine whether the time of the day has any influence on iop measurements , we selected b6 mice as the representative strain and measured iop at six different time points during the light cycle . the iop values of the b6 mice ranged between 15.10.3 and 15.50.2 mmhg in each age group and did not differ significantly throughout the day ( figure 1a , p>0.05 ) . other groups have shown a distinct circadian fluctuation of iop over 24 h that included a dark period ; however , the present study measured iop only during normal working hours ( 8 am to 6 pm ) . during that time interval , we detected no statistically significant change in iop , although there was a trend toward a reduction in iop during the middle to the latter part of the light cycle , in agreement with published reports . it is also possible that the use of anesthesia blunted larger iop differences that may have been present , as has been described previously . nonetheless , the study measurements detected no statistically significant iop variation from 8 am to 6 pm , and therefore , we pooled the iop data from the different age groups in each bxd strain into a single data set that was stratified only by age . a : the intraocular pressure ( iop ) values in the b6 mice ranged between 15.10.3 and 15.50.2 mmhg and did not differ significantly between 8 am and 6 pm ( p>0.05 ; n = 5 mice at each time of day ) . b : the elevated iop phenotype in the bxd mice is independent of sidedness . within each age cohort , no significant difference was observed between the right and left eyes ( p>0.05 ; the n values , superimposed on each bar of the graph , represent the number of mice ) . c : the elevated iop phenotype in bxd mice is independent of gender . within each age group , no significant differences were observed between female and male mice ( p>0.05 ; the n values , superimposed on each bar of the graph , represent the number of mice ) . within each age cohort , no significant difference was observed between the right and left eyes in each age group ( figure 1b , p>0.05 ) . likewise , no significant differences were observed between females and males of both eyes within each age group ( figure 1c , p>0.05 ) . we have also shown in our previous work that the tid phenotype showed no significant differences between females and males or between right and left eyes . although there may be individual strains of bxd mice in which the iop segregates with the genotypes at tyrp1 and/or gpnmb , on average , there is no association in this genetic reference panel . although some investigations using d2 mice have demonstrated that there may be sex differences in basal iop , other studies from the same group have illustrated that the data to support a universal discrepancy in iop based upon sex is inconsistent , especially when working with genetically diverse mice . consistent with this conclusion , we did not observe a disparity between sexes regarding iop or tid values in the bxd mice , thus allowing us to average the phenotypic data for each age group . if iop and tid are linked by shared genetic determinants , we would expect a similar age of peak expression in both phenotypes . to test this hypothesis , we plotted the iop and the tid across the five age cohorts in separate panels along with the respective heritability values . the mean iop values ranged between 14.310.36 ( the > 13-month - old cohort ) and 16.4560.28 mmhg ( the 6- to 9-month - old cohort ) across the bxd mice ( figure 2a ) . iop increased progressively with age until 69 months , followed by a reduction in iop with age ( figure 2a ) , which is likely due to ciliary body atrophy , as well as the changes in the plasticity and viscoelastic properties of the mouse eye . a : intraocular pressure ( iop ) values increased with age until the 6- to 9-month - old cohort and then progressively decreased in the older cohorts . the mean iop values ranged between 14.140.42 ( the > 13-month - old cohort ) and 16.480.29 mmhg ( the 6- to 9-month - old cohort ) across the bxd mice . heritability of the iop phenotype ranged between 28.07% and 39.87% , with the highest values obtained in the oldest cohort . the average tid grade ranged between 0.710.10 ( the 1- to 2-month - old cohort ) and 1.290.15 ( the > 13-month - old cohort ) . the heritability of the tid phenotype ranged between 88.59% and 97.22% , with the highest values obtained in the 10- to 13-month - old cohort . in previously published work , we demonstrated that the tid phenotype develops in a progressive fashion within this bxd family . we measured a progressive increase in the tid among the five age cohorts , in which the average tid grade within the bxd mice ranged from 0.710.10 ( the 1- to 2-month - old cohort ) to 1.290.15 ( the > 13-month - old cohort ; figure 2b ) . the tid average grade of the d2 and b6 parents was significantly different at all ages . in contrast , the d2 parents had defects that averaged a grade of 2.020.21 as early as 12 months of age and increased to a maximum of 3.450.13 in the mice older than 13 months of age . figure 2 also highlights the heritability of both phenotypes . as seen in figure 2a , heritability of the iop phenotype ranged between 28.07% and 39.87% , with the highest values obtained in the oldest cohort . the tid phenotype , however ( figure 2b ) , ranged between 88.59% and 97.22% , with the highest values obtained in the 10- to 13-month - old cohort . the lower heritability values of the iop phenotype compared to the tid phenotype also likely explain the more complex polygenetic nature of iop as a phenotype compared to the tid phenotype . the lower values also indicate the extent of the influence of genetic and environmental factors on the phenotypic variations in iop and tid across the bxd mice population used in these studies . interestingly , in the bxd mice , the tid grade increased with age similar to what has been observed by various groups for the d2 iris disease , highlighting the importance of the mutations in tyrp1 and gpnmb for the existence of iris disease in the bxd family . we have previously demonstrated that mutations in tyrp1 and gpnmb alter the functional categories of the transcripts with which the mutations are correlated . specifically , the wild - type alleles of both genes are correlated with melanin synthesis . in contrast , the mutant alleles of both genes lose many of those associations and correlate with transcripts that have little or no functional relationship to pigmentation , including glycoprotein metabolism and amino acid phosphorylation . these functional categories have no apparent influence on iop , and thus , it can be postulated that the genetic regulatory networks of iop and tids are composed of distinct groups of gene products that are active in functions within the eye . to test for possible genetic correlations between the iop and tid phenotypes , we examined the influence of mutations in tyrp1 and gpnmb on the iop values and tid grades in the 6- to 9-month - old cohort . after the iop and tid bar graphs were stratified and color - coded based on the genotypes at tyrp1 or gpnmb , important differences were observed ( figure 3a , b ) . the bxd strains carrying wild - type alleles of tyrp1 and gpnmb ( wt / wt ) had iops that varied greatly with some as high as 22.52.7 mmhg in some strains ( e.g. , bxd66 ) , while others had iop values as low as 12.01.1 mmhg ( e.g. , bxd42 ) . if iop were influenced only by the tyrp1 and gpnmb genotype , we would expect to see higher iop values in strains that have mutations in both tyrp1 and gpnmb ( mut / mut ) . however , the iop of the bxd strains that are mut / mut did not show such a trend . specifically , the bxd98 mice had iop of 12.170.55 mmhg , while the bxd95 mice had higher iop of 19.941.4 mmhg . the lack of association between iop elevation and increased tid is also evident in the low correlation values noted between these two traits ( r=0.173 at 6 - 9 months ) . a : bxd strains carrying wild - type alleles of tyrp1 and gpnmb ( wt / wt ) had intraocular pressure ( iop ) values that ranged between 10 and 21 mmhg ( green bars ) . mice with mutant alleles of both genes ( red bars ) had a similar range of iops . b : when plotted in the same rank order as iop , the transillumination defect ( tid ) values had no pattern in ranking . at 6 to 9 months , the only bxd strains that had tid grades above 0 were those that had mutations in tyrp1 . those lacking mutations in either gene had little to no tid , despite having elevated iops in some cases . in contrast , as we reported previously , tids are dependent upon mutations in tyrp1 and , to a more limited degree , gpnmb . high tid values were observed consistently in the bxd strains carrying mutant alleles of both tyrp1 and gpnmb ( mut / mut ) , as opposed to what was seen in the iop values of these strains ( contrast figure 3a , b ) . similarly , we observed high tid values for many bxd strains carrying mutant versions of tyrp1 and wild - type versions of gpnmb ( mut / wt ) in the 6- to 9-month - old mice , yet the iop values did not follow a similar correlation . for example , the bxd21 mice had a high tid value of 2.00 , yet the iop was 13.50 mmhg . based on these findings , we conclude that the genetic modulators of these two phenotypes are likely separate and distinct . these findings also lend strong support to our hypotheses that neither tid nor tyrp1/gpnmb correlate with iop . moreover , this finding also suggests that other genetic modulators or factors may could be responsible for the variation in iop across all bxd strains that may not have a direct role in modulation of tid . to further understand the relationship between iop and tids , we plotted iop versus tids for each strain in the 6- to 9-month - old cohort and color - coded the strains by genotype ( figure 4 ) . some strains that are wild - type at tyrp1 and gpnmb developed high iop ( about 20 mmhg ) but showed negligible tids . in contrast , some strains that are mutant at tyrp1 and gpnmb had low iop values ( about 12 mmhg ) and high tid grades . however , consistently elevated tid grades were observed only in the bxd strains that harbored the mutant alleles of tyrp1 and gpnmb . moreover , in strains that are mut / wt , we observed an inverse relationship , in which higher tid values were present in the strains that had lower iop values . these results suggest that tids do not influence iop directly , and likely the main genetic modulators tyrp1 and gpnmb have no direct roles in modulating iop in the bxd strains . the transillumination defect ( tid ) does not influence intraocular pressure ( iop ) in bxd murine strains when they are stratified based on the tyrp1/gpnmb genotype . some strains that are wild - type at tyrp1 and gpnmb ( green symbols ) developed high iop but showed negligible tid . however , some that are mutant at tyrp1 and gpnmb ( red symbols ) had low iop values and high grades of tid , thus demonstrating that tid does not directly influence iop . to further assess the possible relationship of iop with tids , we performed simple interval mapping for iop and tid for all bxd mice in the 6- to 9-month - old cohort . the interval maps for tids and iop were distinct , and each contained unique peaks . importantly , the iop map does not show a statistically suggestive or statistically significant peak at the chromosomal locations of tyrp1 ( figure 5 , left vertical box ) or gpnmb ( figure 5 , right vertical box ) , suggesting that these genes do not directly modulate iop in bxd mice ( figure 5a ) . in contrast , the tids had a large qtl at the location of tyrp1 in the 6- to 9-month - old bxd strains ( figure 5b ) . we had previously reported that tids also map to the location of gpnmb in the older than 13-month - old cohort of bxd mice . moreover , we have demonstrated conclusively that tyrp1 and gpnmb influence the tid phenotype in a significant manner . tid and iop interval maps are unique and contain non - overlapping peaks . a : intraocular pressure ( iop ) does not show statistically suggestive or statistically significant peaks at the locations of tyrp1 ( left vertical box ) or gpnmb ( right vertical box ) , suggesting that these genes do not modulate iop in bxd mice . b : in contrast , the transillumination defect ( tid ) mapped with a high likelihood ratio statistic ( lrs ) score to tyrp1 in the 6- to 9-month - old cohort of the bxd strains . the role of tyrp1 and gpnmb in tids is well established in the d2 and bxd strains of mice . based on the premise that mutations in tyrp1 and gpnmb that are present in d2 mice are responsible for the development of glaucoma , anderson and colleagues introduced the d2-derived tyrp1b and gpnmb mutations on the widely used b6 background to generate several lines of b6 double - congenic mice . these strains were used in genetic epistasis experiments and compared with d2 regarding clinical indices of iris disease , iop elevation , and optic nerve damage . the researchers discovered that similar to d2 mice , b6 double - congenic mice develop a severe depigmentation iris disease leading to massive pigment dispersion . to their surprise , in comparison to d2 , the b6 double - congenic mice were much less likely to develop increased iop in response to this pigment dispersing disease . these results indicated that initiation ( iris disease ) and progression ( high iop ) of glaucomatous phenotypes in d2 mice are genetically separable and that additional genetic factors relevant to glaucoma remain to be characterized . these data also suggest that additional important allelic variants specific to the b6 strain may mitigate elevations in iop . the identification of susceptibility and resistance loci and alleles in the mouse has tremendous potential to enhance our understanding of glaucoma and may help define corresponding loci and genes in human populations . we evaluated the relationship of tids and iop and further studied the influence of tyrp1 and gpnmb on these traits in the bxd murine panel . we conclude that the elevated iop phenotype in bxd mice is independent of sidedness and sex . we also observed that high iop and the most severe tid phenotypes differed in the age of peak expression . the tyrp1 and gpnmb gene expression levels correlated with the tid phenotype but not with iop . remarkably , the severity of tids did not correlate with iop in any age group . we conclude that the presence of mutations in tyrp1 and gpnmb are a prerequisite for tids but not for elevated iop in the bxd family of mice .

purposeintraocular pressure ( iop ) is currently the only treatable phenotype associated with primary open angle glaucoma ( poag ) . our group has developed the bxd murine panel for identifying genetic modulators of the various endophenotypes of glaucoma , including pigment dispersion , iop , and retinal ganglion cell ( rgc ) death . the bxd family consists of the inbred progeny of crosses between the c57bl/6j ( b6 ) strain and the glaucoma - prone dba/2j ( d2 ) strain that has mutations in tyrp1 and gpnmb . the role of these genes in the iris transillumination defect ( tid ) has been well documented ; however , their possible roles in modulating iop during glaucoma onset and progression are yet not well understood.methodswe used the iop data sets and the eye m430v2 ( sep08 ) rma database available on genenetwork to determine whether mutations in tyrp1 and gpnmb or tids have a direct role in the elevation of iop in the bxd family . we also determined whether tids and iop are coregulated.resultsas expected , tyrp1 and gpnmb expression levels showed a high degree of correlation with tids . however , there was no correlation between the expression of these genes and iop . moreover , unlike tids , iop did not map to either the tyrp1 or gpnmb locus . although the tyrp1 and gpnmb mutations in bxd strains are a prerequisite for the development of tid , they are not required for or associated with elevated iop.conclusionsgenetic modulators of iop thus may be independently identified using the full array of bxd mice without concern for the presence of tids or mutations in typr1 and/or gpnmb .

Introduction Methods The BXD family of strains BXD genotypes database and mapping algorithm IOP measurements TID grading Heritability calculation Stratification of IOP and TID by genotype and Pearson correlation Simple interval mapping Results and Discussion IOP in BXD mice is not influenced by time during the light cycle, sidedness, or sex IOP and TID phenotypes differ in age of peak expression None TID does not influence IOP in the BXD murine strain stratified by the Unlike TID, IOP does not map to Conclusions

the various subtypes of glaucoma share the common clinical pathologies of retinal ganglion cell ( rgc ) damage , optic nerve axonal destruction , and visual field defects . several risk factors are known for glaucoma [ 5 - 7 ] , and elevated intraocular pressure ( iop ) is the major risk factor linked to the development and progression of primary open angle glaucoma ( poag ) [ 3,5,8 - 10 ] . pigment dispersion glaucoma ( pg)a form of secondary open angle glaucoma is the second most common form of glaucoma in young adults . it has been well documented that the pigmentary dispersion syndrome in d2 mice is due to recessive mutations in the gpnmb and tyrp1b genes [ 12,16 - 22 ] with other pigmentation genes modulating the phenotype . in addition to iris stromal atrophy and pigment dispersion , d2 mice develop elevated iop , rgc loss , optic nerve damage , and optic disc cup excavation that is age - related and progressive , yet highly variable in severity with some mice having relatively normal pressure and optic nerves . recombinant inbred ( ri ) strains of mice are a valuable resource for identifying genetic sources of variation in disease risk and progression , in this case , the various molecular and clinical phenotypes associated with glaucoma onset and severity . the largest panel of these strains the bxd family consists of approximately 150 inbred strains generated from crosses between the wild - type normotensive b6 strain and the pg - prone d2s train . the bxd strains have been used extensively over the past two decades in genetic and genomic studies of the eye and the primary visual system [ 21,22,25 - 28 ] . in the present study , we determine whether an increase in the severity of iris transillumination defects ( tids ) or mutations in tyrp1 and gpnmb modulate iop levels within the bxd family . the current study was motivated by initial observations that several bxd strains carrying wild - type alleles of both tyrp1 and gpnmb ( wt / wt ) had elevated iop , while others had low iop . this range of variation in iop values suggested that mutations in tyrp1 and gpnmb are neither necessary nor sufficient to cause iop elevation . given the known time course of glaucoma in the d2 parent , the bxd strains were classified by age and grouped into five categories : 12 months ( immature and without disease ) , 35 months ( possible early onset preclinical symptoms ) , 69 months ( early - stage to mid - stage disease ) , 1013 months ( advanced glaucoma ) , and older than 13 months ( final disease outcome ) . to determine whether iop has a diurnal rhythm , iop was measured in both eyes of the b6 mice every 2 h from 8 am until 6 pm ( i.e. to determine whether iop was influenced by sidedness , we measured and recorded individually the iop values of the left and right eyes of all mice . to assess the possibility of such differences in the bxd mice , we analyzed the iop of each sex separately within each age group . to determine whether the iop values of the left and right eyes of the mice differed among mice of each age group or if iop was influenced by sex of the mouse , two - tailed student s t - tests were performed on data within each age group ( analystsoftstatplus : mac software , version 5.8.3.8 ; walnut , ca ) . to determine the effect of the mutations in tyrp1 and gpnmb on the iop values and tid scores , we stratified the bxd strains based on their genotype . we also plotted the iop and tid values for the 6- to 9-month - old cohort and evaluated whether the iop and tid phenotypes segregate based on the bxd genotypes for the genes tyrp1 and gpnmb . the iop simple interval map was examined for the presence of significant or suggestive expression qtls ( eqtls ) at the locations of tyrp1 ( chr4 at ~80.5 mb ) and gpnmb ( chr6 at ~49 mb ) , as well as any other eqtls . given the known time course of glaucoma in the d2 parent , the bxd strains were classified by age and grouped into five categories : 12 months ( immature and without disease ) , 35 months ( possible early onset preclinical symptoms ) , 69 months ( early - stage to mid - stage disease ) , 1013 months ( advanced glaucoma ) , and older than 13 months ( final disease outcome ) . to determine whether iop has a diurnal rhythm , iop was measured in both eyes of the b6 mice every 2 h from 8 am until 6 pm ( i.e. to determine whether iop was influenced by sidedness , we measured and recorded individually the iop values of the left and right eyes of all mice . to assess the possibility of such differences in the bxd mice , we analyzed the iop of each sex separately within each age group . to determine whether the iop values of the left and right eyes of the mice differed among mice of each age group or if iop was influenced by sex of the mouse , two - tailed student s t - tests were performed on data within each age group ( analystsoftstatplus : mac software , version 5.8.3.8 ; walnut , ca ) . to determine the effect of the mutations in tyrp1 and gpnmb on the iop values and tid scores , we stratified the bxd strains based on their genotype . we also plotted the iop and tid values for the 6- to 9-month - old cohort and evaluated whether the iop and tid phenotypes segregate based on the bxd genotypes for the genes tyrp1 and gpnmb . the iop simple interval map was examined for the presence of significant or suggestive expression qtls ( eqtls ) at the locations of tyrp1 ( chr4 at ~80.5 mb ) and gpnmb ( chr6 at ~49 mb ) , as well as any other eqtls . to determine whether the time of the day has any influence on iop measurements , we selected b6 mice as the representative strain and measured iop at six different time points during the light cycle . during that time interval , we detected no statistically significant change in iop , although there was a trend toward a reduction in iop during the middle to the latter part of the light cycle , in agreement with published reports . a : the intraocular pressure ( iop ) values in the b6 mice ranged between 15.10.3 and 15.50.2 mmhg and did not differ significantly between 8 am and 6 pm ( p>0.05 ; n = 5 mice at each time of day ) . b : the elevated iop phenotype in the bxd mice is independent of sidedness . although there may be individual strains of bxd mice in which the iop segregates with the genotypes at tyrp1 and/or gpnmb , on average , there is no association in this genetic reference panel . consistent with this conclusion , we did not observe a disparity between sexes regarding iop or tid values in the bxd mice , thus allowing us to average the phenotypic data for each age group . a : intraocular pressure ( iop ) values increased with age until the 6- to 9-month - old cohort and then progressively decreased in the older cohorts . interestingly , in the bxd mice , the tid grade increased with age similar to what has been observed by various groups for the d2 iris disease , highlighting the importance of the mutations in tyrp1 and gpnmb for the existence of iris disease in the bxd family . we have previously demonstrated that mutations in tyrp1 and gpnmb alter the functional categories of the transcripts with which the mutations are correlated . these functional categories have no apparent influence on iop , and thus , it can be postulated that the genetic regulatory networks of iop and tids are composed of distinct groups of gene products that are active in functions within the eye . to test for possible genetic correlations between the iop and tid phenotypes , we examined the influence of mutations in tyrp1 and gpnmb on the iop values and tid grades in the 6- to 9-month - old cohort . the bxd strains carrying wild - type alleles of tyrp1 and gpnmb ( wt / wt ) had iops that varied greatly with some as high as 22.52.7 mmhg in some strains ( e.g. however , the iop of the bxd strains that are mut / mut did not show such a trend . a : bxd strains carrying wild - type alleles of tyrp1 and gpnmb ( wt / wt ) had intraocular pressure ( iop ) values that ranged between 10 and 21 mmhg ( green bars ) . b : when plotted in the same rank order as iop , the transillumination defect ( tid ) values had no pattern in ranking . at 6 to 9 months , the only bxd strains that had tid grades above 0 were those that had mutations in tyrp1 . high tid values were observed consistently in the bxd strains carrying mutant alleles of both tyrp1 and gpnmb ( mut / mut ) , as opposed to what was seen in the iop values of these strains ( contrast figure 3a , b ) . similarly , we observed high tid values for many bxd strains carrying mutant versions of tyrp1 and wild - type versions of gpnmb ( mut / wt ) in the 6- to 9-month - old mice , yet the iop values did not follow a similar correlation . moreover , this finding also suggests that other genetic modulators or factors may could be responsible for the variation in iop across all bxd strains that may not have a direct role in modulation of tid . however , consistently elevated tid grades were observed only in the bxd strains that harbored the mutant alleles of tyrp1 and gpnmb . these results suggest that tids do not influence iop directly , and likely the main genetic modulators tyrp1 and gpnmb have no direct roles in modulating iop in the bxd strains . the transillumination defect ( tid ) does not influence intraocular pressure ( iop ) in bxd murine strains when they are stratified based on the tyrp1/gpnmb genotype . however , some that are mutant at tyrp1 and gpnmb ( red symbols ) had low iop values and high grades of tid , thus demonstrating that tid does not directly influence iop . to further assess the possible relationship of iop with tids , we performed simple interval mapping for iop and tid for all bxd mice in the 6- to 9-month - old cohort . importantly , the iop map does not show a statistically suggestive or statistically significant peak at the chromosomal locations of tyrp1 ( figure 5 , left vertical box ) or gpnmb ( figure 5 , right vertical box ) , suggesting that these genes do not directly modulate iop in bxd mice ( figure 5a ) . we had previously reported that tids also map to the location of gpnmb in the older than 13-month - old cohort of bxd mice . a : intraocular pressure ( iop ) does not show statistically suggestive or statistically significant peaks at the locations of tyrp1 ( left vertical box ) or gpnmb ( right vertical box ) , suggesting that these genes do not modulate iop in bxd mice . b : in contrast , the transillumination defect ( tid ) mapped with a high likelihood ratio statistic ( lrs ) score to tyrp1 in the 6- to 9-month - old cohort of the bxd strains . the role of tyrp1 and gpnmb in tids is well established in the d2 and bxd strains of mice . based on the premise that mutations in tyrp1 and gpnmb that are present in d2 mice are responsible for the development of glaucoma , anderson and colleagues introduced the d2-derived tyrp1b and gpnmb mutations on the widely used b6 background to generate several lines of b6 double - congenic mice . we evaluated the relationship of tids and iop and further studied the influence of tyrp1 and gpnmb on these traits in the bxd murine panel . the tyrp1 and gpnmb gene expression levels correlated with the tid phenotype but not with iop . we conclude that the presence of mutations in tyrp1 and gpnmb are a prerequisite for tids but not for elevated iop in the bxd family of mice . during that time interval , we detected no statistically significant change in iop , although there was a trend toward a reduction in iop during the middle to the latter part of the light cycle , in agreement with published reports . a : the intraocular pressure ( iop ) values in the b6 mice ranged between 15.10.3 and 15.50.2 mmhg and did not differ significantly between 8 am and 6 pm ( p>0.05 ; n = 5 mice at each time of day ) . b : the elevated iop phenotype in the bxd mice is independent of sidedness . although there may be individual strains of bxd mice in which the iop segregates with the genotypes at tyrp1 and/or gpnmb , on average , there is no association in this genetic reference panel . consistent with this conclusion , we did not observe a disparity between sexes regarding iop or tid values in the bxd mice , thus allowing us to average the phenotypic data for each age group . a : intraocular pressure ( iop ) values increased with age until the 6- to 9-month - old cohort and then progressively decreased in the older cohorts . interestingly , in the bxd mice , the tid grade increased with age similar to what has been observed by various groups for the d2 iris disease , highlighting the importance of the mutations in tyrp1 and gpnmb for the existence of iris disease in the bxd family . we have previously demonstrated that mutations in tyrp1 and gpnmb alter the functional categories of the transcripts with which the mutations are correlated . these functional categories have no apparent influence on iop , and thus , it can be postulated that the genetic regulatory networks of iop and tids are composed of distinct groups of gene products that are active in functions within the eye . to test for possible genetic correlations between the iop and tid phenotypes , we examined the influence of mutations in tyrp1 and gpnmb on the iop values and tid grades in the 6- to 9-month - old cohort . the bxd strains carrying wild - type alleles of tyrp1 and gpnmb ( wt / wt ) had iops that varied greatly with some as high as 22.52.7 mmhg in some strains ( e.g. if iop were influenced only by the tyrp1 and gpnmb genotype , we would expect to see higher iop values in strains that have mutations in both tyrp1 and gpnmb ( mut / mut ) . however , the iop of the bxd strains that are mut / mut did not show such a trend . a : bxd strains carrying wild - type alleles of tyrp1 and gpnmb ( wt / wt ) had intraocular pressure ( iop ) values that ranged between 10 and 21 mmhg ( green bars ) . b : when plotted in the same rank order as iop , the transillumination defect ( tid ) values had no pattern in ranking . at 6 to 9 months , the only bxd strains that had tid grades above 0 were those that had mutations in tyrp1 . high tid values were observed consistently in the bxd strains carrying mutant alleles of both tyrp1 and gpnmb ( mut / mut ) , as opposed to what was seen in the iop values of these strains ( contrast figure 3a , b ) . similarly , we observed high tid values for many bxd strains carrying mutant versions of tyrp1 and wild - type versions of gpnmb ( mut / wt ) in the 6- to 9-month - old mice , yet the iop values did not follow a similar correlation . moreover , this finding also suggests that other genetic modulators or factors may could be responsible for the variation in iop across all bxd strains that may not have a direct role in modulation of tid . however , consistently elevated tid grades were observed only in the bxd strains that harbored the mutant alleles of tyrp1 and gpnmb . these results suggest that tids do not influence iop directly , and likely the main genetic modulators tyrp1 and gpnmb have no direct roles in modulating iop in the bxd strains . the transillumination defect ( tid ) does not influence intraocular pressure ( iop ) in bxd murine strains when they are stratified based on the tyrp1/gpnmb genotype . however , some that are mutant at tyrp1 and gpnmb ( red symbols ) had low iop values and high grades of tid , thus demonstrating that tid does not directly influence iop . to further assess the possible relationship of iop with tids , we performed simple interval mapping for iop and tid for all bxd mice in the 6- to 9-month - old cohort . importantly , the iop map does not show a statistically suggestive or statistically significant peak at the chromosomal locations of tyrp1 ( figure 5 , left vertical box ) or gpnmb ( figure 5 , right vertical box ) , suggesting that these genes do not directly modulate iop in bxd mice ( figure 5a ) . we had previously reported that tids also map to the location of gpnmb in the older than 13-month - old cohort of bxd mice . a : intraocular pressure ( iop ) does not show statistically suggestive or statistically significant peaks at the locations of tyrp1 ( left vertical box ) or gpnmb ( right vertical box ) , suggesting that these genes do not modulate iop in bxd mice . b : in contrast , the transillumination defect ( tid ) mapped with a high likelihood ratio statistic ( lrs ) score to tyrp1 in the 6- to 9-month - old cohort of the bxd strains . the role of tyrp1 and gpnmb in tids is well established in the d2 and bxd strains of mice . based on the premise that mutations in tyrp1 and gpnmb that are present in d2 mice are responsible for the development of glaucoma , anderson and colleagues introduced the d2-derived tyrp1b and gpnmb mutations on the widely used b6 background to generate several lines of b6 double - congenic mice . we evaluated the relationship of tids and iop and further studied the influence of tyrp1 and gpnmb on these traits in the bxd murine panel . the tyrp1 and gpnmb gene expression levels correlated with the tid phenotype but not with iop . we conclude that the presence of mutations in tyrp1 and gpnmb are a prerequisite for tids but not for elevated iop in the bxd family of mice .

plasmids pcdna3.1-app695myc plasmid has been described previously ( 21 ) . to generate pcdna3.1-sp - c99myc expression vector , an 18-residue - long signal peptide was amplified by pcr from the human app695 cdna and introduced into hindiii and ecori sites , followed by ligation with c - terminal 99 amino acids ( c99 ) of app695 and myc tag . aicdmyc - ires2-egfp construct was generated by pcr amplification from pcdna3.1-app695myc , introduced with an atg start codon , and then cloned into the bglii and bamhi sites in the pires2-egfp vector ( clontech ) . pcdna3.0-c99-gvp , which encodes c99 fused with gal4 dna - binding / vp16 transactivation domains , was kindly provided by dr . site - directed mutagenesis of app695myc and sp - c99myc was conducted by pcr strategy ( 23 ) . the paired mutagenic primers consist of primer forward ( 5-ttgacgccgctgtcgccccagaggagcgccacct-3 ) and primer reverse ( 5-gctcctc tggggcgacagcggcgtcaacctc-3 ) with the underlined nucleotides indicating the changes introduced for the point mutations . cell culture and treatments human embryonic kidney 293 t cells ( hek293 t ) and human neuroblastoma cells ( sh - sy5y ) were cultured in dulbecco 's modified eagle 's medium / ham 's f-12 ( sigma ) with 10% fetal bovine serum ( hyclone ) . transfection experiments were performed using fugene 6 ( roche applied science ) in accordance with the manufacturer 's instructions . to generate human neuroblastoma cell lines stably expressing app695myc , sh - sy5y cells were transfected with the construct and selected in the presence of g418 ( 500 g / ml ) . sh - sy5y / app695myc cells were maintained in the medium containing 200 g / ml of g418 . sh - sy5y cells were routinely treated with 1.0 mm h2o2 for 1 h unless otherwise indicated in figure legends . various pharmacological kinase inhibitors , i.e. sp600125 ( 20 m ; calbiochem ) , u0126 ( 5 m ; calbiochem ) , wortmannin ( 20 nm ; calbiochem ) , and -secretase inhibitor dapt ( 1 - 10 m ; sigma ) , were added into dulbecco 's modified eagle 's medium / ham 's f-12 medium for 3 h before h2o2 treatment . rna interference silencing of jnk was achieved by transfection with stealth sirna duplexes targeting jnk1 or jnk2 ( 24 ) . sijnk1 ( 5-ucacaguccugaaacgauatt-3 ) targets jnk1 and sijnk1/2 ( 5-aaagaauguccuaccuucu tt-3 ) targets a common sequence in both jnk1 and jnk2 mrna . t cells in 12-well cell dish were co - transfected with 50 pmol of sirna and 1.5 g of pcdna3.1-app695myc with lipofectamine2000 ( invitrogen ) . a elisa to determine the production of intracellular a , sh - sy5y / app695myc cells , or pcdna3.1-sp - c99myc transiently expressed hek293 t cells were treated with 1.0 mm h2o2 for 1 h. the cells were lysed with cold radioimmune precipitation assay buffer containing 50 mm tris - hcl ( ph 8.0 ) , 150 mm nacl , 5 mm edta , 1% nonidet p-40 , 0.25 mm phenylmethanesulfonyl fluoride , and a mixture of protease inhibitors ( roche applied science ) . the cell extracts were centrifuged at 13,000 g for 30 min to remove cell debris . the same amounts of protein extracts were subjected to sandwich a elisa kits according to the manufacturer 's instruction ( biosource ) . to detect secreted a40 and a42 , conditioned medium of h2o2-treated cells ( 300 l / well ) the cells or tissues were lysed in cell lysis buffer containing 50 mm tris - hcl ( ph 8.0 ) , 150 mm nacl , 0.5% nadoc , 0.1% sds , 1% nonidet p-40 , 5 mm edta , 0.25 mm phenylmethanesulfonyl fluoride , and a mixture of protease inhibitors . protein extracts ( 30 g ) were subjected to immunoblotting with the following primary antibodies : anti - myc ( 1:3,000 ; covance ) , anti - phospho - jnk ( 1:1,000 ; cell signaling ) , anti - pan jnk ( 1:2,000 ; pharmingen ) , anti - phospho - erk ( 1:2,000 ; cell signaling ) , anti - erk ( 1:1,000 ; santa cruz ) , antiphospho - pkb ( 1:1,000 ; santa cruz ) , anti - pkb ( 1:1,000 ; santa cruz ) , and anti--actin ( 1:7,000 ; sigma ) . to detect sapp and sapp , conditioned medium of h2o2-treated cells was collected and analyzed by immunoblotting using the following primary antibodies : 22c11 recognizing app n - terminal amino acid residues 66 - 81 ( 1:2,000 ; chemicon ) , 6e10 reacting with the a4 - 9 region in the c terminus of sapp ( 1:1,000 ; signet laboratory ) , and anti - sapp antibody recognizing the potion of c terminus of human sapp ( isevkm ) ( 1:200 ; ibl ) . the autoradiography of x - ray film and the band intensity were processed using labworks software version 4.5 ( uvp ) . fluorogenic substrate assay fluorogenic substrate assay was performed as described previously ( 25 ) . in brief , h2o2-treated cells were homogenized in 50 mm hepes ( ph 7.4 ) , 150 mm nacl , 1 mm edta solution . the homogenate was centrifuged for 15 min at 1,000 g at 4 c to prepare a post - nuclear supernatant fraction . the cell membranes were pelleted from the post - nuclear supernatant by centrifugation for 30 min at 13,000 g. the pellet was resuspended and incubated at 37 c for 2 h in 30 l of reaction buffer ( -secretase reaction buffer : 100 mm sodium acetate , ph 7.0 ; -secretase reaction buffer : 100 mm sodium acetate , ph 4.5 ) containing 2 g of fluorogenic substrates ( calbiochem ) . the fluorescence values were measured by spectramax m5 spectrometer ( molecular devices ) with an excitation wavelength at 340 nm and an emission wavelength at 490 nm . figure 1.h2o2 significantly induces intracellular and secreted a. a , sh - sy5y / app695myc cells were treated with 1.0 mm h2o2 in serum - free medium for 1 h or left untreated as indicated . conditioned medium was collected and analyzed by sandwich a elisa to measure secreted a40 and a42 level . b , sh - sy5y / app695myc cells were lysed in ice - cold radioimmune precipitation assay buffer , and the same cell extracts were subjected to a elisa . h2o2 significantly induces intracellular and secreted a. a , sh - sy5y / app695myc cells were treated with 1.0 mm h2o2 in serum - free medium for 1 h or left untreated as indicated . conditioned medium was collected and analyzed by sandwich a elisa to measure secreted a40 and a42 level . b , sh - sy5y / app695myc cells were lysed in ice - cold radioimmune precipitation assay buffer , and the same cell extracts were subjected to a elisa . figure 2.h2o2 does not induce app expression or stimulate the activity of - and -secretase . a , after h2o2 treatment , sh - sy5y / app695myc cells were analyzed by immunoblotting ( ib ) using anti - myc antibody . b - d , conditioned medium was collected and analyzed by immunoblotting , using 22c11 antibody to detect total sapp ( b ) , 6e10 antibody to detect sapp ( c ) , and anti - sapp antibody to detect sapp ( d ) . lysates of h2o2-treated cells were incubated with secretase substances at 37 c for 2 h , and emission was measured . a , after h2o2 treatment , sh - sy5y / app695myc cells were analyzed by immunoblotting ( ib ) using anti - myc antibody . b - d , conditioned medium was collected and analyzed by immunoblotting , using 22c11 antibody to detect total sapp ( b ) , 6e10 antibody to detect sapp ( c ) , and anti - sapp antibody to detect sapp ( d ) . lysates of h2o2-treated cells were incubated with secretase substances at 37 c for 2 h , and emission was measured . cell - based -secretase assay the activity of endogenous -secretase was measured by c99-gal4/vp16 luciferase reporter assay as described previously ( 22 ) . for each well of the 24-well cell culture plate , 100 ng of c99-gvp , 200 ng of pfr - luc ( stratagene ) and 50 ng of prl - tk plasmids ( promega ) were co - transfected into hek293 t cells . dapt was added into cell medium at a final concentration of 10 m for 12 h. the cells were stimulated with h2o2 for 1 h and analyzed for luciferase activity with the dual luciferase reporter system using a luminometer ( promega ) . human brain tissues acquisition fresh frozen human cortex tissues from four individuals with ad and four age - matched nondemented control individuals were obtained via the rapid autopsy system of the netherlands brain bank , netherlands institute for neuroscience , amsterdam , the netherlands , which supplies post - mortem specimens from clinically well documented and neuropathologically confirmed cases . all of the material was collected from donors from whom a written informed consent for brain autopsy , and the use of the material and clinical information for research purposes had been obtained by the netherlands brain bank . the research protocol with human tissues in this study was reviewed and approved by the review board of shanghai institutes for biological sciences , chinese academy of sciences . twelve - month old tg2576 mice ( 26 ) and control nontransgenic littermates were perfused after deep anesthesia . the brains were fixed in phosphate - buffered saline containing 4% para - formaldehyde for 4 h , followed by the equilibration in 20% sucrose overnight at 4 c . embedded tissues were cryosectioned coronally into 10-m sections and treated with 70% formic acid for 15 min , followed by immunostaining . the primary antibodies were anti - phospho - jnk ( 1:600 ) and 6e10 ( 1:300 ) . secondary antibodies were cy3-conjugated goat anti - rabbit igg ( 1:500 ; jackson immunoresearch laboratories ) and fluorescein isothiocyanate - conjugated goat anti - mouse igg ( 1:500 ; jackson immunoresearch laboratories ) . normal rabbit igg ( 1:500 ; zymed laboratories inc . ) was used as the negative control . the images were taken with olympus bx50 fluorescence microscopy and leica dm re confocal microscopy . a , sh - sy5y / app695myc cells were treated with 1.0 mm h2o2 , followed by immunoblotting ( ib ) using anti - myc antibody . quantification of aicd and ctfs was performed through normalization of app level ( bottom panel ) . the cells were treated with h2o2 up to 50 min and harvested at indicated time points . app processing was analyzed as described in a. c , cells were pretreated with the -secretase inhibitor dapt ( 1 and 10 m ) or the control me2so for 3 h , followed by h2o2 treatment . a , sh - sy5y / app695myc cells were treated with 1.0 mm h2o2 , followed by immunoblotting ( ib ) using anti - myc antibody . quantification of aicd and ctfs was performed through normalization of app level ( bottom panel ) . b , a time course of h2o2-induced -secretase - mediated app processing . the cells were treated with h2o2 up to 50 min and harvested at indicated time points . app processing was analyzed as described in a. c , cells were pretreated with the -secretase inhibitor dapt ( 1 and 10 m ) or the control me2so for 3 h , followed by h2o2 treatment . app processing was monitored by immunoblotting as described in a. ns , nonspecific . statistics the differences were considered statistically significant as : , p < 0.05 ; , p < 0.01 ; and , p < 0.001 . h2o2 enhances intracellular and secreted aprevious studies have shown that the level of ros was abnormally high in brain areas that surround senile plaques , because of microglia activation or a accumulation ( 12 , 13 ) . h2o2 is an important endogenous product of ros and is often used as a pro - oxidant in vitro studies ( 27 ) . to determine whether a high level of ros affects a generation , sh - sy5y / app695myc cells were treated with 1 mm h2o2.a elisa showed that h2o2 significantly increased the level of a40 and a42 in cell culture medium when compared with the control ( fig . 1a ) . to determine that a accumulation in cell medium was a result of enhanced a generation but not facilitated a secretion , intracellular a40 and a42 in h2o2-treated cells were also measured . we found that h2o2 also significantly increased the level of intracellular a40 and a42 ( fig . these data indicate that pro - oxidant h2o2 elevates intracellular a generation and increases its accumulation in cell culture medium . h2o2 has no effect on app expression or /-secretase activity a derives from its precursor app through two proteolytic events that are mediated by - and -secretase . deregulation of app processing can cause a dysmetabolism , leading to pathological deposition ( 4 ) . to study the underlying mechanism of h2o2-induced a production , we first determined whether h2o2 induces app expression , thereby promoting a production . immunoblot analysis showed that there were no detectable changes in the level of mature or immature app695 proteins in sh - sy5y / app695myc cells upon h2o2 treatment ( fig . 2a ) , suggesting that enhanced a was not caused by alteration of app synthesis or maturation . next we examined the activity of - and -secretase in response to h2o2 treatment by measuring the products of - and -secretase - mediated app cleavage ( sapp and sapp ) ( 28 ) . we used anti - app antibody ( 22c11 ) and found that there were no detectable changes in total soluble app ( sapp+ ) ( fig . neither the level of sapp nor sapp was affected by h2o2 , as analyzed by immunoblotting using 6e10 and anti - sapp antibodies , which recognize sapp and sapp in the cell medium , respectively ( fig . similar results were obtained when the activity of - or -secretase was measured by fluorogenic substrate assay ( fig . thus , h2o2 does not significantly affect app expression or the activity of - and -secretase . h2o2 promotes -secretase - mediated app695 cleavage in addition to - and -secretase , -secretase is another key secretase that directly determines a production ( 5 ) . to determine whether -secretase - mediated app cleavage is stimulated by h2o2 , thereby promoting a production , we examined app processing by immunoblotting . as previously reported ( 29 ) , the expression level of app695 in sh - sy5y cells was quite abundant , and it was cleaved by - or -secretase into a small quantity of ctfs ( including c83 and c99 ) spanning in the membrane . ctfs were further hydrolyzed by -secretase , releasing app intracellular domain ( aicd ) into cytoplasm ( supplemental fig . , we found that both c99 and c83 were decreased , whereas aicd was evidently elevated ( fig . a time course study revealed that ctfs in sh - sy5y / app695myc cells were reduced gradually , accompanied with the increase in aicd . h2o2 induced the conversion of c83 to aicd as early as 25 min after h2o2 treatment ( fig . hek293 t cells were transiently transfected with pcdna3.1-sp - c99myc for 24 h. conditioned medium of h2o2-treated cells was collected and analyzed by elisa to measure secreted a40 and a42 levels . c , immunoblotting analysis of h2o2-induced aicd production in a dose - dependent manner in above cells ( 0.2 and 1.0 mm h2o2 ) . d , hek293 t cells were transfected with pcdna3.1-aicdmyc or empty vector and treated with various doses of h2o2 ( 0.2 and 1.0 mm h2o2 ) . e , schematic representation of -secretase - dependent luciferase reporter assay ( left panel ) . hek293 t cells were co - transfected with expression plasmids encoding c99-gvp , pfr - luc , and prl - tk . the cells were pretreated with dapt ( 10 m ) for 12 h , followed by h2o2 for another hour . relative luciferase activity was analyzed as described under experimental procedures ( right panel ) . hek293 t cells were transiently transfected with pcdna3.1-sp - c99myc for 24 h. conditioned medium of h2o2-treated cells was collected and analyzed by elisa to measure secreted a40 and a42 levels . c , immunoblotting analysis of h2o2-induced aicd production in a dose - dependent manner in above cells ( 0.2 and 1.0 mm h2o2 ) . d , hek293 t cells were transfected with pcdna3.1-aicdmyc or empty vector and treated with various doses of h2o2 ( 0.2 and 1.0 mm h2o2 ) . e , schematic representation of -secretase - dependent luciferase reporter assay ( left panel ) . hek293 t cells were co - transfected with expression plasmids encoding c99-gvp , pfr - luc , and prl - tk . the cells were pretreated with dapt ( 10 m ) for 12 h , followed by h2o2 for another hour . relative luciferase activity was analyzed as described under experimental procedures ( right panel ) . , the ratio of aicd/(aicd+ctfs ) might represent the efficiency of -secretase - mediated hydrolysis reaction . quantification of ctfs and aicd bands intensity showed that there was a significant increase in aicd/(aicd+ctfs ) between control and h2o2-treated cells ( fig . whether -secretase is involved in h2o2-induced conversion of ctfs to aicd , sh - sy5y / app695myc cells were pretreated with a specific -secretase inhibitor , dapt , before h2o2 treatment . indeed , the h2o2-enhanced aicd level was decreased after 1 m dapt pretreatment ( fig . 3c , compare lanes 4 - 6 with lanes 1 - 3 ) , and aicd was effectively reduced to the basal level with a higher concentration ( 10 m ) of dapt pretreatment ( fig . the identity of the bands recognized by the anti - myc antibody was further validated by immunoblotting analysis with anti - app c - terminal antibody ( anti - app676 - 695 ) ( supplemental fig . , h2o2 could promote -secretase - mediated normal untagged app695 processing by reducing c83 levels also ( supplemental fig . s1c ) , although we were unable to detect aicd band because it was degraded very quickly without fe65 stabilization ( 30 ) . c99 , which is generated from -secretase - mediated cleavage of app695 , is a direct substrate of-secretase and an immediate precursor of a ( 31 ) . to further determine whether h2o2 induces a elevation through activation of -secretase , but not - or -secretase , hek293 t cells were transfected with pcdna3.1-sp - c99myc to replace full - length app695 . consistent with the results obtained from app695 , elisa showed that h2o2 induced a significant increase in secreted a40 and a42 in cell culture medium ( fig . , western blot showed that the other product of -secretase - mediated cleavage of c99 , aicd , was also correspondingly increased in a dose - dependent manner upon h2o2 treatment ( fig . 4c ) . to exclude the possibility that the h2o2-elevated aicd level is caused by the impairment of aicd degradation , hek293 t cells were transiently transfected with expression plasmids encoding aicdmyc , and its yield was analyzed after h2o2 treatment . immunoblotting analysis showed that there was no significant difference in aicd level between control and h2o2-treated cells ( fig . we next used a cell - based reporter gene assay to measure the endogenous -secretase activity ( 22 ) . transfection of either c99-gvp or pfr - luc plasmids into hek293 t cells could not activate the reporter gene , but co - transfection of these two constructs could activate the luciferase gene expression , indicating that luciferase activity was controlled by endogenous -secretase - mediated c99 cleavage ( fig . when c99-gvp and pfr - luc co - transfected cells were treated with h2o2 , the luciferase activity was significantly increased compared with controls . this increase , however , was abolished by pretreatment of -secretase inhibitor dapt ( fig . figure 5.h2o2 promotes -secretase - mediated app cleavage through jnk activation . a , immunofluorescent staining of jnk activation in h2o2-treated sh - sy5y cells . b , immunoblot ( ib ) analysis of h2o2-induced jnk activation in sh - sy5y cells . the cells were serum - starved for 12 h and pretreated with jnk inhibitor sp600125 ( 20 m ) or me2so control ( ctrl ) for 3 h. after incubation with h2o2 for 15 min , the cells were harvested , and jnk activation was analyzed by immunoblotting with anti - phospho - jnk antibody . c , determination of the inhibitory effect of sp600125 on -secretase - mediated cleavage of app695myc in h2o2-treated sh - sy5y / app695myc cells . d , determination of the inhibitory effect of sp600125 on -secretase - mediated cleavage in h2o2-treated hek293t / sp - c99myc cells . hek293 t cells were co - transfected with app695myc and sirna against jnk1 or jnk1/2 . after h2o2 stimulation , the cells were analyzed by immunoblotting . f and g , immunoblot analysis of h2o2 effect on the processing of app695myc(t668a ) mutant ( f ) or sp - c99myc(t668a ) mutant ( g ) . b , immunoblot ( ib ) analysis of h2o2-induced jnk activation in sh - sy5y cells . the cells were serum - starved for 12 h and pretreated with jnk inhibitor sp600125 ( 20 m ) or me2so control ( ctrl ) for 3 h. after incubation with h2o2 for 15 min , the cells were harvested , and jnk activation was analyzed by immunoblotting with anti - phospho - jnk antibody . c , determination of the inhibitory effect of sp600125 on -secretase - mediated cleavage of app695myc in h2o2-treated sh - sy5y / app695myc cells . d , determination of the inhibitory effect of sp600125 on -secretase - mediated cleavage in h2o2-treated hek293t / sp - c99myc cells . hek293 t cells were co - transfected with app695myc and sirna against jnk1 or jnk1/2 . after h2o2 stimulation , the cells were analyzed by immunoblotting . f and g , immunoblot analysis of h2o2 effect on the processing of app695myc(t668a ) mutant ( f ) or sp - c99myc(t668a ) mutant ( g ) . it has been shown that h2o2 activates jnk , one of the mitogen - activated protein kinases that is readily activated in response to various environmental stresses , to decide the fate of cells ( 32 ) . to test whether jnk is involved in activation of -secretase by h2o2 , we first determined the stimulatory effect of h2o2 on jnk in sh - sy5y cells . immunofluorescence staining and western blot using anti - phospho - jnk antibody showed that jnk was phosphorylated at thr and tyr in the activation t - loop in cells treated with h2o2 when compared with that in control cells ( fig . 5 , a and b , lane 2 ) , as well as in hek293 t cells ( data not shown ) . consistently , pretreatment with jnk inhibitor sp600125 also decreased jnk activation ( fig . 5b , lane 4 ) . next , we wondered whether jnk activation is required for h2o2 to promote -secretase - mediated app cleavage . sh - sy5y / app695myc cells were pretreated with or without sp600125 for 3 h , followed by h2o2 treatment . 5c , compare lane 3 with lane 1 ) but was unable to do so in cells pretreated with sp600125 ( fig . the pretreatment with sp600125 significantly reduced the ratio of h2o2-induced aicd/(aicd+ctfs ) , as measured by the intensity of signals ( fig . similarly , the pretreatment with sp600125 also reduced h2o2-indcued aicd to basal level in hek293 t cells transiently transfected with sp - c99myc ( fig . furthermore , silencing of jnk expression by sijnks ( 24 ) , which efficiently reduced the endogenous jnk protein level ( fig . taken together , these data show that activation of jnk is required for h2o2 to promote -secretase - mediated app cleavage . it has been reported that jnk can phosphorylate the -secretase substrate app at thr , thereby facilitating app processing and a production ( 33 , 34 ) . to determine whether thr phosphorylation of app accounts for h2o2-induced jnk - dependent promotion of -secretase - mediated app cleavage , hek293 t cells were transiently transfected with mammalian expression plasmids encoding wild type app695myc or app(t668a)myc , in which thr has been replaced by ala . immunoblot analysis showed that in both wild type and t668a mutant transfected cells , aicd products were elevated , whereas ctfs products decreased after h2o2 stimulation ( fig . consistently , h2o2 also increased aicd level in hek293 t cells expressing sp - c99myc mutant ( fig . the similarity of proteolytic process between wild type app and app(t668a ) mutant suggests that h2o2-induced , jnk - dependent augmentation of -secretase - mediated app cleavage is likely due to activation of the -secretase rather than thr phosphorylation of app . figure 6.jnk , but not erk or pkb , is required for h2o2 to promote -secretase - mediated app cleavage . the cells were serum - starved for 12 h and pretreated with u0126 ( 5 m ) ( a ) , wortmannin ( 20 nm ) ( b ) , sp600125 ( 20 m ) ( c ) or the control me2so for 3 h. after h2o2 treatment , the cells were analyzed by immunoblotting ( ib ) to determine the effect of the above inhibitors on activation of erk ( a ) , pkb ( b ) , and app processing ( c ) . jnk , but not erk or pkb , is required for h2o2 to promote -secretase - mediated app cleavage . the cells were serum - starved for 12 h and pretreated with u0126 ( 5 m ) ( a ) , wortmannin ( 20 nm ) ( b ) , sp600125 ( 20 m ) ( c ) or the control me2so for 3 h. after h2o2 treatment , the cells were analyzed by immunoblotting ( ib ) to determine the effect of the above inhibitors on activation of erk ( a ) , pkb ( b ) , and app processing ( c ) . jnk , but not erk or pkb , is required for h2o2 to promote -secretase - mediated app cleavage we found that in addition to jnk , erk and pkb were activated by h2o2 ( fig . 6 , a and b ) . to determine whether jnk is mainly responsible for h2o2-induced augmentation of -secretase activity , we used specific pharmacological inhibitors to inhibit individual kinases . immunoblot analysis showed that erk and pkb were significantly inhibited by u0126 or wortmannin , respectively ( fig . 6 , a and b ) . however , only sp600125 , but not u0126 or wortmannin , was able to inhibit h2o2-induced augmentation of aicd production ( fig . thus , these data suggest that h2o2 enhances endogenous -secretase activity through activation of jnk . jnk is activated in brain areas surrounding senile plaques of ad animal model to investigate the relation between jnk and a production in vivo , we examined jnk activation in brain tissues of ad patients . hippocampus and cortices of human brain tissues from ad patients ( n = 4 ) and nondementional controls ( n = 4 ) were examined for jnk activation ( table 1 ) . the level of phosphorylated jnk was evidently increased in the brains of ad patients when compared with control brains , whereas there was no significant difference in jnk expression level ( fig . table 1case details of ad patients and control subjects from the netherlands brain bankgroupgenderagepmdaphbrain weightregionhgalzheimer disease a1 m 92 3:30 7.2 1175 medial frontal gyrus a2 f 94 2:55 6.53 955 superior temporalis gyrus a3 f 80 2:20 6.41 1030 superior temporalis gyrus a4 f 78 6:35 7 1084 angular gyrus nondemented control n1 m 88 7:00 6.84 1398 medial frontal gyrus n2 f 78 6:25 6.5 1135 inferior temporalis gyrus n3 f 73 5:30 6.38 1304 superior temporalis gyrus n4 f 74 5:35 7.04 982 angular gyrus apmd , postmortem delay . case details of ad patients and control subjects from the netherlands brain bank pmd , postmortem delay . to determine the link between amyloid deposits and jnk activation , we performed an immunostaining in brains of the ad animal model ( tg2576 mice ) and found that phospho - jnk immunoreactivity was localized in the surrounding region of senile plaques , which was positive with a antibody 6e10 ( fig . in contrast , both senile plaques and phospho - jnk were negative in the brains of control nontransgenic littermates ( data not shown ) . the immunostaining of jnk by the anti - phospho - jnk antibody was highly specific , because normal rabbit igg was unable to detect any phospho - jnk immunoreactivity ( fig . 7b , panels c and g ) , suggesting that cells were dead and lost in the area of amyloid deposited . taken together , oxidative stress has been implicated in the pathogenesis of ad , because it induces a production and contributes to a neurotoxicity ( 12 - 15 , 19 ) . yet the underlying mechanism is not completely understood . in this study , we report that the prooxidant h2o2 promotes a production through jnk - dependent activation of -secretase . the app processing is a sequential proteolysis that is carried out by - and -secretase , which leads to the generation of a and ultimately neurotoxicity ( 4 ) . although it has been reported that oxidative stress can induce a production , it is not clear whether -secretase activity is regulated by oxidative stress and , if so , what the underlying mechanism is ( 15 - 18 , 20 ) . we found that h2o2 induced a production through enhancement of -secretase activation , resulting in promotion of app cleavage . first , h2o2 induced a significant increase in aicd without the inhibition on aicd degradation ( figs . 3 and 4 ) . this notion is also supported by the observation that the substrates of -secretase , ctfs , were decreased accordingly ( fig . 3 ) . second , h2o2-induced increase in aicd was blocked by dapt , the inhibitor of -secretase ( fig . 3 ) . third , h2o2 accelerated sp - c99 processing , which was only determined by -secretase without -secretase interference ( fig . 4 ) . this excludes the possibility that the increase of a is due to the enhanced -secretase activity to elevate c99 level . in contrast to previous reports ( 16 - 18 , 20 ) , immunoblot analysis and fluorogenic substrate assay showed that app expression or -secretase activity was not affected by h2o2 treatment ( fig . in the previous reports , changes in app expression or -secretase activity occurred several hours after the cells were treated with a low concentration of h2o2 ( 16 , 17 ) . however , we found that -secretase - mediated app cleavage was not affected by a low concentration of h2o2 ( from 1 to 100 m ; data not shown ) . although the exact concentration of h2o2 in vivo is unknown , it has been reported that 100 m is the possible concentration under physiological conditions ( 38 ) . under pathological conditions , however , the concentration of ros may be much higher ( 39 ) . to defend a host against microbial organism infections , immune cells may need to generate millimolar quantities of h2o2 ( 27 , 40 , 41 ) . patients with vitiligo accumulate millimolar concentration of h2o2 in their epidermis ( 42 , 43 ) . in the brains of ad patients , microglia is activated , and inflammation occurs around the senile plaques ( 44 ) , which might produce large amounts of h2o2 . another source of h2o2 might be from the progression of a aggregation ( 14 ) . it is consistent with the observation that ros is abnormally higher in the ad patients , especially in the senile plaque - surrounding areas , than that in normal elder people , ( 12 , 13 ) . thus , the high concentration of h2o2 ( mm ) used in this study might mimic the pathological conditions of the limited areas that surround the senile plaques . taken together , our results show that h2o2 promotes -secretase - mediated app cleavage , thereby contributing to a production . a , human brain samples ( 30 g of total protein extracts ) from ad ( n = 4 ) and age - matched control samples ( n = 4 ) were analyzed for jnk activation using anti - phospho - jnk and anti - pan jnk antibodies . sections were stained with 6e10 ( green , panels a and e ) and antiphospho - jnk antibody ( red , panel b ) . normal rabbit igg was used as a negative control of anti - phospho - jnk ( panel f ) . the nucleus was indicated with 4,6-diamino-2-phenylindole ( dapi , blue , panels c and g ) . c , the model of h2o2 via jnk promotes -secretase - mediated app processing . see discussion for the details . jnk is activated in the brain areas that surround senile plaques in vivo . a , human brain samples ( 30 g of total protein extracts ) from ad ( n = 4 ) and age - matched control samples ( n = 4 ) were analyzed for jnk activation using anti - phospho - jnk and anti - pan jnk antibodies . sections were stained with 6e10 ( green , panels a and e ) and antiphospho - jnk antibody ( red , panel b ) . normal rabbit igg was used as a negative control of anti - phospho - jnk ( panel f ) . the nucleus was indicated with 4,6-diamino-2-phenylindole ( dapi , blue , panels c and g ) . h2o2 activates multiple signaling pathways , including erk , pkb , and jnk ( 35 ) . first , immunofluorescent staining and immunoblotting showed that jnk was activated in the h2o2-treated sh - sy5y cells ( fig . 5 ) . second , the jnk inhibitor sp600125 , but not the inhibitors of erk and pkb , specifically blocked h2o2-promoted -secretase - mediated cleavage of app or c99 ( figs . 5 and 6 ) . third , jnk sirna efficiently reduced h2o2-induced aicd levels ( fig . 5 ) . these observations are consistent with a previous finding that proinflammatory cytokine - activated jnk also contributes to -secretase activity and a production in hek293 cells ( 45 ) . thus , jnk may play a central role in mediating the stimulatory effect of different stress signals on -secretase activity under pathological conditions . the molecular mechanism by which jnk regulates -secretase activity and app processing remains to be determined . it is possible that jnk phosphorylates app and thereby makes it a better substrate for -secretase ( 33 , 34 ) . however , this seems less likely because proteolysis of app(t668a ) mutant and sp - c99(t668a ) mutants was still promoted by h2o2 in our study ( fig . another possibility is that jnk may enhance the enzyme activity of -secretase through protein phosphorylation , either directly or indirectly . in vitro kinase assay showed that activated jnk directly phosphorylated presenilin 1 , which is the core component of -secretase enzyme ( supplemental fig . it has been reported that a high concentration of h2o2 induces cell death via jnk activation ( 46 ) . we found that h2o2-treated cells had typical characteristics of cell death , as analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and trypan blue exclusion ( data not shown ) . furthermore , only sp600125 , but not u0126 or wortmannin , protected cells from h2o2-induced morphological changes ( supplemental fig . thus , it is possible that h2o2-stimulated jnk contributes to cell death , which provides a subtle acid environment that is needed for -secretase aspartyl protease activity , resulting in enhanced a production ( 47 ) . this hypothesis is consistent with the report that a products increase during cell death ( 48 , 49 ) . during the preparation of the current manuscript , it was reported that oxidative stress induces expression of -secretase through jnk - dependent regulation of -secretase , thereby providing a forward feedback between - and -secretase for the cleavage of app ( 50 ) . the classical a hypothesis states that excessive accumulation of a in the brains of ad patients increases oxidative stress and activates protein kinases , resulting in neurofibrillary tangle formation and neuronal loss ( 1 ) . we found that active jnk located around amyloid deposits in brains of ad mouse model ( fig . 7c ) , consistent with the previous report that jnk is strongly activated in mutant app transgenic mice upon extensive oxidative damage but not in mutant app transgenic mice with little oxidative damage ( 51 ) . thus , we hypothesize that at the modest and late stage of ad brains , escalated a accumulation and microglia activation induce high level of ros including endogenous h2o2 . these cells in turn produce more a peptide through jnk - dependent , -secretase - mediated app cleavage . the resultant a is secreted and deposited around the core of original plaque , leading to plaque expansion in the brains of ad patients . such exacerbation of a vicious cycle may explain how the process of ad pathology becomes accelerated and irreversible at the modest and late ad stage ( fig . new therapeutic targets for the intervention of ad pathogenesis might be identified along this signaling cascade .

accumulation of senile plaques composed of amyloid -peptide ( a ) is a pathological hallmark of alzheimer disease ( ad ) , and a is generated through the sequential cleavage of amyloid precursor protein ( app ) by - and -secretase . although oxidative stress has been implicated in the ad pathogenesis by inducing a production , the underlying mechanism remains elusive . here we show that the pro - oxidant h2o2 promotes a production through c - jun n - terminal kinase ( jnk)-dependent activation of -secretase . treatment with h2o2 induced significant increase in the levels of intracellular and secreted a in human neuroblastoma sh - sy5y cells . although -secretase - mediated cleavage of app or c99 was enhanced upon h2o2 treatment , expression of app or its /-secretase - mediated cleavage was not affected . silencing of the stress - activated jnk by small interfering rna or the specific jnk inhibitor sp600125 reduced h2o2-induced -secretase - mediated cleavage of app . jnk activity was augmented in human brain tissues from ad patients and active jnk located surrounding the senile plaques in the brain of ad model mouse . our data suggest that oxidative stress - activated jnk may contribute to senile plaque expansion through the promotion of -secretase - mediated app cleavage and a production .

EXPERIMENTAL PROCEDURES RESULTS DISCUSSION Supplementary Material

cell culture and treatments human embryonic kidney 293 t cells ( hek293 t ) and human neuroblastoma cells ( sh - sy5y ) were cultured in dulbecco 's modified eagle 's medium / ham 's f-12 ( sigma ) with 10% fetal bovine serum ( hyclone ) . to generate human neuroblastoma cell lines stably expressing app695myc , sh - sy5y cells were transfected with the construct and selected in the presence of g418 ( 500 g / ml ) . sh - sy5y / app695myc cells were maintained in the medium containing 200 g / ml of g418 . sh - sy5y cells were routinely treated with 1.0 mm h2o2 for 1 h unless otherwise indicated in figure legends . sp600125 ( 20 m ; calbiochem ) , u0126 ( 5 m ; calbiochem ) , wortmannin ( 20 nm ; calbiochem ) , and -secretase inhibitor dapt ( 1 - 10 m ; sigma ) , were added into dulbecco 's modified eagle 's medium / ham 's f-12 medium for 3 h before h2o2 treatment . a elisa to determine the production of intracellular a , sh - sy5y / app695myc cells , or pcdna3.1-sp - c99myc transiently expressed hek293 t cells were treated with 1.0 mm h2o2 for 1 h. the cells were lysed with cold radioimmune precipitation assay buffer containing 50 mm tris - hcl ( ph 8.0 ) , 150 mm nacl , 5 mm edta , 1% nonidet p-40 , 0.25 mm phenylmethanesulfonyl fluoride , and a mixture of protease inhibitors ( roche applied science ) . to detect secreted a40 and a42 , conditioned medium of h2o2-treated cells ( 300 l / well ) the cells or tissues were lysed in cell lysis buffer containing 50 mm tris - hcl ( ph 8.0 ) , 150 mm nacl , 0.5% nadoc , 0.1% sds , 1% nonidet p-40 , 5 mm edta , 0.25 mm phenylmethanesulfonyl fluoride , and a mixture of protease inhibitors . to detect sapp and sapp , conditioned medium of h2o2-treated cells was collected and analyzed by immunoblotting using the following primary antibodies : 22c11 recognizing app n - terminal amino acid residues 66 - 81 ( 1:2,000 ; chemicon ) , 6e10 reacting with the a4 - 9 region in the c terminus of sapp ( 1:1,000 ; signet laboratory ) , and anti - sapp antibody recognizing the potion of c terminus of human sapp ( isevkm ) ( 1:200 ; ibl ) . figure 1.h2o2 significantly induces intracellular and secreted a. a , sh - sy5y / app695myc cells were treated with 1.0 mm h2o2 in serum - free medium for 1 h or left untreated as indicated . h2o2 significantly induces intracellular and secreted a. a , sh - sy5y / app695myc cells were treated with 1.0 mm h2o2 in serum - free medium for 1 h or left untreated as indicated . b , sh - sy5y / app695myc cells were lysed in ice - cold radioimmune precipitation assay buffer , and the same cell extracts were subjected to a elisa . a , after h2o2 treatment , sh - sy5y / app695myc cells were analyzed by immunoblotting ( ib ) using anti - myc antibody . a , after h2o2 treatment , sh - sy5y / app695myc cells were analyzed by immunoblotting ( ib ) using anti - myc antibody . human brain tissues acquisition fresh frozen human cortex tissues from four individuals with ad and four age - matched nondemented control individuals were obtained via the rapid autopsy system of the netherlands brain bank , netherlands institute for neuroscience , amsterdam , the netherlands , which supplies post - mortem specimens from clinically well documented and neuropathologically confirmed cases . b , a time course of h2o2-induced -secretase - mediated app processing . h2o2 enhances intracellular and secreted aprevious studies have shown that the level of ros was abnormally high in brain areas that surround senile plaques , because of microglia activation or a accumulation ( 12 , 13 ) . these data indicate that pro - oxidant h2o2 elevates intracellular a generation and increases its accumulation in cell culture medium . h2o2 has no effect on app expression or /-secretase activity a derives from its precursor app through two proteolytic events that are mediated by - and -secretase . to study the underlying mechanism of h2o2-induced a production , we first determined whether h2o2 induces app expression , thereby promoting a production . immunoblot analysis showed that there were no detectable changes in the level of mature or immature app695 proteins in sh - sy5y / app695myc cells upon h2o2 treatment ( fig . 2a ) , suggesting that enhanced a was not caused by alteration of app synthesis or maturation . next we examined the activity of - and -secretase in response to h2o2 treatment by measuring the products of - and -secretase - mediated app cleavage ( sapp and sapp ) ( 28 ) . thus , h2o2 does not significantly affect app expression or the activity of - and -secretase . h2o2 promotes -secretase - mediated app695 cleavage in addition to - and -secretase , -secretase is another key secretase that directly determines a production ( 5 ) . to determine whether -secretase - mediated app cleavage is stimulated by h2o2 , thereby promoting a production , we examined app processing by immunoblotting . as previously reported ( 29 ) , the expression level of app695 in sh - sy5y cells was quite abundant , and it was cleaved by - or -secretase into a small quantity of ctfs ( including c83 and c99 ) spanning in the membrane . a time course study revealed that ctfs in sh - sy5y / app695myc cells were reduced gradually , accompanied with the increase in aicd . , the ratio of aicd/(aicd+ctfs ) might represent the efficiency of -secretase - mediated hydrolysis reaction . whether -secretase is involved in h2o2-induced conversion of ctfs to aicd , sh - sy5y / app695myc cells were pretreated with a specific -secretase inhibitor , dapt , before h2o2 treatment . the identity of the bands recognized by the anti - myc antibody was further validated by immunoblotting analysis with anti - app c - terminal antibody ( anti - app676 - 695 ) ( supplemental fig . c99 , which is generated from -secretase - mediated cleavage of app695 , is a direct substrate of-secretase and an immediate precursor of a ( 31 ) . consistent with the results obtained from app695 , elisa showed that h2o2 induced a significant increase in secreted a40 and a42 in cell culture medium ( fig . , western blot showed that the other product of -secretase - mediated cleavage of c99 , aicd , was also correspondingly increased in a dose - dependent manner upon h2o2 treatment ( fig . to exclude the possibility that the h2o2-elevated aicd level is caused by the impairment of aicd degradation , hek293 t cells were transiently transfected with expression plasmids encoding aicdmyc , and its yield was analyzed after h2o2 treatment . transfection of either c99-gvp or pfr - luc plasmids into hek293 t cells could not activate the reporter gene , but co - transfection of these two constructs could activate the luciferase gene expression , indicating that luciferase activity was controlled by endogenous -secretase - mediated c99 cleavage ( fig . figure 5.h2o2 promotes -secretase - mediated app cleavage through jnk activation . b , immunoblot ( ib ) analysis of h2o2-induced jnk activation in sh - sy5y cells . the cells were serum - starved for 12 h and pretreated with jnk inhibitor sp600125 ( 20 m ) or me2so control ( ctrl ) for 3 h. after incubation with h2o2 for 15 min , the cells were harvested , and jnk activation was analyzed by immunoblotting with anti - phospho - jnk antibody . c , determination of the inhibitory effect of sp600125 on -secretase - mediated cleavage of app695myc in h2o2-treated sh - sy5y / app695myc cells . d , determination of the inhibitory effect of sp600125 on -secretase - mediated cleavage in h2o2-treated hek293t / sp - c99myc cells . b , immunoblot ( ib ) analysis of h2o2-induced jnk activation in sh - sy5y cells . the cells were serum - starved for 12 h and pretreated with jnk inhibitor sp600125 ( 20 m ) or me2so control ( ctrl ) for 3 h. after incubation with h2o2 for 15 min , the cells were harvested , and jnk activation was analyzed by immunoblotting with anti - phospho - jnk antibody . c , determination of the inhibitory effect of sp600125 on -secretase - mediated cleavage of app695myc in h2o2-treated sh - sy5y / app695myc cells . d , determination of the inhibitory effect of sp600125 on -secretase - mediated cleavage in h2o2-treated hek293t / sp - c99myc cells . it has been shown that h2o2 activates jnk , one of the mitogen - activated protein kinases that is readily activated in response to various environmental stresses , to decide the fate of cells ( 32 ) . to test whether jnk is involved in activation of -secretase by h2o2 , we first determined the stimulatory effect of h2o2 on jnk in sh - sy5y cells . next , we wondered whether jnk activation is required for h2o2 to promote -secretase - mediated app cleavage . sh - sy5y / app695myc cells were pretreated with or without sp600125 for 3 h , followed by h2o2 treatment . taken together , these data show that activation of jnk is required for h2o2 to promote -secretase - mediated app cleavage . it has been reported that jnk can phosphorylate the -secretase substrate app at thr , thereby facilitating app processing and a production ( 33 , 34 ) . to determine whether thr phosphorylation of app accounts for h2o2-induced jnk - dependent promotion of -secretase - mediated app cleavage , hek293 t cells were transiently transfected with mammalian expression plasmids encoding wild type app695myc or app(t668a)myc , in which thr has been replaced by ala . the similarity of proteolytic process between wild type app and app(t668a ) mutant suggests that h2o2-induced , jnk - dependent augmentation of -secretase - mediated app cleavage is likely due to activation of the -secretase rather than thr phosphorylation of app . figure 6.jnk , but not erk or pkb , is required for h2o2 to promote -secretase - mediated app cleavage . the cells were serum - starved for 12 h and pretreated with u0126 ( 5 m ) ( a ) , wortmannin ( 20 nm ) ( b ) , sp600125 ( 20 m ) ( c ) or the control me2so for 3 h. after h2o2 treatment , the cells were analyzed by immunoblotting ( ib ) to determine the effect of the above inhibitors on activation of erk ( a ) , pkb ( b ) , and app processing ( c ) . jnk , but not erk or pkb , is required for h2o2 to promote -secretase - mediated app cleavage . the cells were serum - starved for 12 h and pretreated with u0126 ( 5 m ) ( a ) , wortmannin ( 20 nm ) ( b ) , sp600125 ( 20 m ) ( c ) or the control me2so for 3 h. after h2o2 treatment , the cells were analyzed by immunoblotting ( ib ) to determine the effect of the above inhibitors on activation of erk ( a ) , pkb ( b ) , and app processing ( c ) . jnk , but not erk or pkb , is required for h2o2 to promote -secretase - mediated app cleavage we found that in addition to jnk , erk and pkb were activated by h2o2 ( fig . thus , these data suggest that h2o2 enhances endogenous -secretase activity through activation of jnk . jnk is activated in brain areas surrounding senile plaques of ad animal model to investigate the relation between jnk and a production in vivo , we examined jnk activation in brain tissues of ad patients . hippocampus and cortices of human brain tissues from ad patients ( n = 4 ) and nondementional controls ( n = 4 ) were examined for jnk activation ( table 1 ) . the level of phosphorylated jnk was evidently increased in the brains of ad patients when compared with control brains , whereas there was no significant difference in jnk expression level ( fig . table 1case details of ad patients and control subjects from the netherlands brain bankgroupgenderagepmdaphbrain weightregionhgalzheimer disease a1 m 92 3:30 7.2 1175 medial frontal gyrus a2 f 94 2:55 6.53 955 superior temporalis gyrus a3 f 80 2:20 6.41 1030 superior temporalis gyrus a4 f 78 6:35 7 1084 angular gyrus nondemented control n1 m 88 7:00 6.84 1398 medial frontal gyrus n2 f 78 6:25 6.5 1135 inferior temporalis gyrus n3 f 73 5:30 6.38 1304 superior temporalis gyrus n4 f 74 5:35 7.04 982 angular gyrus apmd , postmortem delay . case details of ad patients and control subjects from the netherlands brain bank pmd , postmortem delay . to determine the link between amyloid deposits and jnk activation , we performed an immunostaining in brains of the ad animal model ( tg2576 mice ) and found that phospho - jnk immunoreactivity was localized in the surrounding region of senile plaques , which was positive with a antibody 6e10 ( fig . 7b , panels c and g ) , suggesting that cells were dead and lost in the area of amyloid deposited . taken together , oxidative stress has been implicated in the pathogenesis of ad , because it induces a production and contributes to a neurotoxicity ( 12 - 15 , 19 ) . in this study , we report that the prooxidant h2o2 promotes a production through jnk - dependent activation of -secretase . the app processing is a sequential proteolysis that is carried out by - and -secretase , which leads to the generation of a and ultimately neurotoxicity ( 4 ) . although it has been reported that oxidative stress can induce a production , it is not clear whether -secretase activity is regulated by oxidative stress and , if so , what the underlying mechanism is ( 15 - 18 , 20 ) . we found that h2o2 induced a production through enhancement of -secretase activation , resulting in promotion of app cleavage . first , h2o2 induced a significant increase in aicd without the inhibition on aicd degradation ( figs . in contrast to previous reports ( 16 - 18 , 20 ) , immunoblot analysis and fluorogenic substrate assay showed that app expression or -secretase activity was not affected by h2o2 treatment ( fig . however , we found that -secretase - mediated app cleavage was not affected by a low concentration of h2o2 ( from 1 to 100 m ; data not shown ) . in the brains of ad patients , microglia is activated , and inflammation occurs around the senile plaques ( 44 ) , which might produce large amounts of h2o2 . it is consistent with the observation that ros is abnormally higher in the ad patients , especially in the senile plaque - surrounding areas , than that in normal elder people , ( 12 , 13 ) . thus , the high concentration of h2o2 ( mm ) used in this study might mimic the pathological conditions of the limited areas that surround the senile plaques . taken together , our results show that h2o2 promotes -secretase - mediated app cleavage , thereby contributing to a production . c , the model of h2o2 via jnk promotes -secretase - mediated app processing . jnk is activated in the brain areas that surround senile plaques in vivo . first , immunofluorescent staining and immunoblotting showed that jnk was activated in the h2o2-treated sh - sy5y cells ( fig . second , the jnk inhibitor sp600125 , but not the inhibitors of erk and pkb , specifically blocked h2o2-promoted -secretase - mediated cleavage of app or c99 ( figs . these observations are consistent with a previous finding that proinflammatory cytokine - activated jnk also contributes to -secretase activity and a production in hek293 cells ( 45 ) . during the preparation of the current manuscript , it was reported that oxidative stress induces expression of -secretase through jnk - dependent regulation of -secretase , thereby providing a forward feedback between - and -secretase for the cleavage of app ( 50 ) . the classical a hypothesis states that excessive accumulation of a in the brains of ad patients increases oxidative stress and activates protein kinases , resulting in neurofibrillary tangle formation and neuronal loss ( 1 ) . these cells in turn produce more a peptide through jnk - dependent , -secretase - mediated app cleavage . the resultant a is secreted and deposited around the core of original plaque , leading to plaque expansion in the brains of ad patients .

within the last two years , a number of retrospective epidemiological studies have found that chronic use of beta - blocking drugs is associated with lower recurrence and mortality of breast cancer,1,2 or reduced progression and mortality of breast cancer3,4 and malignant melanoma.5,6 three earlier studies also suggested beneficial effects from prolonged use of beta blockers on general cancer risk7 or on prostate cancer risk.8,9 collectively , these findings are both provocative and encouraging . they are provocative because beta blockers , which have been used clinically for decades worldwide , are typically taken to treat heart - related ailments , such as arrhythmias and hypertension , not cancer . they are encouraging because beta blockers may represent a new , and relatively safe , category of drugs for the prevention and possible treatment of a range of cancers , while also shedding light on the pathophysiological basis of some types of cancer . a few years ago , i put forth the hypothesis that the signaling molecule norepinephrine ( ne ) is an etiological factor in some types of cancer.10,11 in support of this hypothesis i cited seven lines of evidence : ( i ) rodent studies of tumorigenesis in the context of ne manipulation , ( ii ) human studies of tricyclic antidepressant use and cancer rate , ( iii ) existence of pheochromocytoma , a cancer of the adrenal glands , ( iv ) cancer rates in families with individuals who have bipolar disorder , ( v ) hypertension and cancer risk , ( vi ) excessive body weight and cancer risk , and ( vii ) psychological stressors and cancer risk . these lines of evidence tend to show that increased ne release in the body , or increased numbers or sensitivity of ne receptors , is associated with increased occurrence of cancer in various organs . norepinephrine , and the related molecule epinephrine ( epi ) , also known as adrenaline , are stress hormones that activate the body s adrenoceptors , which includes the receptors that beta blockers antagonize . there are five major types of adrenoceptors in the body : beta1 , beta2 , beta3 , alpha1 , and alpha2;10 most beta blockers target the beta1 and beta2 receptors . adrenoceptors are g protein - coupled receptors that initiate second messenger signaling processes within cells that bear these receptors . adrenoceptors are found not only in the brain , but also in nearly all , if not all , organs of the body.10 ne and epi have direct access to these organs through localized release by the sympathetic nervous system ( sns ) , as well as through general release into the bloodstream , as part of the body s fight or flight response to psychological stressors . by binding to adrenoceptors in various organs , ne and epi may modulate various intracellular molecular pathways already implicated in cancer ( see below ) . there is a growing preclinical literature suggesting that ne and epi may be etiological factors in cancer . for example , sarkar et al12 have recently shown that functional balance of the sympathetic and parasympathetic branches of the autonomic nervous system may be important in rodent experimentally induced cancers , where pharmacologically increasing sympathetic ne output or blocking cholinergic parasympathetic output tended to promote cancer in their rat model . in particular , sarkar et al12 used an innovative cellular transplantation technique that modulated beta - endorphin signaling in the hypothalamus region of the rat brain . this method may have decreased sympathetic relative to parasympathetic output , thereby reducing mammary tumor growth and metastasis . this and other studies add to data that began several decades ago , showing an effect of ne manipulation on rodent experimentally induced carcinogenesis.13,14 these data also suggest that it would be informative to epidemiologically test the effects of drugs that act on the autonomic nervous system on risk for or outcome of various types of cancer ( see below ) . it should be noted that some recent clinical studies have found that the use of beta blockers , as well as other ne transmission decreasing drugs , is associated with increased cancer risk and poorer survival . for example , a recent epidemiological study found that chronic beta blocker use and use of other ne decreasing drugs is associated with a slightly increased ( odds ratios approximately 1.051.1 ) risk of various types of cancer combined , including colon , lung , breast , and prostate cancer.15 another study found that longterm use ( 6 or more years ) of beta blockers is associated with significantly increased risk ( odds ratio 2.02 ) of stage iv colorectal cancer.16 a recent study of a united kingdom database comparing cancer patients receiving beta blockers with those receiving other antihypertensive medications found slightly poorer survival rates ( hazard ratio approximately 1.2 ) in those on beta blockers ; this outcome appeared to be limited to pancreatic and prostate cancer.17 one interpretation of the data from these three studies is that persons who eventually take beta - blocking medications chronically for hypertension or other cardiac - related abnormalities have elevated endogenous ( possibly genetic ) ne signaling , predisposing them to various types of cancer and/or poorer cancer outcome . in this scenario , beta - blocking drugs , at least in the manner in which they are currently used , may have a smaller effect on cancer risk or outcome than the underlying elevated ne signaling.18 in general , caution should be exercised in inferring from human drug studies that ne plays an etiological role in some cancers . clinical epidemiological studies tend to be correlative and do not necessarily indicate that the factor in question , such as ne or a noradrenergic drug , is causative in a disease process . another point to consider is that different beta - blocking drugs , which vary in their specificity for different beta adrenoceptors , such as beta1 and beta2 , may differentially affect cancer outcome in both preclinical and clinical settings.18 one beta blocker that has been used in a number of preclinical oncology studies , propranolol , blocks both beta1 and beta2 receptors . there is growing evidence that psychological stress is associated with increased cancer risk or poorer treatment outcome ( for a recent review , see sarkar et al19 ) . for example , three recent preclinical studies have shown that stress affects experimentally induced cancers . in one study , tumor growth transiently increased after stress was brought on by housing mice singly ; this process may be affected by transient changes in peripheral ne release.20 chronic stress was found to increase ovarian tumor growth in mice , which may have been in part mediated by ne - stimulated upregulation of the cytokine interleukin-8.21 in a mouse model of social stress , a number of signaling molecules , including ne and erk protein , increased or were upregulated , and tumor progression was prevented , when the intracellular molecule cyclic adenosine monophosphate was reduced by treatment with the inhibitory neurotransmitter gamma - aminobutyric acid.22 if psychological stress affects cancer onset or progression , one possibility is that stress - related increase in the release of ne and epi , and only these two molecules , is responsible for the deleterious effects on cancer risk and outcome . however , other molecules , such as neuropeptide y ( npy ) and cortisol also mediate the body s physiological stress response . a recent study found that npy receptors are present in the 4t1 breast cancer cell line , and that administration of npy to these cells promoted proliferation and migration.23 in addition , it has been shown that cortisol ( also known as hydrocortisone ) downregulates the expression of the breast cancer susceptibility gene brca1 in the nonmalignant mouse mammary cell line eph4 , where such downregulation has been associated with breast cancer development.24 as we come to better understand the genetics of stress - related molecules such as npy in humans , epidemiological investigation of the possible association between various alleles and cancer initiation or progression would be informative . as sarkar et al19 pointed out , the cellular and molecular mechanisms whereby ne and epi may carry out stress - related effects on carcinogenesis are beginning to be elucidated . they suggest that suppression of sns functioning ( via their beta - endorphin transplantation method , noted above ) can result in reduced tumorigenesis via increased peripheral natural killer cell and macrophage activities , elevated levels of anti - inflammatory cytokines , and reduced levels of inflammatory cytokines , as well as changes in tumor microenvironment . in other words , another point is that perhaps both the magnitude and the duration of exposure to psychological stress affect the probability of developing cancer . this is a topic that would benefit from further epidemiological retrospective investigation in persons who have been exposed to various types and duration of psychological stress . while psychological stress , which is partly mediated by the endogenous release of ne and epi , has already been associated with increased cancer risk in humans,25 it is also possible that genetically elevated ne / epi tone is an etiological factor in some cases of cancer . tone refers to the rather steady , baseline output of the sns , rather than the phasic output of the system , where the latter is more closely associated with rapid changes in the system related to the fight or flight response . there appear to be genetic differences ( ie , polymorphisms ) in the ne component of the sns within different persons , such as for the alpha2c receptor subtype,26 which may affect cancer risk or outcome in various individuals . perhaps some individuals have genetically elevated ne and/or epi transmission , including increased steady release of ne and/or increased numbers or sensitivity of adrenoceptors receiving ne input , predisposing them to cancer in a variety of organs . for example , polymorphisms of the beta2 and beta3 adrenoceptor genes may be associated with breast cancer risk.27 a final point on genetics is that gene x environment ( ie , psychological stress ) interactions may play an important role in some types of cancer . in this scenario , an individual with genetically elevated sns ne tone , who is also exposed to significant psychological stress , may be especially susceptible to developing cancer , or have a worse outcome if he or she does develop cancer . the effects of ne / epi genetics , in combination with stress and other environmental inputs , on cancer risk or outcome is a topic that could greatly benefit from epidemiological investigation . work by felitti , anda , and colleagues has suggested that psychological stress or trauma during childhood is associated with later development of cancer.28,29 in one study , they found that psychologically adverse experiences during childhood , such as physical or sexual abuse , are associated with the development of cancer in adulthood.28 more recently , they suggested that adverse childhood experiences may be associated with later development of lung cancer , even after correcting for the effects of smoking.29 how might elevated release of ne and epi , or greater numbers or sensitivity of their receptors , actually cause various types of cancer ? one possibility is that by binding to adrenoceptors on the outside of cells of the body s organs , they activate intracellular molecular pathways already implicated in cancer . for example , there is growing evidence that ne and adrenoceptors interact with the p38/mapk,30 stat3,31 and pi3k / akt32 signaling pathways . if so , this may provide an additional link between extracellular signaling mechanisms and intracellular signaling pathways , such as p38/mapk , which are the focus of intensive study by molecular oncology researchers . ( epidermal growth factor is another example of an extracellular signaling molecule that affects cancer risk . ) as noted above in relation to sarkar et al19 elevated ne signaling by the sns may have deleterious effects on cellular immunity and inflammatory properties . related to this point , ne induces proliferation of rat cardiac fibroblasts , in part by increasing expression of the inflammation - modulating cytokine interleukin-6 , through regulation of mapk.33 it may be that chronically elevated ne signaling has negative effects on the immune system and inflammation , whereas acute increases in ne signaling are a healthy response to such challenges as infection , shock , or sepsis . an additional way that ne and epi may affect cancer is by modulating cellular oxidative damage . for example , a study on application of epi to human leukocytes showed that it produced damaging oxygen species , induced dna strand breakage , and such damage was antagonized by the antioxidant enzymes superoxide dismutase and catalase.34 beta blockers are not the only drugs that interfere with ne and epi signaling . prazosin blocks the alpha 1-adrenoceptor , and it continues to be studied in relation to benign prostatic hyperplasia.35 clonidine and guanfacine , which are alpha 2-adrenoceptor agonists used to treat hypertension , cause a general decrease in ne release , but they have barely been studied in relation to cancer , either preclinically or clinically . clonidine has been studied to some degree in relation to pheochromocytoma ; however , to my knowledge , these studies address its use as a test for the presence of this cancer , not its prevention . it would also be worthwhile to study , preclinically and clinically , the effects of cholinesterase inhibitor drugs , such as galantamine and donepezil , which boost levels of the molecule acetylcholine , as this molecule may oppose the effects of ne in the autonomic nervous system.12 that is , acetylcholine released by the parasympathetic nervous system may oppose the effects of ne released by the sns , thereby possibly having beneficial effects on cancer risk and outcome . it may be even more informative and therapeutic to combine beta blockers ( or clonidine ) with cholinesterase inhibitors , or beta blockers ( or clonidine ) with aspirin,36 to test for additive effects on cancer prevention and/or treatment . holmes et al36 reported beneficial effects from long - term use of aspirin on the survival of nurses who had previously been diagnosed with breast cancer . all of the above drugs have been approved for human use for many years in the united states and elsewhere , making epidemiological studies of them feasible . histone deacetylase inhibitor drugs , which may be useful in treating some cancers , also may achieve this effect , by increasing cellular expression of the ne transporter,37 which could reduce the extracellular concentration of ne . extension of porges polyvagal theory might suggest that brainstem regulation of the release of acetylcholine by the tenth cranial nerve may play a role in peripheral diseases such as cancer.38 long - term , rather than short - term , adrenoceptor - related drug treatment may be necessary to significantly reduce the probability of developing cancer , consistent with the notion that tonic , rather than phasic , release of ne and epi may be more important in carcinogenesis , as tonic output has more of a sustained presence on cells bearing adrenoceptors in various organs . the epidemiological beta blocker studies described at the outset of this review typically measured the effects of years of exposure to these drugs , rather than hours or days . regarding further testing of the ne / epi cancer hypothesis epidemiologically , it would be very informative to carry out prospective studies on the effects of the above drug treatments,39 as , to my knowledge , all of the studies published to date have been retrospective . however , additional retrospective studies also would be informative , and they are readily feasible . as we learn more about the genetics of the ne / epi system , it would be useful to study , in more breadth and detail , the potential associations between genetic polymorphisms and various types of cancer.27 a caveat regarding the use of beta blockers as a potential means for preventing or treating cancer is that at least some of these drugs may be associated with metabolic syndrome or type 2 diabetes mellitus.40 however , vasodilating beta blockers may have beneficial or neutral metabolic effects when compared with nonvasodilating beta blockers.40 this topic would benefit greatly from further investigation , comparing costs and benefits of using these drugs for cancer prevention or treatment . lastly , if beta blockers , as well as other adrenoceptor - based drugs discussed above , really do represent a new and relatively safe category of drugs that may not only prevent but also treat existing cases of various types of cancer , perhaps these drugs could also be considered as a last resort intervention in terminal cases where all conventional treatments have failed . the suggestion here is that beta blockers may actually improve survival in such cases , and not merely be palliative . while i have indicated above that long - term use of beta blockers may be necessary to prevent or treat various types of cancer , perhaps these drugs , to some degree , would start working immediately to reduce further progression of the disease . there is growing evidence that psychological stress is associated with increased cancer risk or poorer treatment outcome ( for a recent review , see sarkar et al19 ) . for example , three recent preclinical studies have shown that stress affects experimentally induced cancers . in one study , tumor growth transiently increased after stress was brought on by housing mice singly ; this process may be affected by transient changes in peripheral ne release.20 chronic stress was found to increase ovarian tumor growth in mice , which may have been in part mediated by ne - stimulated upregulation of the cytokine interleukin-8.21 in a mouse model of social stress , a number of signaling molecules , including ne and erk protein , increased or were upregulated , and tumor progression was prevented , when the intracellular molecule cyclic adenosine monophosphate was reduced by treatment with the inhibitory neurotransmitter gamma - aminobutyric acid.22 if psychological stress affects cancer onset or progression , one possibility is that stress - related increase in the release of ne and epi , and only these two molecules , is responsible for the deleterious effects on cancer risk and outcome . however , other molecules , such as neuropeptide y ( npy ) and cortisol also mediate the body s physiological stress response . a recent study found that npy receptors are present in the 4t1 breast cancer cell line , and that administration of npy to these cells promoted proliferation and migration.23 in addition , it has been shown that cortisol ( also known as hydrocortisone ) downregulates the expression of the breast cancer susceptibility gene brca1 in the nonmalignant mouse mammary cell line eph4 , where such downregulation has been associated with breast cancer development.24 as we come to better understand the genetics of stress - related molecules such as npy in humans , epidemiological investigation of the possible association between various alleles and cancer initiation or progression would be informative . as sarkar et al19 pointed out , the cellular and molecular mechanisms whereby ne and epi may carry out stress - related effects on carcinogenesis are beginning to be elucidated . they suggest that suppression of sns functioning ( via their beta - endorphin transplantation method , noted above ) can result in reduced tumorigenesis via increased peripheral natural killer cell and macrophage activities , elevated levels of anti - inflammatory cytokines , and reduced levels of inflammatory cytokines , as well as changes in tumor microenvironment . in other words , another point is that perhaps both the magnitude and the duration of exposure to psychological stress affect the probability of developing cancer . this is a topic that would benefit from further epidemiological retrospective investigation in persons who have been exposed to various types and duration of psychological stress . while psychological stress , which is partly mediated by the endogenous release of ne and epi , has already been associated with increased cancer risk in humans,25 it is also possible that genetically elevated ne / epi tone is an etiological factor in some cases of cancer . tone refers to the rather steady , baseline output of the sns , rather than the phasic output of the system , where the latter is more closely associated with rapid changes in the system related to the fight or flight response . there appear to be genetic differences ( ie , polymorphisms ) in the ne component of the sns within different persons , such as for the alpha2c receptor subtype,26 which may affect cancer risk or outcome in various individuals . perhaps some individuals have genetically elevated ne and/or epi transmission , including increased steady release of ne and/or increased numbers or sensitivity of adrenoceptors receiving ne input , predisposing them to cancer in a variety of organs . for example , polymorphisms of the beta2 and beta3 adrenoceptor genes may be associated with breast cancer risk.27 a final point on genetics is that gene x environment ( ie , psychological stress ) interactions may play an important role in some types of cancer . in this scenario , an individual with genetically elevated sns ne tone , who is also exposed to significant psychological stress , may be especially susceptible to developing cancer , or have a worse outcome if he or she does develop cancer . the effects of ne / epi genetics , in combination with stress and other environmental inputs , on cancer risk or outcome is a topic that could greatly benefit from epidemiological investigation . work by felitti , anda , and colleagues has suggested that psychological stress or trauma during childhood is associated with later development of cancer.28,29 in one study , they found that psychologically adverse experiences during childhood , such as physical or sexual abuse , are associated with the development of cancer in adulthood.28 more recently , they suggested that adverse childhood experiences may be associated with later development of lung cancer , even after correcting for the effects of smoking.29 how might elevated release of ne and epi , or greater numbers or sensitivity of their receptors , actually cause various types of cancer ? one possibility is that by binding to adrenoceptors on the outside of cells of the body s organs , they activate intracellular molecular pathways already implicated in cancer . for example , there is growing evidence that ne and adrenoceptors interact with the p38/mapk,30 stat3,31 and pi3k / akt32 signaling pathways . if so , this may provide an additional link between extracellular signaling mechanisms and intracellular signaling pathways , such as p38/mapk , which are the focus of intensive study by molecular oncology researchers . ( epidermal growth factor is another example of an extracellular signaling molecule that affects cancer risk . ) as noted above in relation to sarkar et al19 elevated ne signaling by the sns may have deleterious effects on cellular immunity and inflammatory properties . related to this point , ne induces proliferation of rat cardiac fibroblasts , in part by increasing expression of the inflammation - modulating cytokine interleukin-6 , through regulation of mapk.33 it may be that chronically elevated ne signaling has negative effects on the immune system and inflammation , whereas acute increases in ne signaling are a healthy response to such challenges as infection , shock , or sepsis . an additional way that ne and epi may affect cancer is by modulating cellular oxidative damage . for example , a study on application of epi to human leukocytes showed that it produced damaging oxygen species , induced dna strand breakage , and such damage was antagonized by the antioxidant enzymes superoxide dismutase and catalase.34 prazosin blocks the alpha 1-adrenoceptor , and it continues to be studied in relation to benign prostatic hyperplasia.35 clonidine and guanfacine , which are alpha 2-adrenoceptor agonists used to treat hypertension , cause a general decrease in ne release , but they have barely been studied in relation to cancer , either preclinically or clinically . clonidine has been studied to some degree in relation to pheochromocytoma ; however , to my knowledge , these studies address its use as a test for the presence of this cancer , not its prevention . it would also be worthwhile to study , preclinically and clinically , the effects of cholinesterase inhibitor drugs , such as galantamine and donepezil , which boost levels of the molecule acetylcholine , as this molecule may oppose the effects of ne in the autonomic nervous system.12 that is , acetylcholine released by the parasympathetic nervous system may oppose the effects of ne released by the sns , thereby possibly having beneficial effects on cancer risk and outcome . it may be even more informative and therapeutic to combine beta blockers ( or clonidine ) with cholinesterase inhibitors , or beta blockers ( or clonidine ) with aspirin,36 to test for additive effects on cancer prevention and/or treatment . holmes et al36 reported beneficial effects from long - term use of aspirin on the survival of nurses who had previously been diagnosed with breast cancer . all of the above drugs have been approved for human use for many years in the united states and elsewhere , making epidemiological studies of them feasible . histone deacetylase inhibitor drugs , which may be useful in treating some cancers , also may achieve this effect , by increasing cellular expression of the ne transporter,37 which could reduce the extracellular concentration of ne . extension of porges polyvagal theory might suggest that brainstem regulation of the release of acetylcholine by the tenth cranial nerve may play a role in peripheral diseases such as cancer.38 long - term , rather than short - term , adrenoceptor - related drug treatment may be necessary to significantly reduce the probability of developing cancer , consistent with the notion that tonic , rather than phasic , release of ne and epi may be more important in carcinogenesis , as tonic output has more of a sustained presence on cells bearing adrenoceptors in various organs . the epidemiological beta blocker studies described at the outset of this review typically measured the effects of years of exposure to these drugs , rather than hours or days . regarding further testing of the ne / epi cancer hypothesis epidemiologically , it would be very informative to carry out prospective studies on the effects of the above drug treatments,39 as , to my knowledge , all of the studies published to date have been retrospective . however , additional retrospective studies also would be informative , and they are readily feasible . as we learn more about the genetics of the ne / epi system , it would be useful to study , in more breadth and detail , the potential associations between genetic polymorphisms and various types of cancer.27 a caveat regarding the use of beta blockers as a potential means for preventing or treating cancer is that at least some of these drugs may be associated with metabolic syndrome or type 2 diabetes mellitus.40 however , vasodilating beta blockers may have beneficial or neutral metabolic effects when compared with nonvasodilating beta blockers.40 this topic would benefit greatly from further investigation , comparing costs and benefits of using these drugs for cancer prevention or treatment . lastly , if beta blockers , as well as other adrenoceptor - based drugs discussed above , really do represent a new and relatively safe category of drugs that may not only prevent but also treat existing cases of various types of cancer , perhaps these drugs could also be considered as a last resort intervention in terminal cases where all conventional treatments have failed . the suggestion here is that beta blockers may actually improve survival in such cases , and not merely be palliative . while i have indicated above that long - term use of beta blockers may be necessary to prevent or treat various types of cancer , perhaps these drugs , to some degree , would start working immediately to reduce further progression of the disease .

there is growing evidence that the neurotransmitter norepinephrine ( ne ) and its sister molecule epinephrine ( epi ) ( adrenaline ) affect some types of cancer . several recent epidemiological studies have shown that chronic use of beta blocking drugs ( which antagonize ne / epi receptors ) results in lower recurrence , progression , or mortality of breast cancer and malignant melanoma . preclinical studies have shown that manipulation of the levels or receptors of ne and epi with drugs affects experimentally induced cancers . psychological stress may play an etiological role in some cases of cancer ( which has been shown epidemiologically ) , and this could be partly mediated by ne and epi released by the sympathetic nervous system as part of the body s fight or flight response . a less well - appreciated phenomenon is that the genetic tone of ne / epi may play a role in cancer . ne and epi may affect cancer by interacting with molecular pathways already implicated in abnormal cellular replication , such as the p38/mapk pathway , or via oxidative stress . ne / epi - based drugs other than beta blockers also may prevent or treat various types of cancer , as may cholinesterase inhibitors that inhibit the sympathetic nervous system , which could be tested epidemiologically .

Clinical and preclinical findings Psychological stress and genetics Etiological mechanisms Drug studies and treatments

within the last two years , a number of retrospective epidemiological studies have found that chronic use of beta - blocking drugs is associated with lower recurrence and mortality of breast cancer,1,2 or reduced progression and mortality of breast cancer3,4 and malignant melanoma.5,6 three earlier studies also suggested beneficial effects from prolonged use of beta blockers on general cancer risk7 or on prostate cancer risk.8,9 collectively , these findings are both provocative and encouraging . they are provocative because beta blockers , which have been used clinically for decades worldwide , are typically taken to treat heart - related ailments , such as arrhythmias and hypertension , not cancer . they are encouraging because beta blockers may represent a new , and relatively safe , category of drugs for the prevention and possible treatment of a range of cancers , while also shedding light on the pathophysiological basis of some types of cancer . a few years ago , i put forth the hypothesis that the signaling molecule norepinephrine ( ne ) is an etiological factor in some types of cancer.10,11 in support of this hypothesis i cited seven lines of evidence : ( i ) rodent studies of tumorigenesis in the context of ne manipulation , ( ii ) human studies of tricyclic antidepressant use and cancer rate , ( iii ) existence of pheochromocytoma , a cancer of the adrenal glands , ( iv ) cancer rates in families with individuals who have bipolar disorder , ( v ) hypertension and cancer risk , ( vi ) excessive body weight and cancer risk , and ( vii ) psychological stressors and cancer risk . these lines of evidence tend to show that increased ne release in the body , or increased numbers or sensitivity of ne receptors , is associated with increased occurrence of cancer in various organs . norepinephrine , and the related molecule epinephrine ( epi ) , also known as adrenaline , are stress hormones that activate the body s adrenoceptors , which includes the receptors that beta blockers antagonize . there are five major types of adrenoceptors in the body : beta1 , beta2 , beta3 , alpha1 , and alpha2;10 most beta blockers target the beta1 and beta2 receptors . adrenoceptors are found not only in the brain , but also in nearly all , if not all , organs of the body.10 ne and epi have direct access to these organs through localized release by the sympathetic nervous system ( sns ) , as well as through general release into the bloodstream , as part of the body s fight or flight response to psychological stressors . by binding to adrenoceptors in various organs , ne and epi may modulate various intracellular molecular pathways already implicated in cancer ( see below ) . there is a growing preclinical literature suggesting that ne and epi may be etiological factors in cancer . for example , sarkar et al12 have recently shown that functional balance of the sympathetic and parasympathetic branches of the autonomic nervous system may be important in rodent experimentally induced cancers , where pharmacologically increasing sympathetic ne output or blocking cholinergic parasympathetic output tended to promote cancer in their rat model . this and other studies add to data that began several decades ago , showing an effect of ne manipulation on rodent experimentally induced carcinogenesis.13,14 these data also suggest that it would be informative to epidemiologically test the effects of drugs that act on the autonomic nervous system on risk for or outcome of various types of cancer ( see below ) . it should be noted that some recent clinical studies have found that the use of beta blockers , as well as other ne transmission decreasing drugs , is associated with increased cancer risk and poorer survival . for example , a recent epidemiological study found that chronic beta blocker use and use of other ne decreasing drugs is associated with a slightly increased ( odds ratios approximately 1.051.1 ) risk of various types of cancer combined , including colon , lung , breast , and prostate cancer.15 another study found that longterm use ( 6 or more years ) of beta blockers is associated with significantly increased risk ( odds ratio 2.02 ) of stage iv colorectal cancer.16 a recent study of a united kingdom database comparing cancer patients receiving beta blockers with those receiving other antihypertensive medications found slightly poorer survival rates ( hazard ratio approximately 1.2 ) in those on beta blockers ; this outcome appeared to be limited to pancreatic and prostate cancer.17 one interpretation of the data from these three studies is that persons who eventually take beta - blocking medications chronically for hypertension or other cardiac - related abnormalities have elevated endogenous ( possibly genetic ) ne signaling , predisposing them to various types of cancer and/or poorer cancer outcome . in this scenario , beta - blocking drugs , at least in the manner in which they are currently used , may have a smaller effect on cancer risk or outcome than the underlying elevated ne signaling.18 in general , caution should be exercised in inferring from human drug studies that ne plays an etiological role in some cancers . clinical epidemiological studies tend to be correlative and do not necessarily indicate that the factor in question , such as ne or a noradrenergic drug , is causative in a disease process . another point to consider is that different beta - blocking drugs , which vary in their specificity for different beta adrenoceptors , such as beta1 and beta2 , may differentially affect cancer outcome in both preclinical and clinical settings.18 one beta blocker that has been used in a number of preclinical oncology studies , propranolol , blocks both beta1 and beta2 receptors . there is growing evidence that psychological stress is associated with increased cancer risk or poorer treatment outcome ( for a recent review , see sarkar et al19 ) . for example , three recent preclinical studies have shown that stress affects experimentally induced cancers . in one study , tumor growth transiently increased after stress was brought on by housing mice singly ; this process may be affected by transient changes in peripheral ne release.20 chronic stress was found to increase ovarian tumor growth in mice , which may have been in part mediated by ne - stimulated upregulation of the cytokine interleukin-8.21 in a mouse model of social stress , a number of signaling molecules , including ne and erk protein , increased or were upregulated , and tumor progression was prevented , when the intracellular molecule cyclic adenosine monophosphate was reduced by treatment with the inhibitory neurotransmitter gamma - aminobutyric acid.22 if psychological stress affects cancer onset or progression , one possibility is that stress - related increase in the release of ne and epi , and only these two molecules , is responsible for the deleterious effects on cancer risk and outcome . however , other molecules , such as neuropeptide y ( npy ) and cortisol also mediate the body s physiological stress response . a recent study found that npy receptors are present in the 4t1 breast cancer cell line , and that administration of npy to these cells promoted proliferation and migration.23 in addition , it has been shown that cortisol ( also known as hydrocortisone ) downregulates the expression of the breast cancer susceptibility gene brca1 in the nonmalignant mouse mammary cell line eph4 , where such downregulation has been associated with breast cancer development.24 as we come to better understand the genetics of stress - related molecules such as npy in humans , epidemiological investigation of the possible association between various alleles and cancer initiation or progression would be informative . while psychological stress , which is partly mediated by the endogenous release of ne and epi , has already been associated with increased cancer risk in humans,25 it is also possible that genetically elevated ne / epi tone is an etiological factor in some cases of cancer . tone refers to the rather steady , baseline output of the sns , rather than the phasic output of the system , where the latter is more closely associated with rapid changes in the system related to the fight or flight response . there appear to be genetic differences ( ie , polymorphisms ) in the ne component of the sns within different persons , such as for the alpha2c receptor subtype,26 which may affect cancer risk or outcome in various individuals . for example , polymorphisms of the beta2 and beta3 adrenoceptor genes may be associated with breast cancer risk.27 a final point on genetics is that gene x environment ( ie , psychological stress ) interactions may play an important role in some types of cancer . work by felitti , anda , and colleagues has suggested that psychological stress or trauma during childhood is associated with later development of cancer.28,29 in one study , they found that psychologically adverse experiences during childhood , such as physical or sexual abuse , are associated with the development of cancer in adulthood.28 more recently , they suggested that adverse childhood experiences may be associated with later development of lung cancer , even after correcting for the effects of smoking.29 how might elevated release of ne and epi , or greater numbers or sensitivity of their receptors , actually cause various types of cancer ? one possibility is that by binding to adrenoceptors on the outside of cells of the body s organs , they activate intracellular molecular pathways already implicated in cancer . for example , there is growing evidence that ne and adrenoceptors interact with the p38/mapk,30 stat3,31 and pi3k / akt32 signaling pathways . an additional way that ne and epi may affect cancer is by modulating cellular oxidative damage . for example , a study on application of epi to human leukocytes showed that it produced damaging oxygen species , induced dna strand breakage , and such damage was antagonized by the antioxidant enzymes superoxide dismutase and catalase.34 beta blockers are not the only drugs that interfere with ne and epi signaling . prazosin blocks the alpha 1-adrenoceptor , and it continues to be studied in relation to benign prostatic hyperplasia.35 clonidine and guanfacine , which are alpha 2-adrenoceptor agonists used to treat hypertension , cause a general decrease in ne release , but they have barely been studied in relation to cancer , either preclinically or clinically . it would also be worthwhile to study , preclinically and clinically , the effects of cholinesterase inhibitor drugs , such as galantamine and donepezil , which boost levels of the molecule acetylcholine , as this molecule may oppose the effects of ne in the autonomic nervous system.12 that is , acetylcholine released by the parasympathetic nervous system may oppose the effects of ne released by the sns , thereby possibly having beneficial effects on cancer risk and outcome . histone deacetylase inhibitor drugs , which may be useful in treating some cancers , also may achieve this effect , by increasing cellular expression of the ne transporter,37 which could reduce the extracellular concentration of ne . extension of porges polyvagal theory might suggest that brainstem regulation of the release of acetylcholine by the tenth cranial nerve may play a role in peripheral diseases such as cancer.38 long - term , rather than short - term , adrenoceptor - related drug treatment may be necessary to significantly reduce the probability of developing cancer , consistent with the notion that tonic , rather than phasic , release of ne and epi may be more important in carcinogenesis , as tonic output has more of a sustained presence on cells bearing adrenoceptors in various organs . as we learn more about the genetics of the ne / epi system , it would be useful to study , in more breadth and detail , the potential associations between genetic polymorphisms and various types of cancer.27 a caveat regarding the use of beta blockers as a potential means for preventing or treating cancer is that at least some of these drugs may be associated with metabolic syndrome or type 2 diabetes mellitus.40 however , vasodilating beta blockers may have beneficial or neutral metabolic effects when compared with nonvasodilating beta blockers.40 this topic would benefit greatly from further investigation , comparing costs and benefits of using these drugs for cancer prevention or treatment . lastly , if beta blockers , as well as other adrenoceptor - based drugs discussed above , really do represent a new and relatively safe category of drugs that may not only prevent but also treat existing cases of various types of cancer , perhaps these drugs could also be considered as a last resort intervention in terminal cases where all conventional treatments have failed . while i have indicated above that long - term use of beta blockers may be necessary to prevent or treat various types of cancer , perhaps these drugs , to some degree , would start working immediately to reduce further progression of the disease . there is growing evidence that psychological stress is associated with increased cancer risk or poorer treatment outcome ( for a recent review , see sarkar et al19 ) . for example , three recent preclinical studies have shown that stress affects experimentally induced cancers . in one study , tumor growth transiently increased after stress was brought on by housing mice singly ; this process may be affected by transient changes in peripheral ne release.20 chronic stress was found to increase ovarian tumor growth in mice , which may have been in part mediated by ne - stimulated upregulation of the cytokine interleukin-8.21 in a mouse model of social stress , a number of signaling molecules , including ne and erk protein , increased or were upregulated , and tumor progression was prevented , when the intracellular molecule cyclic adenosine monophosphate was reduced by treatment with the inhibitory neurotransmitter gamma - aminobutyric acid.22 if psychological stress affects cancer onset or progression , one possibility is that stress - related increase in the release of ne and epi , and only these two molecules , is responsible for the deleterious effects on cancer risk and outcome . however , other molecules , such as neuropeptide y ( npy ) and cortisol also mediate the body s physiological stress response . a recent study found that npy receptors are present in the 4t1 breast cancer cell line , and that administration of npy to these cells promoted proliferation and migration.23 in addition , it has been shown that cortisol ( also known as hydrocortisone ) downregulates the expression of the breast cancer susceptibility gene brca1 in the nonmalignant mouse mammary cell line eph4 , where such downregulation has been associated with breast cancer development.24 as we come to better understand the genetics of stress - related molecules such as npy in humans , epidemiological investigation of the possible association between various alleles and cancer initiation or progression would be informative . while psychological stress , which is partly mediated by the endogenous release of ne and epi , has already been associated with increased cancer risk in humans,25 it is also possible that genetically elevated ne / epi tone is an etiological factor in some cases of cancer . tone refers to the rather steady , baseline output of the sns , rather than the phasic output of the system , where the latter is more closely associated with rapid changes in the system related to the fight or flight response . there appear to be genetic differences ( ie , polymorphisms ) in the ne component of the sns within different persons , such as for the alpha2c receptor subtype,26 which may affect cancer risk or outcome in various individuals . for example , polymorphisms of the beta2 and beta3 adrenoceptor genes may be associated with breast cancer risk.27 a final point on genetics is that gene x environment ( ie , psychological stress ) interactions may play an important role in some types of cancer . the effects of ne / epi genetics , in combination with stress and other environmental inputs , on cancer risk or outcome is a topic that could greatly benefit from epidemiological investigation . work by felitti , anda , and colleagues has suggested that psychological stress or trauma during childhood is associated with later development of cancer.28,29 in one study , they found that psychologically adverse experiences during childhood , such as physical or sexual abuse , are associated with the development of cancer in adulthood.28 more recently , they suggested that adverse childhood experiences may be associated with later development of lung cancer , even after correcting for the effects of smoking.29 how might elevated release of ne and epi , or greater numbers or sensitivity of their receptors , actually cause various types of cancer ? one possibility is that by binding to adrenoceptors on the outside of cells of the body s organs , they activate intracellular molecular pathways already implicated in cancer . for example , there is growing evidence that ne and adrenoceptors interact with the p38/mapk,30 stat3,31 and pi3k / akt32 signaling pathways . an additional way that ne and epi may affect cancer is by modulating cellular oxidative damage . for example , a study on application of epi to human leukocytes showed that it produced damaging oxygen species , induced dna strand breakage , and such damage was antagonized by the antioxidant enzymes superoxide dismutase and catalase.34 prazosin blocks the alpha 1-adrenoceptor , and it continues to be studied in relation to benign prostatic hyperplasia.35 clonidine and guanfacine , which are alpha 2-adrenoceptor agonists used to treat hypertension , cause a general decrease in ne release , but they have barely been studied in relation to cancer , either preclinically or clinically . it would also be worthwhile to study , preclinically and clinically , the effects of cholinesterase inhibitor drugs , such as galantamine and donepezil , which boost levels of the molecule acetylcholine , as this molecule may oppose the effects of ne in the autonomic nervous system.12 that is , acetylcholine released by the parasympathetic nervous system may oppose the effects of ne released by the sns , thereby possibly having beneficial effects on cancer risk and outcome . histone deacetylase inhibitor drugs , which may be useful in treating some cancers , also may achieve this effect , by increasing cellular expression of the ne transporter,37 which could reduce the extracellular concentration of ne . extension of porges polyvagal theory might suggest that brainstem regulation of the release of acetylcholine by the tenth cranial nerve may play a role in peripheral diseases such as cancer.38 long - term , rather than short - term , adrenoceptor - related drug treatment may be necessary to significantly reduce the probability of developing cancer , consistent with the notion that tonic , rather than phasic , release of ne and epi may be more important in carcinogenesis , as tonic output has more of a sustained presence on cells bearing adrenoceptors in various organs . as we learn more about the genetics of the ne / epi system , it would be useful to study , in more breadth and detail , the potential associations between genetic polymorphisms and various types of cancer.27 a caveat regarding the use of beta blockers as a potential means for preventing or treating cancer is that at least some of these drugs may be associated with metabolic syndrome or type 2 diabetes mellitus.40 however , vasodilating beta blockers may have beneficial or neutral metabolic effects when compared with nonvasodilating beta blockers.40 this topic would benefit greatly from further investigation , comparing costs and benefits of using these drugs for cancer prevention or treatment . lastly , if beta blockers , as well as other adrenoceptor - based drugs discussed above , really do represent a new and relatively safe category of drugs that may not only prevent but also treat existing cases of various types of cancer , perhaps these drugs could also be considered as a last resort intervention in terminal cases where all conventional treatments have failed . while i have indicated above that long - term use of beta blockers may be necessary to prevent or treat various types of cancer , perhaps these drugs , to some degree , would start working immediately to reduce further progression of the disease .

the seminal discovery of adult brain plasticity in animals and humans has hugely influenced the theories and concepts applied in neurorehabilitation research and their translation into practice , in particular with regards to movement deficits in acquired brain injury [ 1 , 2 ] . this work directly translated into new and , in some cases , more efficient interventions for patients with mild to moderate hemiparesis ( e.g. , [ 35 ] ) . however , much less research has specifically investigated the reorganization of the motor system in patients with poor or minimal functional abilities and , most critically , the rehabilitation of these patients [ 6 , 7 ] . firstly , standard rehabilitation approaches are difficult to apply when patients have little voluntary movement . secondly , the lack of funding for regular one - to - one physical therapy sessions beyond the postacute phase , together with the common assumption that substantial improvements in functional motor ability are unlikely once the first 6 months of recovery have passed , primes the health care system and patients to accept the status quo . while the latter is true for the whole range of functional recovery , the impact is particularly grave for patients with poor functional recovery and also affects the research effort . thirdly , the motor system of patients with poor residual recovery can not easily be studied with the paradigms adopted from basic science research , such as finger opposition or grip movements . these methodological challenges can be overcome by excluding patients with high levels of spasm or by limiting the study group to those with relatively good levels of motor control . as a result , the motor system of patients with very poor recovery , in particular in chronic state , has been studied much less than that of patients with mild or moderate impairment . this translates directly into studies on treatment efficacy , and indeed , the availability of suitable treatments per se . based on the aforementioned considerations we argue that research on the mechanisms of long - term recovery , and their interaction with treatment efficacy , needs to widen its focus to the population of stroke survivors with severe long - term motor deficits . hereinafter we briefly summarise the present literature and further discuss the challenges involved in the study and treatment of patients with minimal motor recovery . upper limb paresis occurs in 85% of the patients and substantially impacts disability in the long term . animal models have tried to reproduce stroke lesions that occur in humans , with variable degrees of success . the majority of focal ischemia models involve the middle cerebral artery ( mca ) territory , the most commonly affected arterial territory in ischemic strokes in humans . rat focal ischemia models are frequently used because of low cost , similarities between vasculatures of rats and humans , fewer concerns from the general public compared to nonrodents , and availability of clear behavioral outcomes [ 1113 ] . severe mca infarcts in rodents leading to long - lasting sensorimotor and cognitive deficits can be produced by proximal mca occlusion induced by electrocoagulation . complete or partial mca occlusion can be also achieved by insertion of an intraluminal filament . in the filament model for example , if endothelin-1 , a vasoconstrictor drug , is injected topically or intracerebrally , the forelimb motor cortex is typically spared , but infarcts vary in location and extension depending on the sites and route of drug administration . in models of occlusion of the distal mca or its branches , cortical frontoparietal infarcts are associated with less severe deficits than in the cortico - subcortical infarcts produced by proximal mca occlusion . furthermore , multiple embolic infarcts can be produced in brain areas supplied by the mca , after injection of microspheres , macrospheres , a thrombotic clot , or purified thrombin into the internal carotid artery [ 19 , 20 ] . finally , light activation of photosensitive dyes such as rose bengal makes small cortical infarcts possible , by occlusion of cortical vessels . many of these techniques have not been applied exclusively to small animals such as rats , mice , gerbils , or rabbits , but also to cats , dogs , pigs , and monkeys . larger animals have proportions of gray / white matter that are more similar to those found in human brains , in contrast with lissencephalic brains of rats and mice . however , technical limitations and costs limit widespread use of focal ischemia models in larger animals . it is recommended , for instance , that new drugs be tested first in rodents , before efficacy is investigated in gyrencephalic species . they offer powerful opportunities : the possibility to objectively monitor behavioral outcomes , manipulate experimental conditions , and obtain images ( mri , micropet ) or intracortical recordings of neuronal activity in living animals ; to scrutinize molecular mechanisms of cell death and recovery ; to work with strains and transgenic animals that present hypertension , atherosclerosis and obesity , among other factors that are common in patients with stroke ; and to perform postmortem histological evaluations . still , conclusions based on animal models of focal ischemia must be examined with caution . for instance , small , selective cortical infarcts leading to mild sensorimotor deficits that tend to improve quickly are present in some of the models but are not common in humans , even though similar clinical features can occur in subcortical , lacunar infarcts . in addition , background pathophysiology is not shared between focal cerebral ischemia in rodents and humans . fast arterial recanalization is achieved in several animal models , while in humans recanalization can occur either after r - tpa administration or spontaneously but , unfortunately , does not happen at an early phase after ischemic injury in most patients [ 8 , 2427 ] . lack of arterial recanalization is strongly associated with more severe strokes and lower probabilities of recovery . furthermore , rodent models have often included young healthy animals while , in humans , the bulk of strokes is concentrated in the elderly [ 2832 ] . age is a potent predictor of poor outcome in humans [ 33 , 34 ] . interestingly , despite high mortality rates in old mice , levels of recovery at four weeks have been reported to be similar in aged and young animals . other factors may contribute to poor outcome in humans , ranging from different mechanisms underlying strokes in different age groups to self - fulfilling prophecies in stroke care in the aged , and importantly , to comorbidities that impact recovery . diabetes mellitus and atrial fibrillation , for instance , are significantly associated with more severe outcomes and lower chances of recovery [ 36 , 37 ] . hypertensive rats have poorer collateral blow flow and also worse outcomes after focal ischemia than nonhypertensive animals . the demand for stroke models in hypertensive , obese , and aged rodents has been underlined . contrasts between the compelling efficacy of a myriad of drugs in animal models of neuroprotection and the systematic failure of the same drugs when administered to patients in clinical trials underscore the requirement for animal models that more realistically approach human strokes [ 13 , 22 , 39 ] . however , bigger rates of complications and mortality in aged or unhealthy animals , technical difficulties ( anesthesia , complex surgeries in less flexible arteries , etc . ) , and hence the greater costs involved present challenges for advancements of research in the field . in particular , high mortality rates limit the opportunity to study recovery of animals with severe strokes in the chronic phase . in summary , there is a gap between pathogenesis and clinical features of severe strokes in humans and pathogenesis and clinical features of the most widely used models of focal ischemia in animals . high costs and lower expectations for substantial improvements in activity or quality of life are also major obstacles for rehabilitation of patients with severe strokes . still , as stroke mortality decreases in parallel with advances in health care , it is expected that more patients with severe strokes will survive the acute phase over the next decades . at the moment , thus , animal models of focal ischemia that match severe human strokes more closely are deeply needed . despite these limitations , animal models have provided unique insight on plastic mechanisms underlying sensorimotor recovery after focal ischemia , and on how to enhance beneficial patterns of reorganization to obtain behavioral gains ( e.g. , ) . constraint - induced movement therapy , for instance , shown to improve motor outcomes in humans with different types and sizes of strokes , was developed based on seminal studies that underscored the importance of the amount of use of the paretic limb to promote enhancement of motor function [ 41 , 42 ] . the phenomenon was observed in monkeys with small cortical infarcts submitted to intracortical microstimulation and behavioral testing . as mentioned before , such small infarcts are rarely observed in humans , but still , the information gained by the model was a major step forward in stroke rehabilitation . constraint - induced therapy has now been successfully applied to patients with various types of strokes , with more severe deficits and worse motor prognoses than the monkeys included in the model of forced use of the affected limb [ 1 , 3 , 43 , 44 ] . over the past decades , neuroimaging and neurophysiology techniques have emerged as paramount tools to study brain reorganization in humans [ 4550 ] . overall , functional neuroimaging studies in patients with stroke have shown that good performance is associated with augmented activity in preexisting networks during a motor task , rather than assignment of networks that are not overtly active during these tasks in healthy subjects . furthermore for instance , according to the model of interhemispheric inhibition , an imbalance in activity between the ipsilesional and the contralesional primary motor cortex ( m1 ) can occur after stroke [ 51 , 52 ] a number of studies have shown that this imbalance in activity between the two hemispheres can impair motor performance of the affected hand , at least in some patients in the chronic phase after stroke [ 51 , 52 ] . whether interhemispheric callosal fibers mediate this phenomenon or whether it depends on changes in activity of cerebellar or thalamic pathways , remains an open question . however , the vast majority of published studies that investigated effects of modulation of interhemispheric inhibition only included patients with mild to moderate motor impairments , likely due to difficulties in developing tasks and applying available neurophysiology and neuroimaging tools to more severely affected patients [ 5159 ] . in regard to recruitment of ipsilesional or contralesional secondary motor areas , striking differences in patterns of function were unveiled when paradigms of investigation were applied to patients with poor recovery , compared to those of less affected patients or healthy subjects . recruitment of secondary motor areas occurs when the outflow from m1 is disconnected from the spinal cord in large cortical , cortico - subcortical or subcortical strokes , as well as in strokes that strategically damage the corticospinal tract [ 60 , 61 ] . at least in part , more pronounced patterns of activity in secondary motor areas in the ipsilesional and/or contralesional areas are associated with more severe disruption of the corticospinal pathway , and excessive activation of secondary motor areas correlates with poorer motor behavior [ 48 , 50 , 62 ] . whether excessive activations of secondary motor areas or contralesional m1 are maladaptive , whether they represent the best possible remodeling option after severe injury , or whether they may play hero or villain roles depending on motor tasks / circumstances remains to be determined . there are indications that motor performance may actually rely on activity of contralesional areas in more severely affected patients . for instance , it has been shown that transient disruption of the contralesional dorsal premotor cortex by transcranial magnetic stimulation slows motor performance of the paretic hand to a greater extent in patients with worse motor performance compared to less impaired patients . in addition , activation of the contralesional dorsal premotor cortex has been demonstrated to be greater in patients with more severe hand motor impairment , compared to healthy subjects and to less affected patients . on the other hand , disruption of the ipsilesional dorsal premotor together , these studies provide evidence that ( 1 ) contralesional areas are positively , functionally relevant in at least some well - recovered patients in the chronic phase ; ( 2 ) in patients with severe motor deficits , behavioral gains , albeit small , may occur by augmented activity of networks that are normally either minimally active or not active at all in healthy brains . severity of motor impairment seems to be a key factor influencing patterns of rewiring after stroke , but age , brain status before stroke , intensity , and timing of rehabilitative interventions , among other factors , are also likely to play pivotal roles in the process [ 33 , 34 , 6567 ] . a key concept to develop effective rehabilitation interventions is heterogeneity of mechanisms underlying stroke as well as plastic processes that lead to recovery of function after neuronal injury . mechanisms underlying neurological impairment , recovery of activity , and participation are also distinct . until now , most proof - of - principle studies or clinical trials have excluded patients with severe sensorimotor impairments and the lack of evidence - based effective interventions has nurtured a nihilistic approach for rehabilitation of these patients . moreover , patients with severe sensorimotor impairments often present with cognitive impairments , depression and are faced with massive changes in psychosocial interactions [ 6870 ] . further research efforts must not only address restoration of sensorimotor function but also incorporate an integrative approach to target neuropsychiatric domains , personal experience / expectations , environmental conditions , and psychosocial factors . changes to the functional organization of neural representations and their behavioral concomitants have been described for a number of human study models such as amputates ( e.g. , [ 71 , 72 ] , musicians [ 73 , 74 ] , blind ( e.g. , [ 7578 ] ) and deaf persons ( e.g. , [ 79 , 80 ] ) , as well as learning paradigms ( e.g. , [ 8184 ] ) . together these studies suggest that sensory representations are sensitive to enhanced or altered sensory stimulation . this knowledge has the prospect to devise interventions that capitalize on the plastic capacities of the adult brain , mainly training- or practice - based interventions . the mechanisms of brain plasticity interact with psychological processes and behavior and together provide a number of considerations for the conceptualization of interventions . injury to the peripheral or central nervous system changes receptive field characteristics of neurons and neural representations through deprivation of the original afferent inputs these neurons receive [ 85 , 86 ] . the key mechanisms driving this change are the disinhibition of silent synapses , and the loss of inhibitory or excitatory inputs from lesioned neural populations connected to none - lesioned regions , including homologous areas in the two hemispheres . in both cases , it is important to appreciate the effects of functional changes in nonlesioned areas and their influence on the control of the impaired behavior . as a consequence , rehabilitation efforts should not only focus on the impaired function per se , for example , the affected upper - limb in the case of hemiplegia , but also consider the effects of use or nonuse of the lesser - affected extremities . this is particularly relevant for patients with poor recovery as they will most likely entirely rely on the less - affected extremity in everyday behavior . this might , at least in theory , increase the interhemispheric inhibition exerted by the nonlesioned hemisphere . sensory stimulation and these changes are most likely driven through hebbian mechanisms as well as dendritic and axonal processes . critically , use - related reorganization is not driven by increased neural activation alone but heavily depends on the behavioral relevance of the activity [ 9092 ] . this association of sensory stimulation and consequential changes in neural representations and receptive field parameters has been demonstrated most elegantly in monkeys who received auditory and tactile simulation within the same protocol . behaviorally , the animals were only rewarded for responses in one of the two stimulation modalities , and subsequent changes in the brain were only observed for the rewarded modality . thus this data indicates that neural representations are only susceptible to stimulation - induced reconfigurations if this stimulation is attended to and of behavioral significance . therefore , we argue that interventions aiming to enhance recovery through the induction of practice - induced plasticity not only need to focus on the actual practice element of the intervention but also consider how the intervention characteristics enforce attention and provide tangible and motivating feedback . motor control is a complex behavior that goes far beyond motor execution and the processes typically associated with primary motor cortex function . cognitive processes such as motor planning , error - monitoring and attention can heavily influence motor performance . motor rehabilitation research , however , typically conceptualizes motor function as the ability to execute a movement with little consideration being given to the cognitive processes that might influence this ability . for example , few studies have investigated how much patients with hemiplegia benefit from advanced movement preparation . the respective behavioral costs or benefits result from perceptual , cognitive , and motoric components of the stimulus - response cascade , such as stimulus - response mapping and response selection . few studies have investigated advanced movement preparation in patients . a study by verleger et al . suggests that well - recovered patients show little difference to controls in a motor priming task . using a similar paradigm in patients with poor recovery , our group found marked behavioural and electrophysiological difference between patients and matched control . most strikingly , the data suggests that patients are more sensitive to advance information ( manuscript submitted ) . thus , it appears that visual precues can facilitate or hinder apparent affected arm abilities , and that the magnitude of this effect is modulated by the severity of the motor deficit . while more research is needed to fully understand the interaction of cognitive processes , stroke severity , and motor performance , the findings summarised previously suggest that using advance movement information in a cognizant and explicit manner may be a beneficial addition to rehabilitation interventions . in order to translate improvements in motor ability into real - world benefits , treatments have to obtain not only better motor control over the affected arm , but also a transfer of these newly acquired abilities into the curriculum of everyday behaviors . behavior change can be facilitated through a number of measures falling under the cbt ( cognitive behavioral therapy ) umbrella . these measures use not only learning principles , which in themselves are likely to facilitate use - related plasticity processes , but also tools that enhance motivation , adherence , and coping . the latter , again , are likely to increase the patient 's engagement with the intervention , which in turn is likely to improve outcome . conceptualizing the rehabilitation of motor function as changing motor behavior rather than improving motor ability represents a significant shift in theoretical perspective . it implies that practice - based interventions should include treatment strategies that actively support sustained learning and behavior change , through the explicit use of learning principles and cbt elements . the latter facilitates not only real - world benefit but also enhances the processes of use - related functional reorganization that drive improvements in motor control . interview data ( unpublished ) from patients with severe chronic hemiplegia indicates that these patients experience disproportionate psychological and service - related barriers . i hate it ( the hand ) and want to make it invisible , and it ( the hand ) looks horrible and is no good ; i 'd cut it off if i could suggest that patients do not only face the actual physical impairment but also face psychological barriers to using any residual ability . this aspect is particularly important for interventions that rely on motor practice because the patient 's engagement with the intervention is directly linked to their ability to cope with their disability . however , more often than not physiotherapy is provided without considering the psychological barriers associated with the use of the hemiplegic hand . we therefore argue that in order to improve the prospects for patients with low - functioning hemiparesis it is necessary to better understand the psychology of poor recovery and build that knowledge into motor rehabilitation interventions . in addition to the psychological barriers low - functioning patients seem to experience , our interview data suggests a strong perception amongst these patients that the care system is presumptuous and does not provide for them . this is illustrated by comments such as my doctors gave up on me after 3 month , my physio tried to work with my hand initially but soon gave up , the ot only taught me how to manage things ( with my good hand ) , i guess this was because she knew that my ( paretic ) hand would be no good , and there is nothing they can do for me because i can not move . these comments highlight not only the need to understand the patient perspective and a holistic approach to long - term care of these patients , but also a critical need to develop motor rehabilitation treatments that help patients to enhance their residual motor ability and enhance its real world benefit . this affects not only the patients ' general levels of activity but , of course , also the level of engagement with the therapy process . not a lot can be gained in a therapy session with a tired patient ! moreover , tiredness and fatigue are linked to sleep , and sleep is likely to be a modulating factor of recovery . moreover , patients with chronic low - functioning hemiparesis seem to suffer from sleep difficulties at least as frequently as the general population . it is therefore likely to further aggravate the difficulties patients already have with activities of daily living and to reduce the benefit patients can get from therapy and other activities . finally , an increasing body of literature suggests that sleep enhances brain plasticity in general and procedural learning specifically ( e.g. , [ 97100 ] ) . assuming that brain plasticity is the main driver for the recovery of function , good and sufficient sleep is likely to facilitate and probably enhance the rehabilitation effort . support for this assumption is provided by a series of studies suggesting that motor learning can be positively influenced by sleep [ 101 , 102 ] . the issues raised previously are relevant to all patients but probably have greater significance in patients with poorer recovery . therefore , treatments for these patients in particular should incorporate measures to counter tiredness and fatigue and monitor the quality of nocturnal sleep as well as daytime sleepiness . jeannerod 's theory of neural simulation suggests a shared neural network for the control of overt and covert movement modalities such as movement observation and motor imagery . this theoretical framework provides a powerful tool for research in patients with poor recovery . generally , evidence from behavioral , neuroimaging , and psychophysiological studies confirms jeannerod 's notion of equivalence in healthy populations . for example , several fmri studies have shown comparable activations in the cortical motor regions when participants observe or imagine hand movements ( e.g. , [ 104107 ] ) . thereby , these activities are similar to the activations obtained when the same movements are actually performed in the scanner . typically , these studies use tasks that can be practiced prior to scanning and can be performed in the scanner . while this approach yields interesting and important insights , it lacks ecological validity , particularly with regard to the rehabilitation context . taking these considerations on board , szameitat and colleagues [ 108 , 109 ] studied motor imagery of complex actions , such as eating with knife and fork or running . using an fmri paradigm , they successfully demonstrated that imagery of such complex movements is feasible in the scanner environment and leads to meaningful activations in the motor system . these findings are not trivial because complex everyday actions can not be practiced prior to the scanning . the person will therefore rely on their motor memory rather than the experience obtained through practicing the actual task ( e.g. , finger tapping ) immediately before the scanning begins . in this sense , the imagery of complex movements , by default , affords less stringent experiment control . however , if motor imagery is to be used as a study tool for patients with poor recovery , their inability to move will prevent practice prior to scanning , and therefore , these patients will also perform the task by relying on their motor memory . evidence showing the feasibility and suitability of a paradigm that omits practice is therefore particularly relevant for the application in patients with poor residual recovery . moreover , the study of complex actions with fmri opens the door to the investigation of the representation of activities of daily living and the alteration of those representations throughout recovery and/or treatment . as mentioned previously , the cognitive processes involved in advance movement preparation are likely to play an important part in the person 's ability to function in everyday life but might also enhance or hinder the rehabilitation effort . understanding to what extend the principle of equivalence also holds for the higher cognitive processes involved in motor planning is therefore important . in a series of eeg experiments , kranczioch and colleagues [ 110 , 111 ] directly compared execution , imagination , and observation of finger movements in an advanced motor preparation paradigm . these studies firstly showed that the eeg - derived erps provide a good tool for the study of covert movements . this is important because the high temporal resolution of eeg typically requires precisely timed stimuli , which is a challenge for the imagery condition . at the same time , not all patients can part - take in mri scanning ( e.g. , because of metal in their body ) and eeg can therefore provide an alternative method to study motor processes . covert movements not only provide a good vehicle to study the reorganized motor system of those patients unable to execute the kind of controlled hand movements used in experimental paradigms requiring overt responses , but can also be employed to induce enhanced neural activation of motor circuitries which aids functional reorganization and recovery . coined by sharma and colleagues as a backdoor to the motor system after stroke , therapeutic approaches using covert movement modalities have recently been tested [ 7 , 113116 ] . while initial evidence is promising , there are a number of questions that need to be answered in due course . for example , literally all our knowledge on covert movement has been obtained in younger persons , typically university students . aging is known to affect the motor system ( e.g. , [ 117119 ] ) ; in fact , the way a person moves is quite indicative of older age and frailty . it is therefore not inconceivable that motor - specific mechanisms of covert movement , determined in younger populations , are differentially affected by age . similarly , the ability to imagine , or to focus on stimuli during observation , relies heavily on cognitive and perceptual processes and may therefore , again , be modulated by age . the latter may of course also be affected by the stroke [ 121 , 122 ] . more research that characterizes covert movement in healthy older persons is therefore needed to help tailor treatment development . in addition , it is presently unclear whether imagery and observation provide equally suitable treatment pathways , and how this interacts with lesion location . while several studies have explored the effects of mental practice on recovery , there is , to the best of our knowledge , no direct comparison between these methods . pilot data from our group suggests that motor imagery is best able to activate the reorganized motor system in patients with chronic sever hemiparesis . poor long - term recovery of motor function after stroke is a major public health issue and a big problem for patients and their families . but treatment provision is not satisfactory , research in this area is limited , and ( at least some ) patients feel abandoned by the health care system . a deeper understanding of the complexities involved in motor control and their interaction with the mechanisms of brain plasticity as well as psychological aspects of recovery is needed not only to maximize the treatment outcome for patients but also to tailor health service provisions and support infrastructures accordingly . despite the incredible advancements in brain imaging and rehabilitation research , and the growth of knowledge on brain plasticity over the last 20 years , there is little we can offer to patients with minimal recovery at present . a targeted and interdisciplinary research effort is required to meet the need for research and treatment development . this is necessary for the sake of the individuals affected as well as those who fund the health and welfare systems .

functional reorganization forms the critical mechanism for the recovery of function after brain damage . these processes are driven by inherent changes within the central nervous system ( cns ) triggered by the insult and further depend on the neural input the recovering system is processing . therefore these processes interact with not only the interventions a patient receives , but also the activities and behaviors a patient engages in . in recent years , a wide range of research programs has addressed the association between functional reorganization and the spontaneous and treatment - induced recovery . the bulk of this work has focused on upper - limb and hand function , and today there are new treatments available that capitalize on the neuroplasticity of the brain . however , this is only true for patients with mild to moderated impairments ; for those with very limited hand function , the basic understanding is much poorer and directly translates into limited treatment opportunities for these patients . the present paper aims to highlight the knowledge gap on severe stroke with a brief summary of the literature followed by a discussion of the challenges involved in the study and treatment of severe stroke and poor long - term outcome .

1. Background 2. Animal Models of Focal Ischemia versus Human Strokes 3. Capitalizing on Adult Brain Plasticity to Enhance Motor Recovery: Neural and Behavioral Considerations 4. Overt and Covert Movement: Alternative Ways of Stimulating the Motor System 5. Concluding Remarks

this work directly translated into new and , in some cases , more efficient interventions for patients with mild to moderate hemiparesis ( e.g. however , much less research has specifically investigated the reorganization of the motor system in patients with poor or minimal functional abilities and , most critically , the rehabilitation of these patients [ 6 , 7 ] . secondly , the lack of funding for regular one - to - one physical therapy sessions beyond the postacute phase , together with the common assumption that substantial improvements in functional motor ability are unlikely once the first 6 months of recovery have passed , primes the health care system and patients to accept the status quo . while the latter is true for the whole range of functional recovery , the impact is particularly grave for patients with poor functional recovery and also affects the research effort . thirdly , the motor system of patients with poor residual recovery can not easily be studied with the paradigms adopted from basic science research , such as finger opposition or grip movements . these methodological challenges can be overcome by excluding patients with high levels of spasm or by limiting the study group to those with relatively good levels of motor control . as a result , the motor system of patients with very poor recovery , in particular in chronic state , has been studied much less than that of patients with mild or moderate impairment . this translates directly into studies on treatment efficacy , and indeed , the availability of suitable treatments per se . based on the aforementioned considerations we argue that research on the mechanisms of long - term recovery , and their interaction with treatment efficacy , needs to widen its focus to the population of stroke survivors with severe long - term motor deficits . hereinafter we briefly summarise the present literature and further discuss the challenges involved in the study and treatment of patients with minimal motor recovery . upper limb paresis occurs in 85% of the patients and substantially impacts disability in the long term . rat focal ischemia models are frequently used because of low cost , similarities between vasculatures of rats and humans , fewer concerns from the general public compared to nonrodents , and availability of clear behavioral outcomes [ 1113 ] . severe mca infarcts in rodents leading to long - lasting sensorimotor and cognitive deficits can be produced by proximal mca occlusion induced by electrocoagulation . in the filament model for example , if endothelin-1 , a vasoconstrictor drug , is injected topically or intracerebrally , the forelimb motor cortex is typically spared , but infarcts vary in location and extension depending on the sites and route of drug administration . in models of occlusion of the distal mca or its branches , cortical frontoparietal infarcts are associated with less severe deficits than in the cortico - subcortical infarcts produced by proximal mca occlusion . furthermore , multiple embolic infarcts can be produced in brain areas supplied by the mca , after injection of microspheres , macrospheres , a thrombotic clot , or purified thrombin into the internal carotid artery [ 19 , 20 ] . many of these techniques have not been applied exclusively to small animals such as rats , mice , gerbils , or rabbits , but also to cats , dogs , pigs , and monkeys . however , technical limitations and costs limit widespread use of focal ischemia models in larger animals . they offer powerful opportunities : the possibility to objectively monitor behavioral outcomes , manipulate experimental conditions , and obtain images ( mri , micropet ) or intracortical recordings of neuronal activity in living animals ; to scrutinize molecular mechanisms of cell death and recovery ; to work with strains and transgenic animals that present hypertension , atherosclerosis and obesity , among other factors that are common in patients with stroke ; and to perform postmortem histological evaluations . for instance , small , selective cortical infarcts leading to mild sensorimotor deficits that tend to improve quickly are present in some of the models but are not common in humans , even though similar clinical features can occur in subcortical , lacunar infarcts . furthermore , rodent models have often included young healthy animals while , in humans , the bulk of strokes is concentrated in the elderly [ 2832 ] . other factors may contribute to poor outcome in humans , ranging from different mechanisms underlying strokes in different age groups to self - fulfilling prophecies in stroke care in the aged , and importantly , to comorbidities that impact recovery . contrasts between the compelling efficacy of a myriad of drugs in animal models of neuroprotection and the systematic failure of the same drugs when administered to patients in clinical trials underscore the requirement for animal models that more realistically approach human strokes [ 13 , 22 , 39 ] . , and hence the greater costs involved present challenges for advancements of research in the field . in particular , high mortality rates limit the opportunity to study recovery of animals with severe strokes in the chronic phase . in summary , there is a gap between pathogenesis and clinical features of severe strokes in humans and pathogenesis and clinical features of the most widely used models of focal ischemia in animals . high costs and lower expectations for substantial improvements in activity or quality of life are also major obstacles for rehabilitation of patients with severe strokes . still , as stroke mortality decreases in parallel with advances in health care , it is expected that more patients with severe strokes will survive the acute phase over the next decades . despite these limitations , animal models have provided unique insight on plastic mechanisms underlying sensorimotor recovery after focal ischemia , and on how to enhance beneficial patterns of reorganization to obtain behavioral gains ( e.g. constraint - induced movement therapy , for instance , shown to improve motor outcomes in humans with different types and sizes of strokes , was developed based on seminal studies that underscored the importance of the amount of use of the paretic limb to promote enhancement of motor function [ 41 , 42 ] . as mentioned before , such small infarcts are rarely observed in humans , but still , the information gained by the model was a major step forward in stroke rehabilitation . constraint - induced therapy has now been successfully applied to patients with various types of strokes , with more severe deficits and worse motor prognoses than the monkeys included in the model of forced use of the affected limb [ 1 , 3 , 43 , 44 ] . overall , functional neuroimaging studies in patients with stroke have shown that good performance is associated with augmented activity in preexisting networks during a motor task , rather than assignment of networks that are not overtly active during these tasks in healthy subjects . furthermore for instance , according to the model of interhemispheric inhibition , an imbalance in activity between the ipsilesional and the contralesional primary motor cortex ( m1 ) can occur after stroke [ 51 , 52 ] a number of studies have shown that this imbalance in activity between the two hemispheres can impair motor performance of the affected hand , at least in some patients in the chronic phase after stroke [ 51 , 52 ] . however , the vast majority of published studies that investigated effects of modulation of interhemispheric inhibition only included patients with mild to moderate motor impairments , likely due to difficulties in developing tasks and applying available neurophysiology and neuroimaging tools to more severely affected patients [ 5159 ] . in regard to recruitment of ipsilesional or contralesional secondary motor areas , striking differences in patterns of function were unveiled when paradigms of investigation were applied to patients with poor recovery , compared to those of less affected patients or healthy subjects . at least in part , more pronounced patterns of activity in secondary motor areas in the ipsilesional and/or contralesional areas are associated with more severe disruption of the corticospinal pathway , and excessive activation of secondary motor areas correlates with poorer motor behavior [ 48 , 50 , 62 ] . there are indications that motor performance may actually rely on activity of contralesional areas in more severely affected patients . for instance , it has been shown that transient disruption of the contralesional dorsal premotor cortex by transcranial magnetic stimulation slows motor performance of the paretic hand to a greater extent in patients with worse motor performance compared to less impaired patients . in addition , activation of the contralesional dorsal premotor cortex has been demonstrated to be greater in patients with more severe hand motor impairment , compared to healthy subjects and to less affected patients . on the other hand , disruption of the ipsilesional dorsal premotor together , these studies provide evidence that ( 1 ) contralesional areas are positively , functionally relevant in at least some well - recovered patients in the chronic phase ; ( 2 ) in patients with severe motor deficits , behavioral gains , albeit small , may occur by augmented activity of networks that are normally either minimally active or not active at all in healthy brains . severity of motor impairment seems to be a key factor influencing patterns of rewiring after stroke , but age , brain status before stroke , intensity , and timing of rehabilitative interventions , among other factors , are also likely to play pivotal roles in the process [ 33 , 34 , 6567 ] . a key concept to develop effective rehabilitation interventions is heterogeneity of mechanisms underlying stroke as well as plastic processes that lead to recovery of function after neuronal injury . mechanisms underlying neurological impairment , recovery of activity , and participation are also distinct . until now , most proof - of - principle studies or clinical trials have excluded patients with severe sensorimotor impairments and the lack of evidence - based effective interventions has nurtured a nihilistic approach for rehabilitation of these patients . moreover , patients with severe sensorimotor impairments often present with cognitive impairments , depression and are faced with massive changes in psychosocial interactions [ 6870 ] . further research efforts must not only address restoration of sensorimotor function but also incorporate an integrative approach to target neuropsychiatric domains , personal experience / expectations , environmental conditions , and psychosocial factors . this knowledge has the prospect to devise interventions that capitalize on the plastic capacities of the adult brain , mainly training- or practice - based interventions . the mechanisms of brain plasticity interact with psychological processes and behavior and together provide a number of considerations for the conceptualization of interventions . injury to the peripheral or central nervous system changes receptive field characteristics of neurons and neural representations through deprivation of the original afferent inputs these neurons receive [ 85 , 86 ] . the key mechanisms driving this change are the disinhibition of silent synapses , and the loss of inhibitory or excitatory inputs from lesioned neural populations connected to none - lesioned regions , including homologous areas in the two hemispheres . in both cases , it is important to appreciate the effects of functional changes in nonlesioned areas and their influence on the control of the impaired behavior . as a consequence , rehabilitation efforts should not only focus on the impaired function per se , for example , the affected upper - limb in the case of hemiplegia , but also consider the effects of use or nonuse of the lesser - affected extremities . this is particularly relevant for patients with poor recovery as they will most likely entirely rely on the less - affected extremity in everyday behavior . this might , at least in theory , increase the interhemispheric inhibition exerted by the nonlesioned hemisphere . critically , use - related reorganization is not driven by increased neural activation alone but heavily depends on the behavioral relevance of the activity [ 9092 ] . this association of sensory stimulation and consequential changes in neural representations and receptive field parameters has been demonstrated most elegantly in monkeys who received auditory and tactile simulation within the same protocol . behaviorally , the animals were only rewarded for responses in one of the two stimulation modalities , and subsequent changes in the brain were only observed for the rewarded modality . thus this data indicates that neural representations are only susceptible to stimulation - induced reconfigurations if this stimulation is attended to and of behavioral significance . therefore , we argue that interventions aiming to enhance recovery through the induction of practice - induced plasticity not only need to focus on the actual practice element of the intervention but also consider how the intervention characteristics enforce attention and provide tangible and motivating feedback . motor control is a complex behavior that goes far beyond motor execution and the processes typically associated with primary motor cortex function . motor rehabilitation research , however , typically conceptualizes motor function as the ability to execute a movement with little consideration being given to the cognitive processes that might influence this ability . for example , few studies have investigated how much patients with hemiplegia benefit from advanced movement preparation . the respective behavioral costs or benefits result from perceptual , cognitive , and motoric components of the stimulus - response cascade , such as stimulus - response mapping and response selection . most strikingly , the data suggests that patients are more sensitive to advance information ( manuscript submitted ) . thus , it appears that visual precues can facilitate or hinder apparent affected arm abilities , and that the magnitude of this effect is modulated by the severity of the motor deficit . while more research is needed to fully understand the interaction of cognitive processes , stroke severity , and motor performance , the findings summarised previously suggest that using advance movement information in a cognizant and explicit manner may be a beneficial addition to rehabilitation interventions . in order to translate improvements in motor ability into real - world benefits , treatments have to obtain not only better motor control over the affected arm , but also a transfer of these newly acquired abilities into the curriculum of everyday behaviors . these measures use not only learning principles , which in themselves are likely to facilitate use - related plasticity processes , but also tools that enhance motivation , adherence , and coping . the latter facilitates not only real - world benefit but also enhances the processes of use - related functional reorganization that drive improvements in motor control . interview data ( unpublished ) from patients with severe chronic hemiplegia indicates that these patients experience disproportionate psychological and service - related barriers . i hate it ( the hand ) and want to make it invisible , and it ( the hand ) looks horrible and is no good ; i 'd cut it off if i could suggest that patients do not only face the actual physical impairment but also face psychological barriers to using any residual ability . however , more often than not physiotherapy is provided without considering the psychological barriers associated with the use of the hemiplegic hand . we therefore argue that in order to improve the prospects for patients with low - functioning hemiparesis it is necessary to better understand the psychology of poor recovery and build that knowledge into motor rehabilitation interventions . in addition to the psychological barriers low - functioning patients seem to experience , our interview data suggests a strong perception amongst these patients that the care system is presumptuous and does not provide for them . this is illustrated by comments such as my doctors gave up on me after 3 month , my physio tried to work with my hand initially but soon gave up , the ot only taught me how to manage things ( with my good hand ) , i guess this was because she knew that my ( paretic ) hand would be no good , and there is nothing they can do for me because i can not move . these comments highlight not only the need to understand the patient perspective and a holistic approach to long - term care of these patients , but also a critical need to develop motor rehabilitation treatments that help patients to enhance their residual motor ability and enhance its real world benefit . this affects not only the patients ' general levels of activity but , of course , also the level of engagement with the therapy process . not a lot can be gained in a therapy session with a tired patient ! assuming that brain plasticity is the main driver for the recovery of function , good and sufficient sleep is likely to facilitate and probably enhance the rehabilitation effort . support for this assumption is provided by a series of studies suggesting that motor learning can be positively influenced by sleep [ 101 , 102 ] . the issues raised previously are relevant to all patients but probably have greater significance in patients with poorer recovery . therefore , treatments for these patients in particular should incorporate measures to counter tiredness and fatigue and monitor the quality of nocturnal sleep as well as daytime sleepiness . jeannerod 's theory of neural simulation suggests a shared neural network for the control of overt and covert movement modalities such as movement observation and motor imagery . this theoretical framework provides a powerful tool for research in patients with poor recovery . generally , evidence from behavioral , neuroimaging , and psychophysiological studies confirms jeannerod 's notion of equivalence in healthy populations . thereby , these activities are similar to the activations obtained when the same movements are actually performed in the scanner . typically , these studies use tasks that can be practiced prior to scanning and can be performed in the scanner . however , if motor imagery is to be used as a study tool for patients with poor recovery , their inability to move will prevent practice prior to scanning , and therefore , these patients will also perform the task by relying on their motor memory . evidence showing the feasibility and suitability of a paradigm that omits practice is therefore particularly relevant for the application in patients with poor residual recovery . moreover , the study of complex actions with fmri opens the door to the investigation of the representation of activities of daily living and the alteration of those representations throughout recovery and/or treatment . as mentioned previously , the cognitive processes involved in advance movement preparation are likely to play an important part in the person 's ability to function in everyday life but might also enhance or hinder the rehabilitation effort . understanding to what extend the principle of equivalence also holds for the higher cognitive processes involved in motor planning is therefore important . in a series of eeg experiments , kranczioch and colleagues [ 110 , 111 ] directly compared execution , imagination , and observation of finger movements in an advanced motor preparation paradigm . these studies firstly showed that the eeg - derived erps provide a good tool for the study of covert movements . this is important because the high temporal resolution of eeg typically requires precisely timed stimuli , which is a challenge for the imagery condition . covert movements not only provide a good vehicle to study the reorganized motor system of those patients unable to execute the kind of controlled hand movements used in experimental paradigms requiring overt responses , but can also be employed to induce enhanced neural activation of motor circuitries which aids functional reorganization and recovery . while initial evidence is promising , there are a number of questions that need to be answered in due course . aging is known to affect the motor system ( e.g. similarly , the ability to imagine , or to focus on stimuli during observation , relies heavily on cognitive and perceptual processes and may therefore , again , be modulated by age . the latter may of course also be affected by the stroke [ 121 , 122 ] . in addition , it is presently unclear whether imagery and observation provide equally suitable treatment pathways , and how this interacts with lesion location . pilot data from our group suggests that motor imagery is best able to activate the reorganized motor system in patients with chronic sever hemiparesis . poor long - term recovery of motor function after stroke is a major public health issue and a big problem for patients and their families . but treatment provision is not satisfactory , research in this area is limited , and ( at least some ) patients feel abandoned by the health care system . a deeper understanding of the complexities involved in motor control and their interaction with the mechanisms of brain plasticity as well as psychological aspects of recovery is needed not only to maximize the treatment outcome for patients but also to tailor health service provisions and support infrastructures accordingly . despite the incredible advancements in brain imaging and rehabilitation research , and the growth of knowledge on brain plasticity over the last 20 years , there is little we can offer to patients with minimal recovery at present . a targeted and interdisciplinary research effort is required to meet the need for research and treatment development . this is necessary for the sake of the individuals affected as well as those who fund the health and welfare systems .

emergency hematopoiesis defines the production of functional hematopoietic cells under nonhomeostatic , proinflammatory , or biologically stressed conditions [ 14 ] . blood cell production is a tightly regulated process that , after birth and throughout life , starts in a conspicuous hematopoietic stem cell ( hsc ) subset residing within the bone marrow ( bm ) . our current understanding of how hsc early differentiation is governed by the microenvironment indicates that , besides the stromal cell components of the various hematopoietic niches , not only essential growth and differentiation factors but also microbes and their products can influence differentiation fate decisions [ 3 , 5 , 6 ] . of note , emergency hematopoiesis is regulated at the stem and progenitor cell ( hspc ) level , where conditions such as infection demand the expedited production and activation of innate immune cells to combat noxious extrinsic agents , and the resulting proinflammatory conditions can at the time regulate the earliest steps of the hematopoietic development in favor of the clearance of insulting cues and to further maintain homeostasis . we have previously shown that pathogens and damaged tissue products and proinflammatory cytokines promote emergency hematopoiesis and alter patterns of early lymphoid differentiation in mouse and human [ 35 , 710 ] . in mice , pathogen recognition through toll like receptors ( tlr ) and the resulting cytokine release induce the expansion of hsc and instruct lineage differentiation fates so immediate innate cell development is guaranteed [ 6 , 7 ] . in general , ligation of tlr2 and tlr4 on these seminal cells promotes redirection toward myeloid cell production , while the sole tlr9 stimulation of primitive common lymphoid progenitors ( clp ) strikingly induces b cell differentiation blockage while development of dendritic cells ( dc ) , plasmacytoid dendritic cells ( pdc ) , and nk - related interferon killer dendritic cells ( ikdc ) is substantially enforced [ 5 , 8 ] . in humans , most findings relate to strengthening of myeloid lineage cell production under emergent scenarios , whereas adjustments within the lymphoid branch of the hematopoiesis have been poorly addressed [ 2 , 6 ] . according to what mouse research has shown , human multilymphoid progenitors ( mlp ) are capable of responding to tlr stimulation by producing dendritic cells , and our recent work suggests that primitive early lymphoid progenitor populations are also capable of microbial components discrimination through tlr , a mechanism that mostly facilitates their differentiation to innate lymphoid lineage cells . of special interest , tlr9 ligation on adult bm progenitors promotes the quick development of nk lineage cells by using mechanisms that involve il-15r upregulation [ 4 , 9 ] . thus , innate immune quick responses against viral threatening infections start in earlier developmental stages than we previously thought . whether the actual tlr - emergent hematopoiesis contributes to innate immunity under pathological conditions and other biological stress settings , including malignant diseases , interestingly , not only conventional pathogen associated molecular patterns ( pamps ) but also the damage associated molecular patterns- ( damps- ) like molecules can trigger innate immune sensors and prr signals , including micrornas , histones , fibronectin , and bacterial second messengers like di - gmp ( reviewed in [ 3 , 12 ] ) . even though efficient therapeutic agents have been developed that improve infectious and malignant disease outcomes and increase the overall survival rates , the adjuvant effect of molecules capable of remodeling hematopoietic pathways thus , the possibility of having extensive means of replenishing innate cells opens additional venues for receptor - ligand axes of clinical significance . disruption of normal peripheral blood leukocytes results in the release of heterogeneous mixtures of peptides , among other complex molecules . upon dialysis , the enriched mixture of low - molecular - weight polar and hydrophilic peptides ( < 10 kda ) , named dialyzable leukocyte extracts ( dle ) , has shown a number of therapeutic and adjuvant properties through modulation of immune responses [ 13 , 14 ] . although the precise molecular mechanisms underlying its positive experimental and clinical effects are currently unknown , critical signaling pathways for survival and cellular activation states , including toll like receptor ( tlr ) and nfb , are apparently involved and often trigger the production of proinflammatory cytokines [ 1418 ] . of note , a recent investigation using in vitro controlled models of tlr - mediated proinflammation suggested the content of tlr-2 agonist ligands within dle . the exposure of human peripheral mononuclear cells to dle induced the copious secretion of tnfalpha by the monocyte fraction . whether this phenomenon is due to damps or damp - like related peptides within dle is still a matter in question . using an elegant mouse model of experimental tuberculosis , fabre and colleagues demonstrated that the administration of dle ( formerly denominated transfer factor ) evokes an efficient reconstitution of the cell - mediated immunity , concomitant with a substantial production of ifnalpha , il-2 , and inos , an immune protective profile provoking inhibition of bacterial proliferation [ 19 , 20 ] . in contrast , inflammatory injuries of human ocular tissues where limbal epithelial stem cells giving rise to corneal epithelium are compromised have been shown to respond to dle treatment by downregulating the secretion of il-8 and il-6 . the same is true for atopic dermatitis , where dle contribute the decrease of inflammatory cells and the severity of the disease . then , despite the proinflammatory potential induction of dle , the net balance induction or suppression may depend on additional biological settings of the damaged tissues , where the dose of dle would be absolutely crucial to get a beneficial result . resolution of herpes virus infections is remarkably benefited from the adjuvant effect of dle treatment [ 13 , 16 ] . in fact , a herpes murine model has become a powerful biological assay to test the functional activity of dle . of special interest , a direct effect on viral replication or infected target cell viability could not been recorded . instead , its protective effects correlated with serum cytokine levels and , most likely , with changes in the cellular immune system . accordingly , as cell - mediated immunity plays a central role in controlling viral - infected and tumor cells , and the capability of dle of strengthening the cellular immunity has been suggested from several studies , dle is considered as a presumptive instrument with adjuvant potential for treating virally induced cancer . moreover , a growing list of viral , parasitic , or fungal infections , as well as acute and chronic diseases , including immunodeficiencies , malignancies , allergies , and autoimmune disorders , seems to favorably respond to dle . however , a more comprehensive understanding of its biological mechanisms is yet needed and will be benefited from less heterogeneous and highly controlled extracts . dle transferon , a blood product licensed for clinical use , has been shown to exhibit batch - to - batch- reproducibility and relatively high homogeneity when ultra - performance liquid chromatography ( se - uplc ) is used to characterize its content . due to the putative tlr agonist elements within dle and their biological capability of inducing proinflammatory microenvironments , here we sought to examine the dle 's contribution to innate immune replenishment through emergent hematopoiesis . by using in vitro functional assays and controlled early differentiation culture systems , our findings define a powerful route to promote development of a unique cd11c nk cell subset with immune - surveillance capacity . umbilical cord blood ( ucb ) samples were obtained from normal full - term neonates upon mothers ' written informed consent , while adult bone marrow ( abm ) was collected from healthy adult donors who entered orthopedic surgery and according to institutional guidelines . mononuclear cells ( mncs ) were prepared by ficoll - paque plus ( ge healthcare bioscience ) gradient centrifugation and preserved at 80c until use . all procedures were approved by the ethics and scientific committee of health research at imss ( r-2006 - 3602 - 16 ) . cd34 cells from abm and ucb were enriched using the human cd34 progenitor cell isolation kit ( miltenyi biotec ) according to manufacturer instructions and our previous reports . after staining with pe - conjugated antilineage markers ( cd3 , cd8 , tcr , cd56 , cd14 , cd11b , cd20 , cd19 , and cd235a ) , anti - cd34-apc , anti - cd38-fitc , and anti - cd45ra - pe - txr conjugated antibodies , primitive cell populations were highly purified by multicolor flow cytometry using a facsaria sorter ( bd biosciences ) . hematopoietic stem cells ( hsc ) were separated as lincd34cd38cd45ra , while multipotent progenitors ( mpp ) were sorted as lincd34cd38cd45ra , and early lymphoid progenitors ( elp ) as lincd34cd38cd45ra , as described . upon harvesting from culture , anti - cd56-pe , anti - cd11c - fitc , and anti - cd16-apc ( bd biosciences ) nk cells were identified by flow cytometry in a facscanto ii equipment ( bd biosciences ) as cd56cd11c or cd56cd16cd11c cells , while dendritic cells ( dc ) were detected as cd56cd11c . analysis of flow cytometry data was performed using the flowjo 10 software ( treestar inc . dle transferon is manufactured by udimeb at gmp facilities in the national school of biological sciences , national polytechnic institute ( ipn ) , as described [ 14 , 15 ] . cells were disrupted by 5 cycles of freezing and thawing followed by dialysis against water using spectra / pore membranes with a cut - off of 12 kda ( spectrum labs , usa ) . the quality control of transferon comprised ( a ) endotoxin content , quantified using the endosafe - portable test ( charles river laboratories , charleston , sc , usa ) according to the manufacturer 's instructions ( the specification for endotoxin was established in mexican pharmacopeia , section mga-0316 ( 4.0 eu / ml ) ) ; ( b ) microbiological tests , according to mexican pharmacopeia , section mga-0571 ; and ( c ) physicochemical characterization by a validated ultra - performance liquid chromatography ( uplc ) method that analyzes molecular weights and the time of retention of the main peaks compared with those of an internal batch pattern . peptide content per final dose was measured by bicinchoninic acid ( bca ) method using the pierce bca kit ( thermo fisher scientific , waltham ma , usa ) according to the manufacturer 's instructions . batch - to - batch reproducibility is consistently analyzed by se - uplc chromatographic profiles , while ifn production from dle - stimulated jurkat cells is quantified as a biological activity test . enriched cd34 cells from ucb and abm were cultured for 24 hours in -mem medium supplemented with 10% fetal bovine serum , 100 u / ml penicillin , and 100 mg / ml streptomycin . dle transferon was used for cell culture stimulation at 5 g / ml . ms-5 stromal cells were grown in presence or not of dle transferon 24 hours before coculturing with cd34 hspc . on the other hand , and upon 24 hours of dle prestimulation , hspc ( including the whole enriched cd34 fraction or lincd34cd38cd45ra hsc , lincd34cd38cd45ra mpp , or lincd34cd38cd45ra elp ) were centrifuged to remove the medium and cocultured in the presence of ms-5 stromal cells for 30 more days . cocultures were performed using -modified essential medium ( -mem ) ( gibco ) supplemented with 10% fetal bovine serum and 100 u / ml penicillin and 100 mg / ml streptomycin . lymphoid lineage cytokines and growth factors were contained throughout coculture : 1 ng / ml flt3-l ( fl ) , 2 ng / ml scf , 5 ng / ml il-7 , and 10 ng / ml il-15 ( preprotech ) . coculture systems were incubated at 37c in a humidified atmosphere of 5% co2 . enriched cd34 cells from ucb and abm were cultured for 72 hours with 5 g / ml of dle transferon and bromo-2-deoxyuridine ( brdu ) to a final concentration of 10 m , in -mem serum - free medium supplemented with lymphoid cytokines . after stimulation , cells were stained for the identification of cd34 cell progenitors by multicolor flow cytometry followed by intracellular staining with a monoclonal antibody to brdu according to an established protocol ( brdu flow kit , bd biosciences ) . ucb cd34 cells were pretreated with 20 m of myd88 control or inhibitory peptides ( imgenex img-200501 ) for two hours , followed by 2x washing and staining with 5 m cell trace violet ( ctv ) dye . dle transferon was included or not at 5 g / ml in a 72 h culture . the dilution of fluorescence intensity was estimated using the application for cell proliferation from the flowjo 7.6.2 software . to investigate the influence of tlr signals in the dle - mediated nk - like cell differentiation , ucb cd34 cells were pretreated with 20 m of myd88 control or inhibitory peptides ( imgenex img-200501 ) for 24 hours , followed by 2x washing and coculturing on ms-5 stromal cells for 15 more days . cocultures were performed using -modified essential medium ( -mem ) ( gibco ) supplemented with 10% fetal bovine serum and 100 u / ml penicillin and 100 mg / ml streptomycin ( as described above in section 2.4 ) . natural killer cytolytic activity was evaluated using a fluorescence - based assay , as described [ 9 , 23 ] . this assay uses the dye carboxyfluorescein succinimidyl ester ( cfse ) to distinguish target from effector cells , and the dna intercalating dye 7-aminoactinomycin d ( 7-aad ) ( bd pharmingen ) for dead / live cells distinction . briefly , the myeloid leukemia k562 cell line ( atcc ) was kept in log phase growth in rpmi 1640 + glutamax supplemented with 10% of fetal calf serum ( fcs ) . k562 cells were stained with 5 m carboxyfluorescein succinimidyl ester ( cfse ) . in vitro differentiated nk cells from dle - cultures were enumerated by flow cytometry and cocultured with cfse - labeled k562 cells , according to an effector : target ratio curve . il-2 ( 40 ng / ml ) ( preprotech ) was added to induce nk cell activation , followed by 4 hours of incubation at 37c . cells were washed and 7-aad ( bd pharmingen ) was incorporated into the cell suspension . multiparametric flow cytometry ( facscanto ii , bd biosciences ) was conducted to determine functionality . to investigate the capability of dle - newly derived nk - like cells of producing ifn - gamma ( ifn ) upon stimulation , an ifn assay was performed . after 30-day coculture , the produced cells were stimulated with il-12 ( 10 ng / ml ) and il-18 ( 50 ng / ml ) for 12 hours . nk - like cells were harvested and brefeldin a ( bfa , 10 g / ml ) was added for 4 hours to inhibit protein intracellular transport . after bfa blockage , cells were carefully washed , followed by incubation with anti - cd56-apc , anti - cd11c - fitc ( bd biosciences ) , and anti - ifn-pe ( biolegend ) conjugated antibodies to perform extra- and intracellular staining , respectively . gammadelta t peripheral blood lymphocytes ( tcr-1 ) from healthy donors were enriched by the t lymphocyte isolation kit ( miltenyibiotec , igg1 clone 11f2 ) according to manufacturer instructions , followed by staining with 5 m cell trace violet ( ctv ) . simultaneously , newly differentiated cd11ccd56 nk cells from dle - stimulated 30-day cocultures were harvested and purified by flow cytometry sorting in bd facsaria equipment . postsort cell purity was investigated by using the anti - human tcr / mouse igg1 antibody clone b1 . the purified t cells were then cocultured with the nk cell population of interest , at a ratio of 2 : 1 cd56cd11c nk : t lymphocyte . supplemented -mem medium with 10% fetal bovine serum , 100 u / ml penicillin , and 100 mg / ml streptomycin was used . t cells proliferation was assessed at 72 hours by dilution of ctv ( within the pacific blue channel ) . the prism software , version 5.01 ( graphpad , usa ) , was used for statistical analysis . comparisons between groups were performed with either the unpaired t - test or the one - way anova . our previous research on mouse and human early hematopoietic fate redirection in response to extrinsic prr agonists prompted us to investigate whether this phenomenon may occur upon exposure to dle transferon . to first determine sensitivity of primitive populations to dle components , we performed brdu incorporation analyses on cd34 cell fractions from cord blood and adult bone marrow . notably , we recorded significant increases of proliferative cell frequencies from both sources after 72 h of dle stimulation ( figure 1 ) . furthermore , bone marrow cell stimulation under lymphoid or myeloid culture conditions revealed that adult lymphoid progenitors preferentially expand in response to dle ( supplemental figure 1 in supplementary material available online at http://dx.doi.org/10.1155/2016/4097642 ) . natural killer ( nk ) cells constitute a granular innate immune cell type comprising 320% of the human peripheral blood mononuclear cell ( pbmc ) lymphocyte fraction , which function as major players in innate responses by producing cytokines and chemokines and exerting cytolytic activity against virus - infected or tumor cells [ 2426 ] . here , dle stimulation of cb cd34 cells rapidly influenced early cell fate decisions and accelerated the differentiation toward a special population of cd56cd11c nk cell ( figure 2 ) . although final yields per input progenitor were not indicative of higher net production when the full cd34 compartment is considered , substantially discrepant cd56cd11c cell frequencies suggest the speeding up of such specialized cells ( figures 2(a ) and 2(b ) ) . in line with the observations in the context of tlr signaling , cells harboring early lymphoid progenitor phenotype were the major producers of dle - newly differentiated nk cells ( figure 2(c ) ) . the identity of cd11c nk cells has been a matter in question and a debate point for years . the various studies from dematteo have indicated that cd11c expression defines a distinct subset of murine liver nk cells that respond to adenoviral infection by producing ifn - gamma in a tlr9-il-12-il-18-dependent way . moreover , the same nk dendritic cells ( nkdc ) phenotype was found to be multifunctional cells with pleiotropic functions , including the ifn-mediated response against bacterial infections and malignant processes , and the migration to central nervous system for viral clearance [ 2832 ] . belonging to the hardy fraction a of the mouse b cell developmental pathway , the discrete b220cd11cnk1.1 nk subset seemed to meet the biological properties of nkdc , that is , dependency on il-15 and ability of producing highest amounts of ifn . of relevance , two simultaneous reports recently revealed the hybrid phenotypic and functional characteristics of dc and nk within a novel subset of interferon - producing killer dendritic cell ( ikdc ) population , endowed with strong cytotoxic and antitumor activities , and antigen presenting competence [ 3437 ] . ikdcs share some properties with other innate lymphoid cells , but their development is unique and is most efficiently generated from l - selectin bm progenitors , while common lymphoid progenitors ( clp ) are the most effective source of conventional nk cells . even though a ikdc subset has not been formally described in humans , expression of cd11c in nk lineage cells is induced by combination of il-15 and inflammatory cytokines and distinguishes a conspicuous cell subtype participating in immune regulation of chronic diseases . starting from the hsc fraction , dle stimulation could efficiently trigger their production ( supplemental figure 2 ) , emphasizing its potential to aim at innate responses but suggesting the divergent origin of dle - derived nk and dc . accordingly , animals treated with dle have showed increased nk cell frequencies and numbers , among other hematopoietic cells [ 19 , 20 ] . due to their fundamental position in viral and tumor immune - surveillance , extensive efforts our data suggest a preliminary strategy to produce high numbers of these cells and , most of all , to robust the in vivo production by remodeling central hematopoiesis . of note , inhibition of myd88 adaptor molecule reduced proliferation and nk - differentiation potentials of dle - stimulated cd34 cells , suggesting the partial contribution of tlr signaling pathway in this emergent phenomenon ( supplemental figure 3 ) . despite the wealth of information relevant to transcription factors- and phenotype - associated biological functions of nk lineage cells , we still need to understand the role of environmental niches in their early differentiation under steady - state and pathological conditions . it is well known that they are generated and perform their critical functions in the context of hematopoietic niches within the bone marrow [ 24 , 39 ] or in extramedullar tissues , respectively . indeed , besides the identified developmental niches , an immune response niche , an inflammatory or stress niche , a tumor niche , and a pregnancy niche have been proposed to regulate the biology of nk cells . such niches are presumably controlled by nonhematopoietic cells providing signals triggered from adhesion molecules , chemokines , and cytokines / growth factors [ 3941 ] . generation of nk cells requires il-15 and fms - like tyrosine kinase 3 ligand , but not il-7 . strikingly , the cxcr4/cxcl12 axis conformed by cxcl12-abundant reticular ( car ) mesenchymal cells and nk precursors might function as the major niche element and is crucial for nk development within bm [ 39 , 41 ] . moreover , production of il-15 is the highest in car cells . our data confirm that microenvironment cues affect the development and biological roles of distinct nk subsets and suggest a substantial positive effect of dle on stromal cell activation that , in turn , may promote cd11cnk and dc expeditious differentiation ( figure 3 ) . the additive differentiation effect by stromal cells may result from the action of cytokines and nk growth factors . certainly , a multiplex type assay has shown the differential production of lymphoid factors and vegf ( our preliminary unpublished observations ) . two main subtypes of human nk cells are recognized : cd56cd16 and cd56cd16 . while the abundant peripheral cd56cd16 nk cells are phenotypically classified as the most cytotoxic ones , the less copious cd56cd16 subset has been shown to be endowed with noticeable cytokine production capability . their activation is mediated by the net balance between activating and inhibitory receptor engagement signals and in response to cytokines such as il-2 , il-12 , il-15 , and il-18 . among activating molecules displayed by the cd56cd16 nk cells , cd16 plays a critical role in targeting antibody fc - bound cells and executing antibody dependent cell - mediated cytotoxicity ( adcc ) . predominantly in tissues and secondary lymphoid organs , the cd56cd16 subset is responsible for production of ifn , tnf , gm - csf , and rantes after activation . the recent reevaluation of functional properties of both cell categories has changed the binary paradigm of nk biology , providing clear evidence of the rapid and simultaneous effector activities exerted by the same cd56cd16 nk cell subset . thus , a prompt and abundant ifn production overlaps with their cytolytic ability and may lead to amplification of macrophage and dc - mediated responses . interestingly , we have now found that most dle - newly produced cells expressed cd16 and that microenvironmental prestimulation induces the density of this molecule ( figure 4 ) . the functional competence of nk cells produced from dle - stimulated lymphoid progenitors was tested , demonstrating a very efficient cytolytic potential when a nonradioactive method was used ( figure 5(a ) ) . we also observed a copious production of ifn only from the specialized cd11c nk population ( figure 5(b ) ) , which is highly relevant to antitumoral and antimicrobial immune protection as this proinflammatory cytokine is pivotal in initiating th1 immune responses against pathogens and tumors [ 30 , 31 ] . thus , these data suggest that early progenitors are promoted to differentiate toward both cytotoxic and regulatory cells in response to dle . recently , cd56cd11c human nk - like cells were shown to induce the proliferation of t cells in an il-18 dependent manner [ 42 , 43 ] . because v2v2 t lymphocytes are key components of the innate immune system that function against infections and human tumor cells , we explore the faculty of dle - nk cells of activating them . of note , dilution of the dye ctv was significantly apparent when t cells were cultured with cd11cnk that are produced upon stimulation with dle . surprisingly , neither unstimulated t cells nor dle - directly stimulated t cells got activated ( figures 6 and 7 ) . activation of t cells can be achieved by a number of microorganisms and phosphoantigens and does not require antigen processing , but some atypical costimulatory molecules such as nkg2d play crucial roles in activating them to kill neoplastic or pathogen - infected cells . these innate cells function through a variety of mechanisms , including secretion of cytokines and chemokines , dendritic cell activation , macrophage recruitment , cytolytic activity , and antigen presentation . their contribution to immune - surveillance in a major histocompatibility complex- ( mhc- ) unrestricted way , by their capability of producing ifn and tnf and via an nk - like pathway , has positioned them as attractive targets for immunotherapy strategies [ 4548 ] . taken together , our study provides a rational explanation for the adjuvant contribution of dle to the observed effective immune responses against viral and malignant diseases . dle could play an inductor role in the nk lineage emergent hematopoiesis , suggesting the need of elucidating central mechanisms that govern dle - associated tissue regeneration and give an insight into novel cooperative drug activities . while remarkable progress has long been recorded in identifying targets for cellular - based immunotherapeutic strategies to improve acute and chronic disease outcomes , it is becoming clear that definition of extrinsic factors promoting innate cell differentiation processes from the earliest developmental stages may change our vision of immunomodulation . our findings suggest , for the first time , that , in response to dialyzable leukocyte extracts ( dle ) transferon , human stem and progenitor cells contribute to the emergent production of a special subset of innate cd11c nk cells . of note , this dle - derived nk cell population is endowed with properties such as ifn production , tumor cell cytotoxicity , and the capability of inducing t lymphocyte proliferation that may , in turn , function as coadjuvant component of innate immune responses against virus - infected or tumor cells .

reconstitution of the hematopoietic system during immune responses and immunological and neoplastic diseases or upon transplantation depends on the emergent differentiation of hematopoietic stem / progenitor cells within the bone marrow . although in the last decade the use of dialyzable leukocyte extracts ( dle ) as supportive therapy in both infectious and malignant settings has increased , its activity on the earliest stages of human hematopoietic development remains poorly understood . here , we have examined the ability of dle to promote replenishment of functional lymphoid lineages from cd34 + cells . our findings suggest that dle increases their differentiation toward a conspicuous cd56+cd16+cd11c+ nk - like cell population endowed with properties such as ifny production , tumor cell cytotoxicity , and the capability of inducing t lymphocyte proliferation . of note , long - term coculture controlled systems showed the bystander effect of dle - stromal cells by providing nk progenitors with signals to overproduce this cell subset . thus , by direct effect on progenitor cells and through activation and remodeling of the supporting hematopoietic microenvironment , dle may contribute a robust innate immune response by promoting the emerging lymphopoiesis of functional cd11c+ nk cells in a partially tlr - related manner . unraveling the identity and mechanisms of the involved dle components may be fundamental to advance the nk cell - based therapy field .

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

blood cell production is a tightly regulated process that , after birth and throughout life , starts in a conspicuous hematopoietic stem cell ( hsc ) subset residing within the bone marrow ( bm ) . our current understanding of how hsc early differentiation is governed by the microenvironment indicates that , besides the stromal cell components of the various hematopoietic niches , not only essential growth and differentiation factors but also microbes and their products can influence differentiation fate decisions [ 3 , 5 , 6 ] . of note , emergency hematopoiesis is regulated at the stem and progenitor cell ( hspc ) level , where conditions such as infection demand the expedited production and activation of innate immune cells to combat noxious extrinsic agents , and the resulting proinflammatory conditions can at the time regulate the earliest steps of the hematopoietic development in favor of the clearance of insulting cues and to further maintain homeostasis . in mice , pathogen recognition through toll like receptors ( tlr ) and the resulting cytokine release induce the expansion of hsc and instruct lineage differentiation fates so immediate innate cell development is guaranteed [ 6 , 7 ] . in general , ligation of tlr2 and tlr4 on these seminal cells promotes redirection toward myeloid cell production , while the sole tlr9 stimulation of primitive common lymphoid progenitors ( clp ) strikingly induces b cell differentiation blockage while development of dendritic cells ( dc ) , plasmacytoid dendritic cells ( pdc ) , and nk - related interferon killer dendritic cells ( ikdc ) is substantially enforced [ 5 , 8 ] . in humans , most findings relate to strengthening of myeloid lineage cell production under emergent scenarios , whereas adjustments within the lymphoid branch of the hematopoiesis have been poorly addressed [ 2 , 6 ] . according to what mouse research has shown , human multilymphoid progenitors ( mlp ) are capable of responding to tlr stimulation by producing dendritic cells , and our recent work suggests that primitive early lymphoid progenitor populations are also capable of microbial components discrimination through tlr , a mechanism that mostly facilitates their differentiation to innate lymphoid lineage cells . of special interest , tlr9 ligation on adult bm progenitors promotes the quick development of nk lineage cells by using mechanisms that involve il-15r upregulation [ 4 , 9 ] . thus , innate immune quick responses against viral threatening infections start in earlier developmental stages than we previously thought . whether the actual tlr - emergent hematopoiesis contributes to innate immunity under pathological conditions and other biological stress settings , including malignant diseases , interestingly , not only conventional pathogen associated molecular patterns ( pamps ) but also the damage associated molecular patterns- ( damps- ) like molecules can trigger innate immune sensors and prr signals , including micrornas , histones , fibronectin , and bacterial second messengers like di - gmp ( reviewed in [ 3 , 12 ] ) . even though efficient therapeutic agents have been developed that improve infectious and malignant disease outcomes and increase the overall survival rates , the adjuvant effect of molecules capable of remodeling hematopoietic pathways thus , the possibility of having extensive means of replenishing innate cells opens additional venues for receptor - ligand axes of clinical significance . upon dialysis , the enriched mixture of low - molecular - weight polar and hydrophilic peptides ( < 10 kda ) , named dialyzable leukocyte extracts ( dle ) , has shown a number of therapeutic and adjuvant properties through modulation of immune responses [ 13 , 14 ] . of note , a recent investigation using in vitro controlled models of tlr - mediated proinflammation suggested the content of tlr-2 agonist ligands within dle . the exposure of human peripheral mononuclear cells to dle induced the copious secretion of tnfalpha by the monocyte fraction . using an elegant mouse model of experimental tuberculosis , fabre and colleagues demonstrated that the administration of dle ( formerly denominated transfer factor ) evokes an efficient reconstitution of the cell - mediated immunity , concomitant with a substantial production of ifnalpha , il-2 , and inos , an immune protective profile provoking inhibition of bacterial proliferation [ 19 , 20 ] . in contrast , inflammatory injuries of human ocular tissues where limbal epithelial stem cells giving rise to corneal epithelium are compromised have been shown to respond to dle treatment by downregulating the secretion of il-8 and il-6 . the same is true for atopic dermatitis , where dle contribute the decrease of inflammatory cells and the severity of the disease . then , despite the proinflammatory potential induction of dle , the net balance induction or suppression may depend on additional biological settings of the damaged tissues , where the dose of dle would be absolutely crucial to get a beneficial result . resolution of herpes virus infections is remarkably benefited from the adjuvant effect of dle treatment [ 13 , 16 ] . in fact , a herpes murine model has become a powerful biological assay to test the functional activity of dle . of special interest , a direct effect on viral replication or infected target cell viability could not been recorded . instead , its protective effects correlated with serum cytokine levels and , most likely , with changes in the cellular immune system . accordingly , as cell - mediated immunity plays a central role in controlling viral - infected and tumor cells , and the capability of dle of strengthening the cellular immunity has been suggested from several studies , dle is considered as a presumptive instrument with adjuvant potential for treating virally induced cancer . moreover , a growing list of viral , parasitic , or fungal infections , as well as acute and chronic diseases , including immunodeficiencies , malignancies , allergies , and autoimmune disorders , seems to favorably respond to dle . due to the putative tlr agonist elements within dle and their biological capability of inducing proinflammatory microenvironments , here we sought to examine the dle 's contribution to innate immune replenishment through emergent hematopoiesis . by using in vitro functional assays and controlled early differentiation culture systems , our findings define a powerful route to promote development of a unique cd11c nk cell subset with immune - surveillance capacity . umbilical cord blood ( ucb ) samples were obtained from normal full - term neonates upon mothers ' written informed consent , while adult bone marrow ( abm ) was collected from healthy adult donors who entered orthopedic surgery and according to institutional guidelines . hematopoietic stem cells ( hsc ) were separated as lincd34cd38cd45ra , while multipotent progenitors ( mpp ) were sorted as lincd34cd38cd45ra , and early lymphoid progenitors ( elp ) as lincd34cd38cd45ra , as described . upon harvesting from culture , anti - cd56-pe , anti - cd11c - fitc , and anti - cd16-apc ( bd biosciences ) nk cells were identified by flow cytometry in a facscanto ii equipment ( bd biosciences ) as cd56cd11c or cd56cd16cd11c cells , while dendritic cells ( dc ) were detected as cd56cd11c . the quality control of transferon comprised ( a ) endotoxin content , quantified using the endosafe - portable test ( charles river laboratories , charleston , sc , usa ) according to the manufacturer 's instructions ( the specification for endotoxin was established in mexican pharmacopeia , section mga-0316 ( 4.0 eu / ml ) ) ; ( b ) microbiological tests , according to mexican pharmacopeia , section mga-0571 ; and ( c ) physicochemical characterization by a validated ultra - performance liquid chromatography ( uplc ) method that analyzes molecular weights and the time of retention of the main peaks compared with those of an internal batch pattern . enriched cd34 cells from ucb and abm were cultured for 24 hours in -mem medium supplemented with 10% fetal bovine serum , 100 u / ml penicillin , and 100 mg / ml streptomycin . ms-5 stromal cells were grown in presence or not of dle transferon 24 hours before coculturing with cd34 hspc . on the other hand , and upon 24 hours of dle prestimulation , hspc ( including the whole enriched cd34 fraction or lincd34cd38cd45ra hsc , lincd34cd38cd45ra mpp , or lincd34cd38cd45ra elp ) were centrifuged to remove the medium and cocultured in the presence of ms-5 stromal cells for 30 more days . enriched cd34 cells from ucb and abm were cultured for 72 hours with 5 g / ml of dle transferon and bromo-2-deoxyuridine ( brdu ) to a final concentration of 10 m , in -mem serum - free medium supplemented with lymphoid cytokines . to investigate the influence of tlr signals in the dle - mediated nk - like cell differentiation , ucb cd34 cells were pretreated with 20 m of myd88 control or inhibitory peptides ( imgenex img-200501 ) for 24 hours , followed by 2x washing and coculturing on ms-5 stromal cells for 15 more days . this assay uses the dye carboxyfluorescein succinimidyl ester ( cfse ) to distinguish target from effector cells , and the dna intercalating dye 7-aminoactinomycin d ( 7-aad ) ( bd pharmingen ) for dead / live cells distinction . in vitro differentiated nk cells from dle - cultures were enumerated by flow cytometry and cocultured with cfse - labeled k562 cells , according to an effector : target ratio curve . il-2 ( 40 ng / ml ) ( preprotech ) was added to induce nk cell activation , followed by 4 hours of incubation at 37c . to investigate the capability of dle - newly derived nk - like cells of producing ifn - gamma ( ifn ) upon stimulation , an ifn assay was performed . nk - like cells were harvested and brefeldin a ( bfa , 10 g / ml ) was added for 4 hours to inhibit protein intracellular transport . simultaneously , newly differentiated cd11ccd56 nk cells from dle - stimulated 30-day cocultures were harvested and purified by flow cytometry sorting in bd facsaria equipment . the purified t cells were then cocultured with the nk cell population of interest , at a ratio of 2 : 1 cd56cd11c nk : t lymphocyte . t cells proliferation was assessed at 72 hours by dilution of ctv ( within the pacific blue channel ) . to first determine sensitivity of primitive populations to dle components , we performed brdu incorporation analyses on cd34 cell fractions from cord blood and adult bone marrow . notably , we recorded significant increases of proliferative cell frequencies from both sources after 72 h of dle stimulation ( figure 1 ) . natural killer ( nk ) cells constitute a granular innate immune cell type comprising 320% of the human peripheral blood mononuclear cell ( pbmc ) lymphocyte fraction , which function as major players in innate responses by producing cytokines and chemokines and exerting cytolytic activity against virus - infected or tumor cells [ 2426 ] . here , dle stimulation of cb cd34 cells rapidly influenced early cell fate decisions and accelerated the differentiation toward a special population of cd56cd11c nk cell ( figure 2 ) . in line with the observations in the context of tlr signaling , cells harboring early lymphoid progenitor phenotype were the major producers of dle - newly differentiated nk cells ( figure 2(c ) ) . the identity of cd11c nk cells has been a matter in question and a debate point for years . the various studies from dematteo have indicated that cd11c expression defines a distinct subset of murine liver nk cells that respond to adenoviral infection by producing ifn - gamma in a tlr9-il-12-il-18-dependent way . moreover , the same nk dendritic cells ( nkdc ) phenotype was found to be multifunctional cells with pleiotropic functions , including the ifn-mediated response against bacterial infections and malignant processes , and the migration to central nervous system for viral clearance [ 2832 ] . belonging to the hardy fraction a of the mouse b cell developmental pathway , the discrete b220cd11cnk1.1 nk subset seemed to meet the biological properties of nkdc , that is , dependency on il-15 and ability of producing highest amounts of ifn . of relevance , two simultaneous reports recently revealed the hybrid phenotypic and functional characteristics of dc and nk within a novel subset of interferon - producing killer dendritic cell ( ikdc ) population , endowed with strong cytotoxic and antitumor activities , and antigen presenting competence [ 3437 ] . ikdcs share some properties with other innate lymphoid cells , but their development is unique and is most efficiently generated from l - selectin bm progenitors , while common lymphoid progenitors ( clp ) are the most effective source of conventional nk cells . starting from the hsc fraction , dle stimulation could efficiently trigger their production ( supplemental figure 2 ) , emphasizing its potential to aim at innate responses but suggesting the divergent origin of dle - derived nk and dc . accordingly , animals treated with dle have showed increased nk cell frequencies and numbers , among other hematopoietic cells [ 19 , 20 ] . of note , inhibition of myd88 adaptor molecule reduced proliferation and nk - differentiation potentials of dle - stimulated cd34 cells , suggesting the partial contribution of tlr signaling pathway in this emergent phenomenon ( supplemental figure 3 ) . it is well known that they are generated and perform their critical functions in the context of hematopoietic niches within the bone marrow [ 24 , 39 ] or in extramedullar tissues , respectively . indeed , besides the identified developmental niches , an immune response niche , an inflammatory or stress niche , a tumor niche , and a pregnancy niche have been proposed to regulate the biology of nk cells . such niches are presumably controlled by nonhematopoietic cells providing signals triggered from adhesion molecules , chemokines , and cytokines / growth factors [ 3941 ] . generation of nk cells requires il-15 and fms - like tyrosine kinase 3 ligand , but not il-7 . our data confirm that microenvironment cues affect the development and biological roles of distinct nk subsets and suggest a substantial positive effect of dle on stromal cell activation that , in turn , may promote cd11cnk and dc expeditious differentiation ( figure 3 ) . the additive differentiation effect by stromal cells may result from the action of cytokines and nk growth factors . two main subtypes of human nk cells are recognized : cd56cd16 and cd56cd16 . while the abundant peripheral cd56cd16 nk cells are phenotypically classified as the most cytotoxic ones , the less copious cd56cd16 subset has been shown to be endowed with noticeable cytokine production capability . their activation is mediated by the net balance between activating and inhibitory receptor engagement signals and in response to cytokines such as il-2 , il-12 , il-15 , and il-18 . among activating molecules displayed by the cd56cd16 nk cells , cd16 plays a critical role in targeting antibody fc - bound cells and executing antibody dependent cell - mediated cytotoxicity ( adcc ) . predominantly in tissues and secondary lymphoid organs , the cd56cd16 subset is responsible for production of ifn , tnf , gm - csf , and rantes after activation . the recent reevaluation of functional properties of both cell categories has changed the binary paradigm of nk biology , providing clear evidence of the rapid and simultaneous effector activities exerted by the same cd56cd16 nk cell subset . interestingly , we have now found that most dle - newly produced cells expressed cd16 and that microenvironmental prestimulation induces the density of this molecule ( figure 4 ) . the functional competence of nk cells produced from dle - stimulated lymphoid progenitors was tested , demonstrating a very efficient cytolytic potential when a nonradioactive method was used ( figure 5(a ) ) . thus , these data suggest that early progenitors are promoted to differentiate toward both cytotoxic and regulatory cells in response to dle . recently , cd56cd11c human nk - like cells were shown to induce the proliferation of t cells in an il-18 dependent manner [ 42 , 43 ] . because v2v2 t lymphocytes are key components of the innate immune system that function against infections and human tumor cells , we explore the faculty of dle - nk cells of activating them . of note , dilution of the dye ctv was significantly apparent when t cells were cultured with cd11cnk that are produced upon stimulation with dle . surprisingly , neither unstimulated t cells nor dle - directly stimulated t cells got activated ( figures 6 and 7 ) . activation of t cells can be achieved by a number of microorganisms and phosphoantigens and does not require antigen processing , but some atypical costimulatory molecules such as nkg2d play crucial roles in activating them to kill neoplastic or pathogen - infected cells . these innate cells function through a variety of mechanisms , including secretion of cytokines and chemokines , dendritic cell activation , macrophage recruitment , cytolytic activity , and antigen presentation . their contribution to immune - surveillance in a major histocompatibility complex- ( mhc- ) unrestricted way , by their capability of producing ifn and tnf and via an nk - like pathway , has positioned them as attractive targets for immunotherapy strategies [ 4548 ] . taken together , our study provides a rational explanation for the adjuvant contribution of dle to the observed effective immune responses against viral and malignant diseases . dle could play an inductor role in the nk lineage emergent hematopoiesis , suggesting the need of elucidating central mechanisms that govern dle - associated tissue regeneration and give an insight into novel cooperative drug activities . while remarkable progress has long been recorded in identifying targets for cellular - based immunotherapeutic strategies to improve acute and chronic disease outcomes , it is becoming clear that definition of extrinsic factors promoting innate cell differentiation processes from the earliest developmental stages may change our vision of immunomodulation . our findings suggest , for the first time , that , in response to dialyzable leukocyte extracts ( dle ) transferon , human stem and progenitor cells contribute to the emergent production of a special subset of innate cd11c nk cells . of note , this dle - derived nk cell population is endowed with properties such as ifn production , tumor cell cytotoxicity , and the capability of inducing t lymphocyte proliferation that may , in turn , function as coadjuvant component of innate immune responses against virus - infected or tumor cells .

the high incidence of infections and high rates of hospitalization , together with institutionalized long - term care ( ltc ) , highlights the important role of management to the decrease antibacterial susceptibility of organisms cultured from elderly patients . the reservoir for these microorganisms may be the bacterial flora in patients receiving long - term antimicrobial therapy or health care personnel who are colonized with resistant strains or infected with the same pathogens . multidrug - resistant gram - negative rods ( mdr - gnr ) are rapidly spreading throughout ltcfs around the world , and antibiotics are also commonly prescribed for older adults residing in ltcfs . there is risk of development of multi - drug antibiotic resistance after exposure and risk of drug - related adverse effects . furthermore , the increased use of antibiotics in ltcfs results in significant costs . the rapid spread of antimicrobial - resistant bacteria through health care institutions is considered a serious medical and public health issue . the intensive use of antibiotics promotes a relatively high prevalence of mdr - gnr and pathogens able to produce extended - spectrum -lactamases ( esbl ) in ltcfs . ltcfs have older populations with increased prevalence of resistant bacteria isolated from urinary tract infections ( uti ) . resistant isolates are more frequent in long - term care populations than in the general population . preceding colonization with such microorganisms is a risk factor for the development of infection , but the presence of virulence factors in the pathogens is equally important . a wide range of virulence factors ( vfs ) and a persistence of multidrug resistance can make the treatment of infections challenging . they are essential for the interaction between e. coli and its host , as they facilitate colonization , proliferation , and the transition from uncomplicated to severe infections . knowledge of which vfs are prevalent in specific clinical situations and populations is important for the targeted prevention of e. coli infections , as revealed by other epidemiological studies [ 79 ] . older populations in long - term care facilities have an increased prevalence of resistant bacteria isolated from utis . until now , the prevalence of esbl - positive strains and drug resistance among ltcf - residents in poland has not been studied . for this reason , the aim of this study was to assess the prevalence of mdr - gnr and esbl pathogens isolated from asymptomatic bacteriuria and utis . the second purpose of this study was to look for a relationship between the phylogeny , antimicrobial resistance , and virulence among e. coli isolates from bacteriuria . the proposed methods will be the basis for the development of recommendations for empirical treatment of ltcf residents . the point prevalence study was conducted for cases of asymptomatic bacteriuria and a continuing study is being conducted to measure the rate of infections with esbl etiology . a 1-day prevalence survey ( point prevalence study [ pps ] ) was carried out in october 2009 in 3 ltcfs in krakow , poland : 2 residential homes and 1 nursing home . a resident was defined as a person staying in an ltcf during the study day for at least 48 h. a control patient was a person staying in home care ( hc ) , but requiring home visits of a physician . in a nursing home ( nh ) , residents need 24 h / day medical or skilled nurse supervision and intensive health care is provided . in the residential home ( rh ) , residents are unable to live independently and require supervision or assistance with activities of daily living . the study protocol was approved by the ethics committee of jagiellonian university ( kbet/57/b/2008 ) , conducted in accordance with the declaration of helsinki , and carefully explained to the participants , who then gave their written informed consent . medical charts were reviewed for patient demographic data , comorbidities , and residency in an ltcf . medical documentation of residents was analyzed for the presence of chronic diseases and other medical problems . the barthel index ( bi ) is a 10-item measure of disability based on daily activities ( e.g. , bathing , transfer , dressing , feeding , mobility , stairs , using the toilet , and grooming ) . the score corresponds to the sum of all the points obtained , and can range from 0 to 100 points . the katz index of independence in activities of daily living is an instrument used to assess functional status as a measurement of the patient s ability to perform activities of daily living independently . the index ranks the adequacy of performance in 6 functions : bathing , dressing , toileting , transferring , continence , and feeding . a score of 6 indicates full function , 4 indicates moderate impairment , and 2 or less indicates severe functional impairment . physical dependence ( mobility ) was classified according to a 5-point scale ( 1 independent , 2 independent with falls , 3 limitations in mobility , 4 confined to bed , self - changing body position , 5 confined to bed , dependent ) . data on hospital exposure longer than 7 days in the 3 months preceding enrollment and antibiotics taken in the last month were collected . infections were defined according to the mcgeer criteria and were detected by trained health personnel in cooperation with a project worker . continuing prospective infection control was performed between 1 december 2009 and 30 november 2010 , using the standard mcgeer definition protocol . urine samples were taken only from residents when the symptoms of infections occurred . in the period between enrollment and each follow - up , data on potential factors that would increase the likelihood of mdro acquisition were also collected ( ie , antibiotic and hospital exposure , and presence of infections ) . the relationship between types of care , socio - demographic characteristics , probability , and epidemiology of esbl were analyzed with 2 main groups of statistical techniques . if the numerical parameters ( e.g. , age , length of stay ) were compared with the nominal characteristics ( e.g. , type of care , form of infection ) , a simple anova test was used . if the distribution of numerical characteristics did not fit the normal distribution , the nonparametric alternative to anova , the kruskal - wallis test , was used . chi - square ( ) frequency tests and a likelihood ratio were used for the contingency of nominal characteristics . all analyses were performed using the sas jmp 7.01 package ( jmp , version 7 . detailed information about epidemiology of infections in the point prevalence study and the continuing study among included ltcfs has been described . samples of urine were routinely collected ( clean - catch method ) and cultured by standard quantitative methods at the beginning of the study ( pps ) . when symptoms of uti in any enrolled resident occurred throughout the study ( continuing study ) , urine samples were also taken . isolates were identified using polymerase chain reaction ( pcr ) with species - specific primers or phenotypic methods ( api i d 32e , biomerieux ) . all strains belonging to the enterobacteriaceae family were tested using disk diffusion antimicrobial susceptibility methods on mueller - hinton agar plates according to current guidelines of the european committee on antimicrobial susceptibility testing ( clinical breakpoints tables v. 1.3 ; http://www.eucast.org v.1.3 ) . antimicrobials included ampicillin ( amp 10 g ) , piperacillin / tazobactam ( tzp 30 g/6 g ) , ceftazidime ( caz 10 g ) , cefotaxime ( ctx 5 g ) , cefepime ( fep 30 g ) , cefuroxime ( 30 g ) ) , imipenem ( ipm 10 g ) , aztreonam ( atm 30 g ) , ciprofloxacin ( cip 5 g ) , gentamicin ( cn 10 g ) , trimethoprim / sulfamethoxazole ( sxt 25 g ) , and nitrofurantoin ( f 100 g ) . esbl activity was detected with a modified double - disk synergy test ( ddst ) using a combination of cefotaxime ( 5 g ) , ceftazidime ( 10 g ) , cefepime ( 30 g ) , and aztreonam ( 30 g ) disks , placed 20 mm apart around a disk containing amoxicillin - clavulanate ( 20 g/10 g ) . enhancement of the inhibition zone toward the amoxicillin - clavulanate disks was taken as presumptive evidence of esbl production . each isolate was classified as susceptible , intermediate , or resistant using the european committee on antimicrobial susceptibility testing breakpoints . criteria for multidrug resistance were defined as resistance or intermediate resistance to 3 or more antimicrobial groups ( co - resistance ) or 4 or more of the antimicrobials . all isolates with esbl activity ( according to ddst ) were screened with multiplex - pcr for the presence of blactx - m , blashv , and blatem genes using previously published primers . the reaction mixture ( 25 l ) was amplified using the following conditions : 15 min at 95c , 30 cycles of 30 s at 94c , 30 s at 55c , and 2 min at 72c , with a final extension of 10 min at 72c . the amplicon sizes were 445 , 593 , and 747 bp for blatem , blactx - m , and blashv , respectively . bands were visualized using the uvp geldocit imaging system after 1.5% tae - agarose electrophoresis ( 90 min , 100 mv ) with ethidium bromide ( biorad ) . the strains with pcr amplicons positive for any of the previously described bla were selected for sequencing . pcr products were purified using a nucleospin extract ii kit ( macherey - nagel ) . the nucleotide sequences were analyzed with the software available at the national center of biotechnology information ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) . e. coli isolates from bacteriuria were checked for the presence of selected virulence genes usually associated with the e. coli strains responsible for extraintestinal infections : papc and papef ( p fimbriae ) , fimh ( type 1 pili ) , hlya ( hemolysin ) , iuta ( aerobactin ) , and sfa ( s and f1c fimbriae ) using pcr with previously published primers . each vf gene was amplified in a total volume of 25 l containing 2 master mix ( a&a biotechnology , poland ) , 0.5 m primer and dna . a negative control ( containing the same mixture , but with water instead of dna ) was included in each pcr run . the amplification products were separated by electrophoresis in 1.5% agarose gel and visualized using the uvp geldocit imaging system . the results were considered positive if the amplification product was of the expected molecular size ( papc 200 bp , papef the strains were classified according to the escherichia coli reference collection ( ecor ) system by the use of the rapid phylogenetic grouping technique described by clermont et al . . this method is based on a multiplex pcr involving the amplification of 2 genes ( chua and yjaa ) and of an anonymous fragment of dna from e. coli ( tspe4c2 ) . the results are interpreting as follows : chua and yjaa positive indicated group b2 ; chua positive and yjaa negative indicated group d ; chua negative and tspe4c2 positive indicated group b1 ; and chua negative and tspe4c2 negative indicated group a. differences were tested using the test . a 1-day prevalence survey ( point prevalence study [ pps ] ) was carried out in october 2009 in 3 ltcfs in krakow , poland : 2 residential homes and 1 nursing home . a resident was defined as a person staying in an ltcf during the study day for at least 48 h. a control patient was a person staying in home care ( hc ) , but requiring home visits of a physician . in a nursing home ( nh ) , residents need 24 h / day medical or skilled nurse supervision and intensive health care is provided . in the residential home ( rh ) , residents are unable to live independently and require supervision or assistance with activities of daily living . the study protocol was approved by the ethics committee of jagiellonian university ( kbet/57/b/2008 ) , conducted in accordance with the declaration of helsinki , and carefully explained to the participants , who then gave their written informed consent . medical charts were reviewed for patient demographic data , comorbidities , and residency in an ltcf . medical documentation of residents was analyzed for the presence of chronic diseases and other medical problems . the barthel index ( bi ) is a 10-item measure of disability based on daily activities ( e.g. , bathing , transfer , dressing , feeding , mobility , stairs , using the toilet , and grooming ) . the score corresponds to the sum of all the points obtained , and can range from 0 to 100 points . the katz index of independence in activities of daily living is an instrument used to assess functional status as a measurement of the patient s ability to perform activities of daily living independently . the index ranks the adequacy of performance in 6 functions : bathing , dressing , toileting , transferring , continence , and feeding . a score of 6 indicates full function , 4 indicates moderate impairment , and 2 or less indicates severe functional impairment . physical dependence ( mobility ) was classified according to a 5-point scale ( 1 independent , 2 independent with falls , 3 limitations in mobility , 4 confined to bed , self - changing body position , 5 confined to bed , dependent ) . data on hospital exposure longer than 7 days in the 3 months preceding enrollment and antibiotics taken in the last month were collected . infections were defined according to the mcgeer criteria and were detected by trained health personnel in cooperation with a project worker . continuing prospective infection control was performed between 1 december 2009 and 30 november 2010 , using the standard mcgeer definition protocol . urine samples were taken only from residents when the symptoms of infections occurred . in the period between enrollment and each follow - up , data on potential factors that would increase the likelihood of mdro acquisition were also collected ( ie , antibiotic and hospital exposure , and presence of infections ) . the relationship between types of care , socio - demographic characteristics , probability , and epidemiology of esbl were analyzed with 2 main groups of statistical techniques . if the numerical parameters ( e.g. , age , length of stay ) were compared with the nominal characteristics ( e.g. , type of care , form of infection ) , a simple anova test was used . if the distribution of numerical characteristics did not fit the normal distribution , the nonparametric alternative to anova , the kruskal - wallis test , was used . chi - square ( ) frequency tests and a likelihood ratio were used for the contingency of nominal characteristics . all analyses were performed using the sas jmp 7.01 package ( jmp , version 7 . detailed information about epidemiology of infections in the point prevalence study and the continuing study among included ltcfs has been described . samples of urine were routinely collected ( clean - catch method ) and cultured by standard quantitative methods at the beginning of the study ( pps ) . when symptoms of uti in any enrolled resident occurred throughout the study ( continuing study ) , urine samples were also taken . isolates were identified using polymerase chain reaction ( pcr ) with species - specific primers or phenotypic methods ( api i d 32e , biomerieux ) . all strains belonging to the enterobacteriaceae family were tested using disk diffusion antimicrobial susceptibility methods on mueller - hinton agar plates according to current guidelines of the european committee on antimicrobial susceptibility testing ( clinical breakpoints tables v. 1.3 ; http://www.eucast.org v.1.3 ) . antimicrobials included ampicillin ( amp 10 g ) , piperacillin / tazobactam ( tzp 30 g/6 g ) , ceftazidime ( caz 10 g ) , cefotaxime ( ctx 5 g ) , cefepime ( fep 30 g ) , cefuroxime ( 30 g ) ) , imipenem ( ipm 10 g ) , aztreonam ( atm 30 g ) , ciprofloxacin ( cip 5 g ) , gentamicin ( cn 10 g ) , trimethoprim / sulfamethoxazole ( sxt 25 g ) , and nitrofurantoin ( f 100 g ) . esbl activity was detected with a modified double - disk synergy test ( ddst ) using a combination of cefotaxime ( 5 g ) , ceftazidime ( 10 g ) , cefepime ( 30 g ) , and aztreonam ( 30 g ) disks , placed 20 mm apart around a disk containing amoxicillin - clavulanate ( 20 g/10 g ) . enhancement of the inhibition zone toward the amoxicillin - clavulanate disks was taken as presumptive evidence of esbl production . each isolate was classified as susceptible , intermediate , or resistant using the european committee on antimicrobial susceptibility testing breakpoints . criteria for multidrug resistance were defined as resistance or intermediate resistance to 3 or more antimicrobial groups ( co - resistance ) or 4 or more of the antimicrobials . all isolates with esbl activity ( according to ddst ) were screened with multiplex - pcr for the presence of blactx - m , blashv , and blatem genes using previously published primers . the reaction mixture ( 25 l ) was amplified using the following conditions : 15 min at 95c , 30 cycles of 30 s at 94c , 30 s at 55c , and 2 min at 72c , with a final extension of 10 min at 72c . the amplicon sizes were 445 , 593 , and 747 bp for blatem , blactx - m , and blashv , respectively . bands were visualized using the uvp geldocit imaging system after 1.5% tae - agarose electrophoresis ( 90 min , 100 mv ) with ethidium bromide ( biorad ) . the strains with pcr amplicons positive for any of the previously described bla were selected for sequencing . pcr products were purified using a nucleospin extract ii kit ( macherey - nagel ) . the nucleotide sequences were analyzed with the software available at the national center of biotechnology information ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) . e. coli isolates from bacteriuria were checked for the presence of selected virulence genes usually associated with the e. coli strains responsible for extraintestinal infections : papc and papef ( p fimbriae ) , fimh ( type 1 pili ) , hlya ( hemolysin ) , iuta ( aerobactin ) , and sfa ( s and f1c fimbriae ) using pcr with previously published primers . each vf gene was amplified in a total volume of 25 l containing 2 master mix ( a&a biotechnology , poland ) , 0.5 m primer and dna . a negative control ( containing the same mixture , but with water instead of dna ) was included in each pcr run . the amplification products were separated by electrophoresis in 1.5% agarose gel and visualized using the uvp geldocit imaging system . the results were considered positive if the amplification product was of the expected molecular size ( papc 200 bp , papef the strains were classified according to the escherichia coli reference collection ( ecor ) system by the use of the rapid phylogenetic grouping technique described by clermont et al . . this method is based on a multiplex pcr involving the amplification of 2 genes ( chua and yjaa ) and of an anonymous fragment of dna from e. coli ( tspe4c2 ) . the results are interpreting as follows : chua and yjaa positive indicated group b2 ; chua positive and yjaa negative indicated group d ; chua negative and tspe4c2 positive indicated group b1 ; and chua negative and tspe4c2 negative indicated group a. differences were tested using the test . samples of urine were routinely collected ( clean - catch method ) and cultured by standard quantitative methods at the beginning of the study ( pps ) . when symptoms of uti in any enrolled resident occurred throughout the study ( continuing study ) , urine samples were also taken . isolates were identified using polymerase chain reaction ( pcr ) with species - specific primers or phenotypic methods ( api i d 32e , biomerieux ) . all strains belonging to the enterobacteriaceae family were tested using disk diffusion antimicrobial susceptibility methods on mueller - hinton agar plates according to current guidelines of the european committee on antimicrobial susceptibility testing ( clinical breakpoints tables v. 1.3 ; http://www.eucast.org v.1.3 ) . antimicrobials included ampicillin ( amp 10 g ) , piperacillin / tazobactam ( tzp 30 g/6 g ) , ceftazidime ( caz 10 g ) , cefotaxime ( ctx 5 g ) , cefepime ( fep 30 g ) , cefuroxime ( 30 g ) ) , imipenem ( ipm 10 g ) , aztreonam ( atm 30 g ) , ciprofloxacin ( cip 5 g ) , gentamicin ( cn 10 g ) , trimethoprim / sulfamethoxazole ( sxt 25 g ) , and nitrofurantoin ( f 100 g ) . esbl activity was detected with a modified double - disk synergy test ( ddst ) using a combination of cefotaxime ( 5 g ) , ceftazidime ( 10 g ) , cefepime ( 30 g ) , and aztreonam ( 30 g ) disks , placed 20 mm apart around a disk containing amoxicillin - clavulanate ( 20 g/10 g ) . enhancement of the inhibition zone toward the amoxicillin - clavulanate disks was taken as presumptive evidence of esbl production . each isolate was classified as susceptible , intermediate , or resistant using the european committee on antimicrobial susceptibility testing breakpoints . criteria for multidrug resistance were defined as resistance or intermediate resistance to 3 or more antimicrobial groups ( co - resistance ) or 4 or more of the antimicrobials . all isolates with esbl activity ( according to ddst ) were screened with multiplex - pcr for the presence of blactx - m , blashv , and blatem genes using previously published primers . the reaction mixture ( 25 l ) was amplified using the following conditions : 15 min at 95c , 30 cycles of 30 s at 94c , 30 s at 55c , and 2 min at 72c , with a final extension of 10 min at 72c . the amplicon sizes were 445 , 593 , and 747 bp for blatem , blactx - m , and blashv , respectively . bands were visualized using the uvp geldocit imaging system after 1.5% tae - agarose electrophoresis ( 90 min , 100 mv ) with ethidium bromide ( biorad ) . the strains with pcr amplicons positive for any of the previously described bla were selected for sequencing . pcr products were purified using a nucleospin extract ii kit ( macherey - nagel ) . the nucleotide sequences were analyzed with the software available at the national center of biotechnology information ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) . e. coli isolates from bacteriuria were checked for the presence of selected virulence genes usually associated with the e. coli strains responsible for extraintestinal infections : papc and papef ( p fimbriae ) , fimh ( type 1 pili ) , hlya ( hemolysin ) , iuta ( aerobactin ) , and sfa ( s and f1c fimbriae ) using pcr with previously published primers . each vf gene was amplified in a total volume of 25 l containing 2 master mix ( a&a biotechnology , poland ) , 0.5 m primer and dna . a negative control ( containing the same mixture , but with water instead of dna ) was included in each pcr run . the amplification products were separated by electrophoresis in 1.5% agarose gel and visualized using the uvp geldocit imaging system . the results were considered positive if the amplification product was of the expected molecular size ( papc 200 bp , papef the strains were classified according to the escherichia coli reference collection ( ecor ) system by the use of the rapid phylogenetic grouping technique described by clermont et al . . this method is based on a multiplex pcr involving the amplification of 2 genes ( chua and yjaa ) and of an anonymous fragment of dna from e. coli ( tspe4c2 ) . the results are interpreting as follows : chua and yjaa positive indicated group b2 ; chua positive and yjaa negative indicated group d ; chua negative and tspe4c2 positive indicated group b1 ; and chua negative and tspe4c2 negative indicated group a. differences were tested using the test . the study was conducted in a group of 217 persons ( 193 residents of ltcfs and 24 from hc as a control group ) . of these residents , 86 ( 39.6% ) stayed at rhs , and 107 stayed at nhs . the studied sample corresponded to 2.6% of the total ltcf population in poland in 2010 . among the 217 patients participating in this research , 1 patient quit being in a ltcf , the population studied was heterogeneous and significantly diverse with regards to : incidence of hospitalization before study , body weight , problems with maintaining personal hygiene ( expressed as urinary / stool incontinence and the katz index ) , and expressed necessity of care ( barthel index , table 1 ) . the mean value of the barthel index was 75.4 in hc ( sd : 27.7 , 95% ci : 63.787.1 ) , 75.6 in rh ( sd : 34.5 , 95% ci : 68.283.0 ) , and 19.5 in nhs ( sd : 17.4 , 95% ci : 16.122.9 ) . in nhs none of the residents had bi higher than 76 points , and 66.7% of hc patients and 69.8% of rh residents had scores above 76 points . the mean value of katz index was 4.4 for hc patients ( sd : 2.2 , 95% ci : 3.45.3 ) , 4.7 for rh residents ( sd : 2.1 , 95% ci : 4.25.1 ) , and 1.3 for nh residents ( sd : 1.6 , 95% ci : 0.91.6 ) . considerably older patients ( aged 80 ) stayed in hc , while younger ( age < 80 ) patients were more likely to be residents of rhs . the average age for the population ( 63.2% female ) was 76.2 years in rh ( standard deviation sd : 10.5 , 95% ci : 73.978.4 ) , 76.8 years in nhs ( sd : 11.1 , 95% ci : 74.678.9 ) , and 82.9 in hc ( sd : 9.9 , 95% ci : 78.787.0 ) . the average length of ltcf stay was 6.5 years ( sd 5.9 ; 95%ci 4.15.9 ) . differences were also observed in the physical activity of residents nh residents were more likely to have limited ability to walk independently . the mean mobility was 2.4 for hc patients ( sd : 1.6 , 95% ci : 1.73.1 ) , 1.9 for rh residents ( sd : 1.2 , 95% ci : 1.72.2 ) , and 3.7 for nh residents ( sd : 1.1 , 95% ci : 3.53.9 ) . independent mobility ( with no restrictions ) was characteristic for 12 ( 50.0% ) hc patients , 48 ( 55.8% ) rh residents , and 5 ( 4.7% ) nh residents . there were 5 ( 20.8% ) hc residents who were confined to bed and dependent , 4 ( 4.7% ) in rh , and 33 ( 30.8% ) in nhs . the general prevalence rate of infections was 22.1/100 residents and general incidence density rate was 2.7/1000 residents per day for both groups ( residents and home care patients ) . the general prevalence rate of esbl was 4.6/100 in residents with bacteriuria and 0.0/100 in the control group . the incidence density of esbl - producing isolates was 0.3/1000 residents per day ( only in nhs and rh ) . gram - negative rods were identified in 101 cultures of urine : 9 from utis and 92 from asymptomatic bacteriuria . the most commonly isolated pathogen was e. coli ( 58.4% , n=59 , including infections ) . other isolated species included : klebsiella pneumonia ( 13.9% , n=14 ) , proteus spp . accordingly , 60 isolates were ampicillin resistant ( 58.7% from bacteriuria and 75.0% from uti ) ( figure 1 . ) . resistance to trimethoprim / sulfamethoxazole was identified in 45 isolates ( 43.5% from bacteriuria and 71.4% uti ) . a significant proportion of isolates were resistant to fluoroquinolones , and 33 isolates were resistant to ciprofloxacin ( 32.7% ) . the prevalence of pathogens resistant to more than 4 antimicrobials was 16.3% among asymptomatic bacteriuria samples and 33.3% among utis . the most common co - resistance pattern was gentamycin and trimethoprim / sulfamethoxazole , present among 9.1% of isolates . ( n=6 ) , but there was 1 citrobacter spp . and 1 enterobacter aerogenes that were also esbl - positive asymptomatic bacteriuria was not a risk factor for the occurrence of an esbl - positive strain ( p=0.09 ) . among residents who were confined to bed , the frequency of isolation of esbl - positive strains was 3 times higher than in residents able to walk independently . the factors independently associated with esbl were urinary tract infections ( or 5.4 , p=0.0012 ) , urinary and/or fecal incontinence ( or 13.5 , p<0.001 ) , being wheelchair - dependent or being confined to bed ( or 6.2 , p<0.001 ) , low value on the barthel ( or 3.6 , p<0.001 ) and katz indexes ( or 2.8 , p<0.015 ) , and prior hospitalization ( or 3.1 , p<0.045 ) ( table 2 ) . the mortality of residents with esbl - positive strains was significantly higher ( or 4.1 , p<0.001 ) . any antibiotic used in the preceding month also remained a significant risk factor ( or 3.2 , p=0.03 ) associated with esbls . from the group of ltcf resident isolates , pcr experiments performed on bacterial dna with primers specific for 3 bla genes showed that blactx - m was present in 9 strains ( 75.0% ) , blatem in 9 strains ( 75.0% ) , and blashv in 1 ( 7.1% ) . among the isolates , 9 ( 64.3% ) were characterized by the presence of a single -lactamase gene , whereas 5 ( 35.7% ) had a combination of 2 different bla genes ( blactx - m and blatem ) . furthermore , sequencing of the pcr products revealed the presence of 8 blactx - m-15 ( 92.9% ) and 1 blactx - m-3 ( 7.1% ) . the only blactx - m-3 -lactamase was identified in klebsiella oxytoca , in a sample taken from a case of asymptomatic bacteriuria . all blatem were identified as blatem-1 and 1 blashv as blashv-12 . among e. coli isolates , 13 clustered in the ecor group a , 2 clustered in the b1 group , 31 in the b2 group , and 10 in the d group ( table 3 ) . of these , 4 esbl - positive strains belonged to the b2 group , 1 to the d group , and 1 to the a group . vf genes were detected in strains from all of the ecor groups , and no relationship between the number of vf genes and the ecor group was found . the most prevalent virulence factor was fimh , which occurred in 46 isolates ( 82.1% ) . the potential ability of hemolysin production ( expressed by the presence of hlya gene ) was characteristic for 4 strains ( 7.1% ) and 3 of these belonged to the b2 group . the papc gene was found in 16 isolates ( 28.6% ) , papef in 6 ( 10.7% ) , and sfa / foc was found in 13 ( 23.2% ) strains . e. coli able to produce esbl possessed up to 2 different virulence factors ( median : 1.5 , range : 02 ) , whereas non - esbl e. coli possessed up to 6 ( median : 2 , range : 06 ) . isolates belonging to the b2 group were more likely to be resistant to fluoroquinolones than those from the a or d group ( 41.9% , 23.1% , and 30.0% , respectively ) . the presented results are derived from the first polish study concerning urinary tract infections among long - term care facility residents . the study was performed using 2 independent approaches : a prevalence study ( pps ) and an incidence study . these kinds of research programs are carried out in europe and worldwide the population of polish nh and rh residents was slightly different from the population of institutionalized elderly in other countries . the mean age of such residents in italy is 81 years and 83 in germany . in norwegian ltcfs , furthermore , the participation of residents confined to bed and residents with low barthel and katz index scores was relatively lower compared to norwegian or italian surveys . the use of invasive diagnostic and therapeutic procedures ( e.g. , catheterization ) was also lower in this study than in italy ( 18.4% vs. 60.6% ) . the published literature on the epidemiology of esbls in ltcfs is not conclusive ; for instance , the incidence density of esbl - positive enterobacteriaceae in our study was not comparable with data from a swiss tertiary care hospital ( 0.7/1000 patients per day ) . this suggests the need for further multicentre studies involving a larger number of residents over the spread of the studied group of microorganisms . the most commonly isolated pathogen was e. coli ( 58.4% , n=59 , including infections ) , similarly to other studies , for hospital patients , patients with cauti , and outpatients . patients colonized with esbl - positive strains and mdro are considered as reservoirs and are potential sources of infection . it is important to note that colonization often precedes an infection , and about one - fourth of colonized residents may develop an infection with the same factor . the prevalence of bacteriuria in residents without chronic long - term indwelling catheters reaches 2550% for women and 1540% for men , and approximately 40% of nosocomial infections originate in the urinary tract . in this study , bacteriuria was found in 42.5% of residents , and infection developed in 4.6% . in this study , residents infected with esbl - positive strains were 2.6 times more frequently transferred from ltcfs to hospitals and treated under hospital conditions . their attendance at hospital units requires special rules for isolation , common for patients with mdros ( contact isolation ) . the genes responsible for drug resistance are frequently located on plasmids , which contributes to their fast spread . this study identified a relatively high prevalence of esbl - positive strains among residents of ltcfs . another polish survey , conducted in 13 polish hospitals , found a 11.1% prevalence of esbl - positive pathogens , which is consistent with our study . the identification of esbl types revealed the very common occurrence of ctx - m-15 -lactamase , as far as tem-1 , whereas shv appeared rarely ( only 1 case ) . the rapid increase in the prevalence of ctx - m producers can not be questioned and it is the most prevalent esbl in other countries . predictors of esbl - positive strains found by other authors include permanent urinary catheter , pressure sores , and any recent invasive procedures . other strong risk factors that increased the likelihood of esbl , like being wheelchair - dependent or confined to bed , and low scores on the barthel or katz indexes , have not yet been investigated . resistance to gentamicin and trimethoprim / sulfamethoxazole frequently occurs among esbl - positive isolates [ 17,30,3537 ] . our study confirmed that 35.7% of esbl - positive strains were resistant to both of these antibiotics . the majority of the studied isolates were resistant to commonly prescribed antibiotics , including ampicillin , trimethoprim / sulfamethoxazole , and ciprofloxacin . this data raises serious concern about the therapeutic options available for physicians treating ltcf residents . the prevalence of pathogens resistant to more than 4 antimicrobials was 16.2% , which is less than in the ofallon report from boston ltcfs . resistance to piperacillin / tazobactam , which are counted as broad - spectrum antimicrobials , was very low ( 1.4% ) . piperacillin - tazobactam was the most active agent for out - patient urinary isolates in turkey . it has been shown that the number of vfs is proportional to its pathogenic potential . fimh protein recognizes its receptor on uroplakin , which is disseminated on the surface of uroepithelium . it acts as an adhesin but also as an invasin , because it is necessary to initiate the response signalling pathways of the host , leading to internalization of e. coli . its high frequency shows that it is important in the early stage of urinary tract infection . the prevalence of the fimh gene , which is an adhesin important in the early phase of uti , was lower than in studies conducted by narciso et al . . p fimbriae are the second common virulence factor of uropathogenic escherichia coli , which plays an important role in the pathogenesis of ascending utis and pyelonephritis in humans . in our study we show that e. coli isolates from bacteriuria are very often equipped in such fimbriae , as the papc gene was found in 28.6% isolates . s fimbriae and f1c fimbriae are implicated in the process of uti as they show binding to epithelial and endothelial cells . the s fimbriae are also associated with e. coli strains that cause sepsis , meningitis , and ascending utis . sfa / foc was found in 23.2% of isolates , which is also a large proportion of the isolates . the reason for the lower prevalence of hlya in this population than in uroseptic strains could be that those strains came from asymptomatic bacteriuria cases . the studied strains did not possess high levels of virulence , as the selection may favor strains of intermediate resistance . this study shows that the prevalence of esbl - producing rods isolated from urine samples from ltcf residents was lower then in other similar studies published recently . the findings derived from this study emphasize the need for further research on epidemiology of mdro and esbl in ltcfs , including transmission patterns , rates of infection , and factors associated with infections . it may be necessary to extend the requirements and precautions to mdr - gnr and esbl - producers . first , the number of patients included was very low ( only 193 residents from 3 ltcfs ) . this was due to mistrust on the part of residents ( who had to agree to participate ) and the insufficiently enthusiastic attitude of personnel . the control group was also very small , as these patients also had to agree to participate in the study . however , this is the first polish surveillance conducted in ltcfs , and further research should be done because it might benefit physicians in poland in the treatment of ltcf patients .

backgroundthe aim of this study was to assess the prevalence of multidrug - resistant escherichia coli and extended - spectrum -lactamases ( esbl ) pathogens isolated from asymptomatic bacteriuria and urinary tract infections ( utis ) , and the relationship between the phylogeny , antimicrobial resistance , and virulence among isolates in residents of 3 long - term care facilities ( ltcf ) in krakow , poland.material/methodsthis was point prevalence study and prospective infection control in a group of 217 people . urine samples were examined with standard microbiological methods and screened for the presence of blactx - m , blashv , and blatem . e. coli isolates were screened for 6 common virulence factors ( vfs ) and classified according to the rapid phylogenetic grouping technique.resultsamong all the strains tested , 14 isolates ( 13.9% ) expressed esbl activity . a significant proportion of isolates were resistant to ciprofloxacin ( 32.7% , n=33 ) . resistance to trimethoprim / sulfamethoxazole was identified among 45 isolates ( 44.5% ) . independent risk factors for the presence of an esbl - producing strain were : uti , urinary and/or fecal incontinence , bedridden , and low values of the barthel and katz indexes . gene sequencing identified 8 blactx - m-15 , 1 blactx - m-3 , 9 blatem-1 , and 1 blashv-12 . among e. coli , no relationship between number of vf genes and phylogeny was found . the most prevalent virulence factor was fimh ( 82.1%).conclusionsthe findings of this study emphasize the need for further research on the epidemiology of multi - drug resistant organisms ( mdro ) and esbl in ltcf , including transmission patterns , rates of infection , and factors associated with infections . it may be necessary to extend the requirements and precautions to mdro and esbl - producers .

Background Material and Methods Part I. Point prevalence study, PPS Part II. Continuing study Bacterial isolates Antimicrobial susceptibility Polymerase chain reaction (PCR) screening for extended-spectrum -lactamase genes and sequencing of bla genes Virulence factor screening Results Discussion Conclusions

the high incidence of infections and high rates of hospitalization , together with institutionalized long - term care ( ltc ) , highlights the important role of management to the decrease antibacterial susceptibility of organisms cultured from elderly patients . the reservoir for these microorganisms may be the bacterial flora in patients receiving long - term antimicrobial therapy or health care personnel who are colonized with resistant strains or infected with the same pathogens . there is risk of development of multi - drug antibiotic resistance after exposure and risk of drug - related adverse effects . the intensive use of antibiotics promotes a relatively high prevalence of mdr - gnr and pathogens able to produce extended - spectrum -lactamases ( esbl ) in ltcfs . ltcfs have older populations with increased prevalence of resistant bacteria isolated from urinary tract infections ( uti ) . resistant isolates are more frequent in long - term care populations than in the general population . preceding colonization with such microorganisms is a risk factor for the development of infection , but the presence of virulence factors in the pathogens is equally important . a wide range of virulence factors ( vfs ) and a persistence of multidrug resistance can make the treatment of infections challenging . they are essential for the interaction between e. coli and its host , as they facilitate colonization , proliferation , and the transition from uncomplicated to severe infections . older populations in long - term care facilities have an increased prevalence of resistant bacteria isolated from utis . for this reason , the aim of this study was to assess the prevalence of mdr - gnr and esbl pathogens isolated from asymptomatic bacteriuria and utis . the second purpose of this study was to look for a relationship between the phylogeny , antimicrobial resistance , and virulence among e. coli isolates from bacteriuria . the point prevalence study was conducted for cases of asymptomatic bacteriuria and a continuing study is being conducted to measure the rate of infections with esbl etiology . a 1-day prevalence survey ( point prevalence study [ pps ] ) was carried out in october 2009 in 3 ltcfs in krakow , poland : 2 residential homes and 1 nursing home . the study protocol was approved by the ethics committee of jagiellonian university ( kbet/57/b/2008 ) , conducted in accordance with the declaration of helsinki , and carefully explained to the participants , who then gave their written informed consent . medical documentation of residents was analyzed for the presence of chronic diseases and other medical problems . the relationship between types of care , socio - demographic characteristics , probability , and epidemiology of esbl were analyzed with 2 main groups of statistical techniques . detailed information about epidemiology of infections in the point prevalence study and the continuing study among included ltcfs has been described . when symptoms of uti in any enrolled resident occurred throughout the study ( continuing study ) , urine samples were also taken . antimicrobials included ampicillin ( amp 10 g ) , piperacillin / tazobactam ( tzp 30 g/6 g ) , ceftazidime ( caz 10 g ) , cefotaxime ( ctx 5 g ) , cefepime ( fep 30 g ) , cefuroxime ( 30 g ) ) , imipenem ( ipm 10 g ) , aztreonam ( atm 30 g ) , ciprofloxacin ( cip 5 g ) , gentamicin ( cn 10 g ) , trimethoprim / sulfamethoxazole ( sxt 25 g ) , and nitrofurantoin ( f 100 g ) . esbl activity was detected with a modified double - disk synergy test ( ddst ) using a combination of cefotaxime ( 5 g ) , ceftazidime ( 10 g ) , cefepime ( 30 g ) , and aztreonam ( 30 g ) disks , placed 20 mm apart around a disk containing amoxicillin - clavulanate ( 20 g/10 g ) . all isolates with esbl activity ( according to ddst ) were screened with multiplex - pcr for the presence of blactx - m , blashv , and blatem genes using previously published primers . the amplicon sizes were 445 , 593 , and 747 bp for blatem , blactx - m , and blashv , respectively . e. coli isolates from bacteriuria were checked for the presence of selected virulence genes usually associated with the e. coli strains responsible for extraintestinal infections : papc and papef ( p fimbriae ) , fimh ( type 1 pili ) , hlya ( hemolysin ) , iuta ( aerobactin ) , and sfa ( s and f1c fimbriae ) using pcr with previously published primers . the results were considered positive if the amplification product was of the expected molecular size ( papc 200 bp , papef the strains were classified according to the escherichia coli reference collection ( ecor ) system by the use of the rapid phylogenetic grouping technique described by clermont et al . a 1-day prevalence survey ( point prevalence study [ pps ] ) was carried out in october 2009 in 3 ltcfs in krakow , poland : 2 residential homes and 1 nursing home . the study protocol was approved by the ethics committee of jagiellonian university ( kbet/57/b/2008 ) , conducted in accordance with the declaration of helsinki , and carefully explained to the participants , who then gave their written informed consent . medical documentation of residents was analyzed for the presence of chronic diseases and other medical problems . the relationship between types of care , socio - demographic characteristics , probability , and epidemiology of esbl were analyzed with 2 main groups of statistical techniques . detailed information about epidemiology of infections in the point prevalence study and the continuing study among included ltcfs has been described . antimicrobials included ampicillin ( amp 10 g ) , piperacillin / tazobactam ( tzp 30 g/6 g ) , ceftazidime ( caz 10 g ) , cefotaxime ( ctx 5 g ) , cefepime ( fep 30 g ) , cefuroxime ( 30 g ) ) , imipenem ( ipm 10 g ) , aztreonam ( atm 30 g ) , ciprofloxacin ( cip 5 g ) , gentamicin ( cn 10 g ) , trimethoprim / sulfamethoxazole ( sxt 25 g ) , and nitrofurantoin ( f 100 g ) . all isolates with esbl activity ( according to ddst ) were screened with multiplex - pcr for the presence of blactx - m , blashv , and blatem genes using previously published primers . the amplicon sizes were 445 , 593 , and 747 bp for blatem , blactx - m , and blashv , respectively . e. coli isolates from bacteriuria were checked for the presence of selected virulence genes usually associated with the e. coli strains responsible for extraintestinal infections : papc and papef ( p fimbriae ) , fimh ( type 1 pili ) , hlya ( hemolysin ) , iuta ( aerobactin ) , and sfa ( s and f1c fimbriae ) using pcr with previously published primers . each vf gene was amplified in a total volume of 25 l containing 2 master mix ( a&a biotechnology , poland ) , 0.5 m primer and dna . the results were considered positive if the amplification product was of the expected molecular size ( papc 200 bp , papef the strains were classified according to the escherichia coli reference collection ( ecor ) system by the use of the rapid phylogenetic grouping technique described by clermont et al . this method is based on a multiplex pcr involving the amplification of 2 genes ( chua and yjaa ) and of an anonymous fragment of dna from e. coli ( tspe4c2 ) . when symptoms of uti in any enrolled resident occurred throughout the study ( continuing study ) , urine samples were also taken . all strains belonging to the enterobacteriaceae family were tested using disk diffusion antimicrobial susceptibility methods on mueller - hinton agar plates according to current guidelines of the european committee on antimicrobial susceptibility testing ( clinical breakpoints tables v. 1.3 ; http://www.eucast.org v.1.3 ) . antimicrobials included ampicillin ( amp 10 g ) , piperacillin / tazobactam ( tzp 30 g/6 g ) , ceftazidime ( caz 10 g ) , cefotaxime ( ctx 5 g ) , cefepime ( fep 30 g ) , cefuroxime ( 30 g ) ) , imipenem ( ipm 10 g ) , aztreonam ( atm 30 g ) , ciprofloxacin ( cip 5 g ) , gentamicin ( cn 10 g ) , trimethoprim / sulfamethoxazole ( sxt 25 g ) , and nitrofurantoin ( f 100 g ) . esbl activity was detected with a modified double - disk synergy test ( ddst ) using a combination of cefotaxime ( 5 g ) , ceftazidime ( 10 g ) , cefepime ( 30 g ) , and aztreonam ( 30 g ) disks , placed 20 mm apart around a disk containing amoxicillin - clavulanate ( 20 g/10 g ) . all isolates with esbl activity ( according to ddst ) were screened with multiplex - pcr for the presence of blactx - m , blashv , and blatem genes using previously published primers . the amplicon sizes were 445 , 593 , and 747 bp for blatem , blactx - m , and blashv , respectively . e. coli isolates from bacteriuria were checked for the presence of selected virulence genes usually associated with the e. coli strains responsible for extraintestinal infections : papc and papef ( p fimbriae ) , fimh ( type 1 pili ) , hlya ( hemolysin ) , iuta ( aerobactin ) , and sfa ( s and f1c fimbriae ) using pcr with previously published primers . each vf gene was amplified in a total volume of 25 l containing 2 master mix ( a&a biotechnology , poland ) , 0.5 m primer and dna . the results were considered positive if the amplification product was of the expected molecular size ( papc 200 bp , papef the strains were classified according to the escherichia coli reference collection ( ecor ) system by the use of the rapid phylogenetic grouping technique described by clermont et al . the study was conducted in a group of 217 persons ( 193 residents of ltcfs and 24 from hc as a control group ) . among the 217 patients participating in this research , 1 patient quit being in a ltcf , the population studied was heterogeneous and significantly diverse with regards to : incidence of hospitalization before study , body weight , problems with maintaining personal hygiene ( expressed as urinary / stool incontinence and the katz index ) , and expressed necessity of care ( barthel index , table 1 ) . the mean value of the barthel index was 75.4 in hc ( sd : 27.7 , 95% ci : 63.787.1 ) , 75.6 in rh ( sd : 34.5 , 95% ci : 68.283.0 ) , and 19.5 in nhs ( sd : 17.4 , 95% ci : 16.122.9 ) . resistance to trimethoprim / sulfamethoxazole was identified in 45 isolates ( 43.5% from bacteriuria and 71.4% uti ) . a significant proportion of isolates were resistant to fluoroquinolones , and 33 isolates were resistant to ciprofloxacin ( 32.7% ) . the prevalence of pathogens resistant to more than 4 antimicrobials was 16.3% among asymptomatic bacteriuria samples and 33.3% among utis . the most common co - resistance pattern was gentamycin and trimethoprim / sulfamethoxazole , present among 9.1% of isolates . and 1 enterobacter aerogenes that were also esbl - positive asymptomatic bacteriuria was not a risk factor for the occurrence of an esbl - positive strain ( p=0.09 ) . the factors independently associated with esbl were urinary tract infections ( or 5.4 , p=0.0012 ) , urinary and/or fecal incontinence ( or 13.5 , p<0.001 ) , being wheelchair - dependent or being confined to bed ( or 6.2 , p<0.001 ) , low value on the barthel ( or 3.6 , p<0.001 ) and katz indexes ( or 2.8 , p<0.015 ) , and prior hospitalization ( or 3.1 , p<0.045 ) ( table 2 ) . from the group of ltcf resident isolates , pcr experiments performed on bacterial dna with primers specific for 3 bla genes showed that blactx - m was present in 9 strains ( 75.0% ) , blatem in 9 strains ( 75.0% ) , and blashv in 1 ( 7.1% ) . among the isolates , 9 ( 64.3% ) were characterized by the presence of a single -lactamase gene , whereas 5 ( 35.7% ) had a combination of 2 different bla genes ( blactx - m and blatem ) . furthermore , sequencing of the pcr products revealed the presence of 8 blactx - m-15 ( 92.9% ) and 1 blactx - m-3 ( 7.1% ) . the only blactx - m-3 -lactamase was identified in klebsiella oxytoca , in a sample taken from a case of asymptomatic bacteriuria . among e. coli isolates , 13 clustered in the ecor group a , 2 clustered in the b1 group , 31 in the b2 group , and 10 in the d group ( table 3 ) . of these , 4 esbl - positive strains belonged to the b2 group , 1 to the d group , and 1 to the a group . vf genes were detected in strains from all of the ecor groups , and no relationship between the number of vf genes and the ecor group was found . the most prevalent virulence factor was fimh , which occurred in 46 isolates ( 82.1% ) . the potential ability of hemolysin production ( expressed by the presence of hlya gene ) was characteristic for 4 strains ( 7.1% ) and 3 of these belonged to the b2 group . the papc gene was found in 16 isolates ( 28.6% ) , papef in 6 ( 10.7% ) , and sfa / foc was found in 13 ( 23.2% ) strains . e. coli able to produce esbl possessed up to 2 different virulence factors ( median : 1.5 , range : 02 ) , whereas non - esbl e. coli possessed up to 6 ( median : 2 , range : 06 ) . the presented results are derived from the first polish study concerning urinary tract infections among long - term care facility residents . the study was performed using 2 independent approaches : a prevalence study ( pps ) and an incidence study . the published literature on the epidemiology of esbls in ltcfs is not conclusive ; for instance , the incidence density of esbl - positive enterobacteriaceae in our study was not comparable with data from a swiss tertiary care hospital ( 0.7/1000 patients per day ) . this suggests the need for further multicentre studies involving a larger number of residents over the spread of the studied group of microorganisms . the most commonly isolated pathogen was e. coli ( 58.4% , n=59 , including infections ) , similarly to other studies , for hospital patients , patients with cauti , and outpatients . the prevalence of bacteriuria in residents without chronic long - term indwelling catheters reaches 2550% for women and 1540% for men , and approximately 40% of nosocomial infections originate in the urinary tract . this study identified a relatively high prevalence of esbl - positive strains among residents of ltcfs . the rapid increase in the prevalence of ctx - m producers can not be questioned and it is the most prevalent esbl in other countries . other strong risk factors that increased the likelihood of esbl , like being wheelchair - dependent or confined to bed , and low scores on the barthel or katz indexes , have not yet been investigated . resistance to gentamicin and trimethoprim / sulfamethoxazole frequently occurs among esbl - positive isolates [ 17,30,3537 ] . our study confirmed that 35.7% of esbl - positive strains were resistant to both of these antibiotics . the majority of the studied isolates were resistant to commonly prescribed antibiotics , including ampicillin , trimethoprim / sulfamethoxazole , and ciprofloxacin . the prevalence of pathogens resistant to more than 4 antimicrobials was 16.2% , which is less than in the ofallon report from boston ltcfs . the prevalence of the fimh gene , which is an adhesin important in the early phase of uti , was lower than in studies conducted by narciso et al . p fimbriae are the second common virulence factor of uropathogenic escherichia coli , which plays an important role in the pathogenesis of ascending utis and pyelonephritis in humans . in our study we show that e. coli isolates from bacteriuria are very often equipped in such fimbriae , as the papc gene was found in 28.6% isolates . sfa / foc was found in 23.2% of isolates , which is also a large proportion of the isolates . the reason for the lower prevalence of hlya in this population than in uroseptic strains could be that those strains came from asymptomatic bacteriuria cases . this study shows that the prevalence of esbl - producing rods isolated from urine samples from ltcf residents was lower then in other similar studies published recently . the findings derived from this study emphasize the need for further research on epidemiology of mdro and esbl in ltcfs , including transmission patterns , rates of infection , and factors associated with infections . it may be necessary to extend the requirements and precautions to mdr - gnr and esbl - producers . this was due to mistrust on the part of residents ( who had to agree to participate ) and the insufficiently enthusiastic attitude of personnel .

as protein structures become increasingly available and structural genomics provide structural models in a genome - wide strategy ( 1 ) , proteins with unassigned functions are accumulating and the number of protein structures in the protein data bank ( pdb ) is rapidly rising ( 2 ) . the evolutionary classification databases , such as scop ( 3 ) and cath ( 4 ) , are valuable resources for understanding protein functions , structural similarity and evolutionary relationships . however , these two widely used databases are updated intermittently using manual and semi - automated methods . this current structure function gap clearly reveals the need for powerful automated methods to classify protein domains based on their tertiary structures and is important in producing manually tuned classification databases . many automatic domain classification approaches have been developed to determine similar structures and structural classification ( 5,6 ) of a query structure . protein sequence database search tools , such as blast ( 7 ) , psi - blast and superfamily ( 5 ) , are useful computational tools . however , these tools are commonly unreliable in detecting remote homologous relationships that are indicated by such structural alignment tools as dali ( 8) , mammoth ( 9 ) and ssm ( 10 ) . structural alignment tools typically take several seconds to align two known structures . at this speed , about one day is required to compare a single protein structure with those in pdb . scopmap ( 6 ) , which is computationally more expensive , combines sequence and structural information for scop superfamily assignment . recently , we have proposed a fast and efficient tool , called 3d - blast ( 11 ) , to quickly search similar structures . this tool is as fast as blast and provides the statistical significance ( e - value ) of an alignment to indicate the reliability of a structure . 3d - blast outperformed fast structural search methods ( topscan ( 12 ) and yakusa ( 13 ) ) and approached the performance of detailed structural alignment approaches ( ce ( 14 ) and mammoth ( 9 ) ) . 3d - blast is rapid and accurate in scanning a large protein structural database , and is useful in an initial scan for similar protein structures , which can be refined using detailed structural comparison methods . however , several factors that deteriorate 3d - blast 's performance are ( i ) 3d - blast may have made minor shifts in aligning two local segments with similar letters , because the structural alphabet do not consider actual euclidean distances , ( ii ) the e - values of the hit proteins are insignificant and ( iii ) the query is a multiple - domain protein ( 11 ) . this work presents an automated server ( fastscop ) , which integrates a fast structure database search tool ( 3d - blast ) and a detailed structural alignment tool ( mammoth ) , to recognize scop domains and scop superfamilies of a query structure . mammoth provided the z - score and root - mean - square deviation ( rmsd ) of the c atom positions of the aligned residues between the query structure and the hit structure according to the euclidean distance between corresponding residues rather than the distance between amino acid the classification accuracy of this server is 98% for 464 single - domain queries and 122 multiple - domain queries . to combine 3d - blast and mammoth is able to reduce the ill effects of 3d - blast to improve the assignment accuracy . after a query structure is assigned to a superfamily , this server is able to provide both multiple sequence alignments and multiple structural alignments of the selected members in a scop superfamily . figure 1 presents an overview of the fastscop server for rapidly recognizing scop domains and scop superfamilies . this sever uses 3d - blast to scan quickly the scop 1.71 database and selected the top 10 hit domain structures , which are associated with different scop superfamily entries ( figure 1b ) . mammoth was then adopted to align sequentially the query structure with each structure of the top 10 structures to refine the domain boundaries and to recognize scop superfamilies ( figure 1c and d ) . our previous work ( 11 ) demonstrated that 3d - blast required 1.4 s to scan the structural domains in scop 1.69 and was 16 990 and 1413 times faster than ce ( 14 ) and mammoth , respectively . these two detailed structural alignment tools perform similarly on the test set ; mammoth was 12 times faster than ce . the scop 1.71 database ( october 2006 ) has 75 930 domains that are derived from 27 599 pdb entries ( 18 january 2005 ) . the numbers of folds , superfamilies and families are 971 , 1589 and 3004 , respectively . 3d - blast requires structural alphabet sequence databases ( sadb ) for fast scanning a protein structural database . in this work , we created an sadb derived from known domain structures ( 12 927 domains ) in scop1.71 with < 95% identity to each other based on the ( , ) plot ( 11 ) . the fastscop has ( a ) four main steps , including ( b ) 3d - blast for scanning structural database , ( c ) mammoth for detailed structural alignment and ( d ) domain boundary refinement and reassignment . the fastscop has ( a ) four main steps , including ( b ) 3d - blast for scanning structural database , ( c ) mammoth for detailed structural alignment and ( d ) domain boundary refinement and reassignment . first , 3d - blast was adopted to identify the similar structures ( hit scop domains ) , which are ordered by e - value , of a query structure from an sadb database ( figure 1b ) . 3d - blast is the first tool to provide fast search of a protein structural database using the blast , which searches on a sadb database with a structural alphabet substitution matrix ( sasm ) ( 11 ) . the fastscop then selected the top 10 hit domains that have different scop superfamily entries . based on the structural alphabet alignments between the query and hit scop domains , this sever can identify multiple domains if a multiple - domain structure is queried . for each hit domain , the aligned length should be more than 40 residues and the overlap of two neighboring hit domains should be < 10% of the query protein . after the 10 ten hit scop domains were identified , this server applied mammoth to align sequentially the query structure with each structure of these hit domains , ordered by e - value . for each structural alignment , mammoth yielded the z - score and rmsd of the c atom positions of the aligned residues between the query structure and the hit structure ( figure 1c ) . the query structure ( or one domain of a multiple - domain protein ) was assigned to a scop superfamily when the pair - structure alignment satisfied the following criteria : ( i ) the z - score exceeds 5.5 ; ( ii ) the rmsd value is < 4 ; ( iii ) the value of ( z - score rmsd ) exceeds 4.0 and ( iv ) the number of the aligned residues exceeds 40 and the coverage rate between the query protein ( domain ) and hit domain exceeds 75% . in the third step , the fastscop refined the boundaries ( the start and end positions ) of the assigned domain according to the aligned regions and the sequence length of the hit domain ( figure 1d ) . finally , the fastscop executed steps 13 when the length of the unassigned region of the query structure was more than 40 residues . the fastscop server can identify the structural domains and determine the evolutionary classification of a query structure from evolutionary classification databases . when the query structure is a new protein structure , the fastscop server enables users to input the structure file in pdb format . this server typically yielded structural domains and the scop superfamilies of a query structure in an average of 6 s ( figure 2a ) . the server can present the members of the assigned scop superfamily and provide both multiple sequence alignments and multiple structural alignments ( figure 2b ) based on users requirements . the aligned structures are visualized in png format in molscript and raster3d packages ( figure 2c and d ) . the server allows a user to download the aligned structure coordinates in pdb format . figure 2.evolutionary superfamily assignment and structural alignment of the fastscop server using the structure of multi - domain immunophilin ( atfkbp42 ) from arabidopsis thaliana ( pdb code 2if4-a ) as the query . ( a ) the assigned scop superfamilies are the fkbp - like domain ( scop entry d.26.1 ) and the tpr domain ( scop entry a.118.8 ) . ( b ) multiple structural alphabet and amino acid sequences alignments of fkbp - like domain between the query protein and five homologous proteins . the aligned secondary structures are represented as a continuous color spectrum from red through orange , yellow , green and blue to violet . the color is mapped to ( c ) the structure of the fkbp - like domain . ( d ) structural alignments between the fkbp - like domain of the query protein and that of the homologous protein ( pdb code 1q1c - a ) . evolutionary superfamily assignment and structural alignment of the fastscop server using the structure of multi - domain immunophilin ( atfkbp42 ) from arabidopsis thaliana ( pdb code 2if4-a ) as the query . ( a ) the assigned scop superfamilies are the fkbp - like domain ( scop entry d.26.1 ) and the tpr domain ( scop entry a.118.8 ) . ( b ) multiple structural alphabet and amino acid sequences alignments of fkbp - like domain between the query protein and five homologous proteins . the aligned secondary structures are represented as a continuous color spectrum from red through orange , yellow , green and blue to violet . the color is mapped to ( c ) the structure of the fkbp - like domain . ( d ) structural alignments between the fkbp - like domain of the query protein and that of the homologous protein ( pdb code 1q1c - a ) . figure 2 shows a fastscop result with multi - domain immunophilin ( atfkbp42 ) from arabidopsis thaliana ( pdb code 2if4-a ) ( 15 ) as the query structure . the release date of this protein is 31 , october 2006 , and this protein has not been recorded in scop . as shown in figure 2a , the fastscop recognized two domains and their scop superfamilies , which are the fkbp - like superfamily ( scop entry d.26.1 ) and the tpr - like superfamily ( scop entry a.118.8 ) for this query . the fkbp domain ( figure 2c ) of atfkbp42 consists of a six - stranded anti - parallel -sheet , wrapped around a short -helix , and is similar to those of fkbp52 ( pdb code 1q1c - a ) ( 16 ) , fkbp 25 ( pdb code 1pbk ) ( 17 ) , fkbp 13 ( pdb code 1u79-a ) ( 18 ) and fkbp 12 ( pdb code 1bkf ) ( 19 ) . the fkbp domain has been demonstrated to interact with plasma membrane - localized abc transporters atpgp1 and atpgp , which directly mediate cellular auxin efflux ( 20 ) . the tpr domain of atfkbp42 is completely helical and binds to athsp90 , which is critical to plant development and phenotypic plasticity ( 21,22 ) . after the structural domains and evolutionary superfamilies were recognized , the fastscop server allowed users to browse similar structures of these superfamilies . using this atfkbp42 as a query , the server can identify 13 and 17 similar structures of the fkbp - like domain and tpr domain , respectively . figure 2b illustrates the multiple amino acid sequence alignment and structural alphabet alignment between atfkbp42 and five fkbp - like homologous proteins , including fkbp52 , fkbp 25 , fkbp 13 and fkbp 12 . the aligned secondary structures are represented as a continuous color spectrum from red through orange , yellow , green and blue to violet ( figure 2b and c ) . the structural alphabets were strongly conserved in areas of the secondary structures , which are -strands ( represented by structural alphabets e , f , h , k and n ) or -helices ( represented by structural alphabets a , y , b , c and d ) . these results reveal that the structural alphabet sequences are much better conserved than the amino acid sequences , which result explains why 3d - blast detected these distantly related proteins ( 11 ) . the fastscop server can identify the structural domains and determine the evolutionary classification of a query structure from evolutionary classification databases . when the query structure is a new protein structure , the fastscop server enables users to input the structure file in pdb format . this server typically yielded structural domains and the scop superfamilies of a query structure in an average of 6 s ( figure 2a ) . the server can present the members of the assigned scop superfamily and provide both multiple sequence alignments and multiple structural alignments ( figure 2b ) based on users requirements . the aligned structures are visualized in png format in molscript and raster3d packages ( figure 2c and d ) . the server allows a user to download the aligned structure coordinates in pdb format . figure 2.evolutionary superfamily assignment and structural alignment of the fastscop server using the structure of multi - domain immunophilin ( atfkbp42 ) from arabidopsis thaliana ( pdb code 2if4-a ) as the query . ( a ) the assigned scop superfamilies are the fkbp - like domain ( scop entry d.26.1 ) and the tpr domain ( scop entry a.118.8 ) . ( b ) multiple structural alphabet and amino acid sequences alignments of fkbp - like domain between the query protein and five homologous proteins . the aligned secondary structures are represented as a continuous color spectrum from red through orange , yellow , green and blue to violet . the color is mapped to ( c ) the structure of the fkbp - like domain . ( d ) structural alignments between the fkbp - like domain of the query protein and that of the homologous protein ( pdb code 1q1c - a ) . evolutionary superfamily assignment and structural alignment of the fastscop server using the structure of multi - domain immunophilin ( atfkbp42 ) from arabidopsis thaliana ( pdb code 2if4-a ) as the query . ( a ) the assigned scop superfamilies are the fkbp - like domain ( scop entry d.26.1 ) and the tpr domain ( scop entry a.118.8 ) . ( b ) multiple structural alphabet and amino acid sequences alignments of fkbp - like domain between the query protein and five homologous proteins . the aligned secondary structures are represented as a continuous color spectrum from red through orange , yellow , green and blue to violet . the color is mapped to ( c ) the structure of the fkbp - like domain . ( d ) structural alignments between the fkbp - like domain of the query protein and that of the homologous protein ( pdb code 1q1c - a ) . figure 2 shows a fastscop result with multi - domain immunophilin ( atfkbp42 ) from arabidopsis thaliana ( pdb code 2if4-a ) ( 15 ) as the query structure . the release date of this protein is 31 , october 2006 , and this protein has not been recorded in scop . as shown in figure 2a , the fastscop recognized two domains and their scop superfamilies , which are the fkbp - like superfamily ( scop entry d.26.1 ) and the tpr - like superfamily ( scop entry a.118.8 ) for this query . the fkbp domain ( figure 2c ) of atfkbp42 consists of a six - stranded anti - parallel -sheet , wrapped around a short -helix , and is similar to those of fkbp52 ( pdb code 1q1c - a ) ( 16 ) , fkbp 25 ( pdb code 1pbk ) ( 17 ) , fkbp 13 ( pdb code 1u79-a ) ( 18 ) and fkbp 12 ( pdb code 1bkf ) ( 19 ) . the fkbp domain has been demonstrated to interact with plasma membrane - localized abc transporters atpgp1 and atpgp , which directly mediate cellular auxin efflux ( 20 ) . the tpr domain of atfkbp42 is completely helical and binds to athsp90 , which is critical to plant development and phenotypic plasticity ( 21,22 ) . after the structural domains and evolutionary superfamilies were recognized , the fastscop server allowed users to browse similar structures of these superfamilies . using this atfkbp42 as a query , the server can identify 13 and 17 similar structures of the fkbp - like domain and tpr domain , respectively . figure 2b illustrates the multiple amino acid sequence alignment and structural alphabet alignment between atfkbp42 and five fkbp - like homologous proteins , including fkbp52 , fkbp 25 , fkbp 13 and fkbp 12 . the aligned secondary structures are represented as a continuous color spectrum from red through orange , yellow , green and blue to violet ( figure 2b and c ) . the structural alphabets were strongly conserved in areas of the secondary structures , which are -strands ( represented by structural alphabets e , f , h , k and n ) or -helices ( represented by structural alphabets a , y , b , c and d ) . these results reveal that the structural alphabet sequences are much better conserved than the amino acid sequences , which result explains why 3d - blast detected these distantly related proteins ( 11 ) . a query protein set , scop-586 ( table 1 ) , was selected to evaluate the utility of the fastscop server for recognizing the structural domains and evolutionary superfamilies of a query structure . the scop-586 query set has 464 single - domain proteins and 122 multiple - domain proteins that are in scop 1.69 but not in scop 1.67 , and the search database was scop 1.67 ( 11 001 structures ) . among the 122 multiple - domain queries , 104 proteins have two domains , 14 have three domains and 4 have more than four domains . the total number of domains is 272 in the multiple - domain query set and the total number of domains in the scop-586 is 736 . table 1.accuracy of evolutionary superfamily assignment and average execution time of fastscop , 3d - blast and mammoth on 586 queries in the set scop-586query typenumber of queries ( domains)programnumber of assigned domainsassignment accuracy ( % ) unassigned domain percentage ( % ) average time per query ( s)relative to fastscopsingle domain464 query proteins ( 464 domains)3d - blast46494.4% ( 95.9%)0%1.1660.38mammoth46498.7% ( 98.7%)0%1046.47338.61fastscop45598.5% ( 99.6%)1.94%3.091multiple domain122 query proteins ( 272 domains)3d - blast27586.9%1.8%2.2380.34mammoth23894.1%12.5%1859.80278.40fastscop without reassignment21498.6%19.48%5.110.76fastscop25498%6.6%6.681assignment accuracy at scop fold level.fastscop does not apply the reassignment step , which is step 4 in figure 1a.scop-586 consists of 586 query proteins , which are in scop1.69 but not in scop1.67 ; the search database is scop1.67.time was measured using a personal computer with an intel pentium 2.8 ghz processor with 1024 mb of ram . accuracy of evolutionary superfamily assignment and average execution time of fastscop , 3d - blast and mammoth on 586 queries in the set scop-586 assignment accuracy at scop fold level . fastscop does not apply the reassignment step , which is step 4 in figure 1a . scop-586 consists of 586 query proteins , which are in scop1.69 but not in scop1.67 ; the search database is scop1.67 . time was measured using a personal computer with an intel pentium 2.8 ghz processor with 1024 mb of ram . table 1 presents the accuracy of superfamily assignment and the average execution time of the fastscop , 3d - blast and mammoth on the query set scop-586 . stand - alone fastscop , 3d - blast and mammoth were run on a personal computer with a single pentium 2.8 ghz processor with 1024 mb ram . the 3d - blast and mammoth used e - values and z - scores , respectively , to order the hit proteins . for 3d - blast , the top rank of a hit list of a query was selected as the scop superfamily . for mammoth , the same criteria ( z - score > 5.5 ; rmsd value < 4 and ( z - score rmsd > 4.0 ) of the fastscop were adopted to assign a query protein to a scop superfamily . on average , the fastscop took 3.09 s to recognize the structural domain and classification assignment for a single - domain query protein in the query set scop-586 ( table 1 ) . it was 338 times faster than mammoth and was 2.6 times slower than 3d - blast , because the fastscop required the time of applying mammoth for structure alignments between the query protein and the top 10 hit domains . for multiple - domain query proteins , the fastscop was 278 times faster than mammoth and was 2.7 times slower than 3d - blast . the predicted domain boundaries of the fastscop server were lightly shifted and the accuracy of boundaries accurate within 15 residues was 92% for the set scop-586 . as shown in table 1 , the fastscop server yielded 98.5 and 99.6% assignment accuracies at the superfamily and fold levels , respectively , for 464 single - domain queries . it outperformed 3d - blast ( 94.4 and 95.9% at the superfamily and fold levels , respectively ) and performed similarly to mammoth ( 98.7 and 98.7% ) . the unassignment percentage of the fastscop is 1.94% ( nine query proteins ) , which slightly exceeds those of the other two methods . for 122 multiple - domain queries ( with 272 domains ) , the fastscop yielded a 98.6% ( 214 domains ) assignment accuracy and the unassignment percentage was 19.48% ( 53 domains ) when the reassignment step ( step 4 in figure 1a ) was not applied . however , the assignment accuracy was 98% ( 254 domains ) and the unassignment percentage was reduced to 6.6% ( 18 domains ) when the fastscop used the reassignment step . the accuracy of fastscop significantly exceeded that of mammoth ( 94.1% ) and 3d - blast ( 86.9% ) ; the unassignment percentage was lower than that of mammoth ( 12.5% , 34 domains ) . the fastscop was evaluated using the 8700 pdb entries , which have no annotations in the scop database , and whose publishing date range from 1 january 2006 to 5 december , 2006 . the fastscop used these 8700 protein structures as queries , and the search classification database was scop 1.71 . in this . the fastscop server can automatically assign 7311 ( 84% ) proteins ( 9420 domains ) to the scop superfamilies in 9.6 h. according to the assignment accuracy ( 98% ) of the fastscop applied to the query set scop-586 and the assignment criteria ( step 2 in figure 1a ) , the fastscop server accurately assigns 9000 domains . this work demonstrated the robustness and feasibility of the fastscop server for recognizing the structural domains and the evolutionary classifications of protein structures . the key contribution of this work is the cooperative integration in fastscop of 3d - blast ( a fast structural database search tool ) and mammoth ( a fast detailed structural alignment tool ) ; the former is required for efficiency and the latter for accuracy . future works will adopt the fastscop for other evolutionary classification databases , such as cath ( 4 ) . additionally , the fastscop can be applied to develop structural motifs and sequence motifs from multiple structure and sequence alignments .

the fastscop is a web server that rapidly identifies the structural domains and determines the evolutionary superfamilies of a query protein structure . this server uses 3d - blast to scan quickly a large structural classification database ( scop1.71 with < 95% identity with each other ) and the top 10 hit domains , which have different superfamily classifications , are obtained from the hit lists . mammoth , a detailed structural alignment tool , is adopted to align these top 10 structures to refine domain boundaries and to identify evolutionary superfamilies . our previous works demonstrated that 3d - blast is as fast as blast , and has the characteristics of blast ( e.g. a robust statistical basis , effective search and reliable database search capabilities ) in large structural database searches based on a structural alphabet database and a structural alphabet substitution matrix . the classification accuracy of this server is 98% for 586 query structures and the average execution time is 5 . this server was also evaluated on 8700 structures , which have no annotations in the scop ; the server can automatically assign 7311 ( 84% ) proteins ( 9420 domains ) to the scop superfamilies in 9.6 h. these results suggest that the fastscop is robust and can be a useful server for recognizing the evolutionary classifications and the protein functions of novel structures . the server is accessible at http://fastscop.life.nctu.edu.tw .

INTRODUCTION METHOD AND IMPLEMENTATION Input, output and options Example analysis RESULTS CONCLUSION

as protein structures become increasingly available and structural genomics provide structural models in a genome - wide strategy ( 1 ) , proteins with unassigned functions are accumulating and the number of protein structures in the protein data bank ( pdb ) is rapidly rising ( 2 ) . the evolutionary classification databases , such as scop ( 3 ) and cath ( 4 ) , are valuable resources for understanding protein functions , structural similarity and evolutionary relationships . this current structure function gap clearly reveals the need for powerful automated methods to classify protein domains based on their tertiary structures and is important in producing manually tuned classification databases . many automatic domain classification approaches have been developed to determine similar structures and structural classification ( 5,6 ) of a query structure . protein sequence database search tools , such as blast ( 7 ) , psi - blast and superfamily ( 5 ) , are useful computational tools . however , these tools are commonly unreliable in detecting remote homologous relationships that are indicated by such structural alignment tools as dali ( 8) , mammoth ( 9 ) and ssm ( 10 ) . structural alignment tools typically take several seconds to align two known structures . recently , we have proposed a fast and efficient tool , called 3d - blast ( 11 ) , to quickly search similar structures . this tool is as fast as blast and provides the statistical significance ( e - value ) of an alignment to indicate the reliability of a structure . 3d - blast outperformed fast structural search methods ( topscan ( 12 ) and yakusa ( 13 ) ) and approached the performance of detailed structural alignment approaches ( ce ( 14 ) and mammoth ( 9 ) ) . 3d - blast is rapid and accurate in scanning a large protein structural database , and is useful in an initial scan for similar protein structures , which can be refined using detailed structural comparison methods . however , several factors that deteriorate 3d - blast 's performance are ( i ) 3d - blast may have made minor shifts in aligning two local segments with similar letters , because the structural alphabet do not consider actual euclidean distances , ( ii ) the e - values of the hit proteins are insignificant and ( iii ) the query is a multiple - domain protein ( 11 ) . this work presents an automated server ( fastscop ) , which integrates a fast structure database search tool ( 3d - blast ) and a detailed structural alignment tool ( mammoth ) , to recognize scop domains and scop superfamilies of a query structure . mammoth provided the z - score and root - mean - square deviation ( rmsd ) of the c atom positions of the aligned residues between the query structure and the hit structure according to the euclidean distance between corresponding residues rather than the distance between amino acid the classification accuracy of this server is 98% for 464 single - domain queries and 122 multiple - domain queries . to combine 3d - blast and mammoth is able to reduce the ill effects of 3d - blast to improve the assignment accuracy . after a query structure is assigned to a superfamily , this server is able to provide both multiple sequence alignments and multiple structural alignments of the selected members in a scop superfamily . figure 1 presents an overview of the fastscop server for rapidly recognizing scop domains and scop superfamilies . this sever uses 3d - blast to scan quickly the scop 1.71 database and selected the top 10 hit domain structures , which are associated with different scop superfamily entries ( figure 1b ) . mammoth was then adopted to align sequentially the query structure with each structure of the top 10 structures to refine the domain boundaries and to recognize scop superfamilies ( figure 1c and d ) . our previous work ( 11 ) demonstrated that 3d - blast required 1.4 s to scan the structural domains in scop 1.69 and was 16 990 and 1413 times faster than ce ( 14 ) and mammoth , respectively . these two detailed structural alignment tools perform similarly on the test set ; mammoth was 12 times faster than ce . the scop 1.71 database ( october 2006 ) has 75 930 domains that are derived from 27 599 pdb entries ( 18 january 2005 ) . 3d - blast requires structural alphabet sequence databases ( sadb ) for fast scanning a protein structural database . in this work , we created an sadb derived from known domain structures ( 12 927 domains ) in scop1.71 with < 95% identity to each other based on the ( , ) plot ( 11 ) . the fastscop has ( a ) four main steps , including ( b ) 3d - blast for scanning structural database , ( c ) mammoth for detailed structural alignment and ( d ) domain boundary refinement and reassignment . the fastscop has ( a ) four main steps , including ( b ) 3d - blast for scanning structural database , ( c ) mammoth for detailed structural alignment and ( d ) domain boundary refinement and reassignment . first , 3d - blast was adopted to identify the similar structures ( hit scop domains ) , which are ordered by e - value , of a query structure from an sadb database ( figure 1b ) . 3d - blast is the first tool to provide fast search of a protein structural database using the blast , which searches on a sadb database with a structural alphabet substitution matrix ( sasm ) ( 11 ) . the fastscop then selected the top 10 hit domains that have different scop superfamily entries . based on the structural alphabet alignments between the query and hit scop domains , this sever can identify multiple domains if a multiple - domain structure is queried . for each hit domain , the aligned length should be more than 40 residues and the overlap of two neighboring hit domains should be < 10% of the query protein . after the 10 ten hit scop domains were identified , this server applied mammoth to align sequentially the query structure with each structure of these hit domains , ordered by e - value . for each structural alignment , mammoth yielded the z - score and rmsd of the c atom positions of the aligned residues between the query structure and the hit structure ( figure 1c ) . the query structure ( or one domain of a multiple - domain protein ) was assigned to a scop superfamily when the pair - structure alignment satisfied the following criteria : ( i ) the z - score exceeds 5.5 ; ( ii ) the rmsd value is < 4 ; ( iii ) the value of ( z - score rmsd ) exceeds 4.0 and ( iv ) the number of the aligned residues exceeds 40 and the coverage rate between the query protein ( domain ) and hit domain exceeds 75% . in the third step , the fastscop refined the boundaries ( the start and end positions ) of the assigned domain according to the aligned regions and the sequence length of the hit domain ( figure 1d ) . the fastscop server can identify the structural domains and determine the evolutionary classification of a query structure from evolutionary classification databases . when the query structure is a new protein structure , the fastscop server enables users to input the structure file in pdb format . this server typically yielded structural domains and the scop superfamilies of a query structure in an average of 6 s ( figure 2a ) . the server can present the members of the assigned scop superfamily and provide both multiple sequence alignments and multiple structural alignments ( figure 2b ) based on users requirements . ( a ) the assigned scop superfamilies are the fkbp - like domain ( scop entry d.26.1 ) and the tpr domain ( scop entry a.118.8 ) . ( b ) multiple structural alphabet and amino acid sequences alignments of fkbp - like domain between the query protein and five homologous proteins . evolutionary superfamily assignment and structural alignment of the fastscop server using the structure of multi - domain immunophilin ( atfkbp42 ) from arabidopsis thaliana ( pdb code 2if4-a ) as the query . ( a ) the assigned scop superfamilies are the fkbp - like domain ( scop entry d.26.1 ) and the tpr domain ( scop entry a.118.8 ) . ( b ) multiple structural alphabet and amino acid sequences alignments of fkbp - like domain between the query protein and five homologous proteins . as shown in figure 2a , the fastscop recognized two domains and their scop superfamilies , which are the fkbp - like superfamily ( scop entry d.26.1 ) and the tpr - like superfamily ( scop entry a.118.8 ) for this query . the fkbp domain ( figure 2c ) of atfkbp42 consists of a six - stranded anti - parallel -sheet , wrapped around a short -helix , and is similar to those of fkbp52 ( pdb code 1q1c - a ) ( 16 ) , fkbp 25 ( pdb code 1pbk ) ( 17 ) , fkbp 13 ( pdb code 1u79-a ) ( 18 ) and fkbp 12 ( pdb code 1bkf ) ( 19 ) . after the structural domains and evolutionary superfamilies were recognized , the fastscop server allowed users to browse similar structures of these superfamilies . using this atfkbp42 as a query , the server can identify 13 and 17 similar structures of the fkbp - like domain and tpr domain , respectively . the structural alphabets were strongly conserved in areas of the secondary structures , which are -strands ( represented by structural alphabets e , f , h , k and n ) or -helices ( represented by structural alphabets a , y , b , c and d ) . these results reveal that the structural alphabet sequences are much better conserved than the amino acid sequences , which result explains why 3d - blast detected these distantly related proteins ( 11 ) . the fastscop server can identify the structural domains and determine the evolutionary classification of a query structure from evolutionary classification databases . when the query structure is a new protein structure , the fastscop server enables users to input the structure file in pdb format . this server typically yielded structural domains and the scop superfamilies of a query structure in an average of 6 s ( figure 2a ) . the server can present the members of the assigned scop superfamily and provide both multiple sequence alignments and multiple structural alignments ( figure 2b ) based on users requirements . figure 2.evolutionary superfamily assignment and structural alignment of the fastscop server using the structure of multi - domain immunophilin ( atfkbp42 ) from arabidopsis thaliana ( pdb code 2if4-a ) as the query . ( a ) the assigned scop superfamilies are the fkbp - like domain ( scop entry d.26.1 ) and the tpr domain ( scop entry a.118.8 ) . evolutionary superfamily assignment and structural alignment of the fastscop server using the structure of multi - domain immunophilin ( atfkbp42 ) from arabidopsis thaliana ( pdb code 2if4-a ) as the query . ( a ) the assigned scop superfamilies are the fkbp - like domain ( scop entry d.26.1 ) and the tpr domain ( scop entry a.118.8 ) . ( b ) multiple structural alphabet and amino acid sequences alignments of fkbp - like domain between the query protein and five homologous proteins . the release date of this protein is 31 , october 2006 , and this protein has not been recorded in scop . as shown in figure 2a , the fastscop recognized two domains and their scop superfamilies , which are the fkbp - like superfamily ( scop entry d.26.1 ) and the tpr - like superfamily ( scop entry a.118.8 ) for this query . the fkbp domain ( figure 2c ) of atfkbp42 consists of a six - stranded anti - parallel -sheet , wrapped around a short -helix , and is similar to those of fkbp52 ( pdb code 1q1c - a ) ( 16 ) , fkbp 25 ( pdb code 1pbk ) ( 17 ) , fkbp 13 ( pdb code 1u79-a ) ( 18 ) and fkbp 12 ( pdb code 1bkf ) ( 19 ) . after the structural domains and evolutionary superfamilies were recognized , the fastscop server allowed users to browse similar structures of these superfamilies . using this atfkbp42 as a query , the server can identify 13 and 17 similar structures of the fkbp - like domain and tpr domain , respectively . the structural alphabets were strongly conserved in areas of the secondary structures , which are -strands ( represented by structural alphabets e , f , h , k and n ) or -helices ( represented by structural alphabets a , y , b , c and d ) . these results reveal that the structural alphabet sequences are much better conserved than the amino acid sequences , which result explains why 3d - blast detected these distantly related proteins ( 11 ) . a query protein set , scop-586 ( table 1 ) , was selected to evaluate the utility of the fastscop server for recognizing the structural domains and evolutionary superfamilies of a query structure . the scop-586 query set has 464 single - domain proteins and 122 multiple - domain proteins that are in scop 1.69 but not in scop 1.67 , and the search database was scop 1.67 ( 11 001 structures ) . among the 122 multiple - domain queries , 104 proteins have two domains , 14 have three domains and 4 have more than four domains . table 1.accuracy of evolutionary superfamily assignment and average execution time of fastscop , 3d - blast and mammoth on 586 queries in the set scop-586query typenumber of queries ( domains)programnumber of assigned domainsassignment accuracy ( % ) unassigned domain percentage ( % ) average time per query ( s)relative to fastscopsingle domain464 query proteins ( 464 domains)3d - blast46494.4% ( 95.9%)0%1.1660.38mammoth46498.7% ( 98.7%)0%1046.47338.61fastscop45598.5% ( 99.6%)1.94%3.091multiple domain122 query proteins ( 272 domains)3d - blast27586.9%1.8%2.2380.34mammoth23894.1%12.5%1859.80278.40fastscop without reassignment21498.6%19.48%5.110.76fastscop25498%6.6%6.681assignment accuracy at scop fold level.fastscop does not apply the reassignment step , which is step 4 in figure 1a.scop-586 consists of 586 query proteins , which are in scop1.69 but not in scop1.67 ; the search database is scop1.67.time was measured using a personal computer with an intel pentium 2.8 ghz processor with 1024 mb of ram . accuracy of evolutionary superfamily assignment and average execution time of fastscop , 3d - blast and mammoth on 586 queries in the set scop-586 assignment accuracy at scop fold level . scop-586 consists of 586 query proteins , which are in scop1.69 but not in scop1.67 ; the search database is scop1.67 . table 1 presents the accuracy of superfamily assignment and the average execution time of the fastscop , 3d - blast and mammoth on the query set scop-586 . stand - alone fastscop , 3d - blast and mammoth were run on a personal computer with a single pentium 2.8 ghz processor with 1024 mb ram . the 3d - blast and mammoth used e - values and z - scores , respectively , to order the hit proteins . for 3d - blast , the top rank of a hit list of a query was selected as the scop superfamily . for mammoth , the same criteria ( z - score > 5.5 ; rmsd value < 4 and ( z - score rmsd > 4.0 ) of the fastscop were adopted to assign a query protein to a scop superfamily . on average , the fastscop took 3.09 s to recognize the structural domain and classification assignment for a single - domain query protein in the query set scop-586 ( table 1 ) . it was 338 times faster than mammoth and was 2.6 times slower than 3d - blast , because the fastscop required the time of applying mammoth for structure alignments between the query protein and the top 10 hit domains . for multiple - domain query proteins , the fastscop was 278 times faster than mammoth and was 2.7 times slower than 3d - blast . the predicted domain boundaries of the fastscop server were lightly shifted and the accuracy of boundaries accurate within 15 residues was 92% for the set scop-586 . it outperformed 3d - blast ( 94.4 and 95.9% at the superfamily and fold levels , respectively ) and performed similarly to mammoth ( 98.7 and 98.7% ) . the unassignment percentage of the fastscop is 1.94% ( nine query proteins ) , which slightly exceeds those of the other two methods . for 122 multiple - domain queries ( with 272 domains ) , the fastscop yielded a 98.6% ( 214 domains ) assignment accuracy and the unassignment percentage was 19.48% ( 53 domains ) when the reassignment step ( step 4 in figure 1a ) was not applied . however , the assignment accuracy was 98% ( 254 domains ) and the unassignment percentage was reduced to 6.6% ( 18 domains ) when the fastscop used the reassignment step . the accuracy of fastscop significantly exceeded that of mammoth ( 94.1% ) and 3d - blast ( 86.9% ) ; the unassignment percentage was lower than that of mammoth ( 12.5% , 34 domains ) . the fastscop was evaluated using the 8700 pdb entries , which have no annotations in the scop database , and whose publishing date range from 1 january 2006 to 5 december , 2006 . the fastscop used these 8700 protein structures as queries , and the search classification database was scop 1.71 . the fastscop server can automatically assign 7311 ( 84% ) proteins ( 9420 domains ) to the scop superfamilies in 9.6 h. according to the assignment accuracy ( 98% ) of the fastscop applied to the query set scop-586 and the assignment criteria ( step 2 in figure 1a ) , the fastscop server accurately assigns 9000 domains . this work demonstrated the robustness and feasibility of the fastscop server for recognizing the structural domains and the evolutionary classifications of protein structures . the key contribution of this work is the cooperative integration in fastscop of 3d - blast ( a fast structural database search tool ) and mammoth ( a fast detailed structural alignment tool ) ; the former is required for efficiency and the latter for accuracy . additionally , the fastscop can be applied to develop structural motifs and sequence motifs from multiple structure and sequence alignments .

the case - j trial was a prospective , multicenter , randomized , open - label , active - controlled , two - arm parallel - group comparison with response - dependent dose titration and blinded assessment of end points conducted in high - risk japanese hypertensive patients . the trial protocol was approved by the ethics committee of kyoto university graduate school of medicine in accordance with the principles of the declaration of helsinki . details of the study and the main results have been reported previously ( 14,15 ) . in brief , 4,728 high - risk japanese hypertensive patients aged 2084 years were randomly assigned to either candesartan- or amlodipine - based regimens . high - risk was defined as the presence of any one or more of the following : 1 ) severe hypertension ( sbp / dbp 180/110 mmhg ) ; 2 ) type 2 diabetes ( fasting blood glucose 126 mg / dl , casual blood glucose 200 mg / dl , a1c 6.5% , 2-h blood glucose on a 75-g oral glucose tolerance test 200 mg / dl , or current treatment with a hypoglycemic agent at baseline ) ; 3 ) a history of stroke or transient ischemic attack > 6 months before screening ; 4 ) left ventricular hypertrophy ( lvh ) , angina pectoris , or a history of myocardial infarction > 6 months before screening ; 5 ) proteinuria or renal dysfunction ( serum creatinine 1.3 mg / dl ) ; or 6 ) arteriosclerotic peripheral artery obstruction . enrolled patients were randomly assigned to receive candesartan by oral administration at 412 mg / day or amlodipine by oral administration at 2.510 mg / day . patients already under treatment with diuretics , -blockers , and -blockers at enrollment were allowed to continue taking these drugs , but the new addition of other arbs and ccbs or any ace inhibitors was prohibited . of the 4,703 high - risk hypertensive patients analyzed in the case - j trial , 2,018 who had diabetes at baseline were excluded , leaving 2,685 patients for inclusion in the present study . new - onset diabetes was prespecified as the end point on 17 september 2005 , which was after the beginning but before the completion of the case - j trial ( 15 ) . to detect the occurrence of new - onset diabetes , individual case report forms and adverse - event databases were monitored . a case of new - onset diabetes was defined as a patient reported as having developed diabetes on the adverse event form or a patient who had newly started antidiabetic agent therapy in the case report form . multiple cox regression analysis was used to examine the association between each blood pressure index ( sbp , dbp , and pulse pressure ) at baseline and the risk of new - onset diabetes with adjustment for baseline characteristics ( prior antihypertensive treatment , allocated drug , age , sex , bmi , heart rate , history of cerebrovascular events , lvh , history of ischemic heart disease , renal dysfunction , peripheral vascular disease , hyperlipidemia , and smoking ) as standard covariates and additional drugs ( diuretics , -blockers , and -blockers ) as time - varying covariates . fractional pulse pressure ( ppf ) , which is calculated as pulse pressure divided by mean arterial pressure , has recently been proposed as a new parameter of the pulsatile component of blood pressure ( 16 ) . ppf is thought to more directly reflect arterial stiffness than pulse pressure , because dividing by mean arterial pressure theoretically cancels out the influence of cardiac output and peripheral vascular resistance . we also evaluated the predictive value of this variable for new - onset diabetes by multiple cox regression analysis . because each blood pressure index is affected by aging ( 10 ) , we also conducted subgroup analyses stratified by age ( cutoff point : age 65 years ) , using the median age at baseline of all included patients . the test for interaction in the multiple cox model was evaluated with the interaction term . in addition , to clarify the significance of pulse pressure for new - onset diabetes , the associations of both sbp and dbp with the incidence of new - onset diabetes were examined by multiple cox regression analysis with sbp grouped into two categories ( sbp < 160 mmhg and 160 mmhg sbp ) and dbp plotted as a continuous variable . this model was plotted with the middle 80% of the distribution of dbp for each sbp group , and the hr of a dbp of 90 mmhg in the sbp < 160 mmhg category was assigned a reference value of 1.0 . all statistical tests were two - sided with an level of 0.05 and were performed using sas ( version 9.1 ; sas institute , cary , nc ) . of the 4,703 high - risk hypertensive patients analyzed in the case - j trial , 2,018 who had diabetes at baseline were excluded , leaving 2,685 patients for inclusion in the present study . new - onset diabetes was prespecified as the end point on 17 september 2005 , which was after the beginning but before the completion of the case - j trial ( 15 ) . to detect the occurrence of new - onset diabetes , individual case report forms and adverse - event databases were monitored . a case of new - onset diabetes was defined as a patient reported as having developed diabetes on the adverse event form or a patient who had newly started antidiabetic agent therapy in the case report form . multiple cox regression analysis was used to examine the association between each blood pressure index ( sbp , dbp , and pulse pressure ) at baseline and the risk of new - onset diabetes with adjustment for baseline characteristics ( prior antihypertensive treatment , allocated drug , age , sex , bmi , heart rate , history of cerebrovascular events , lvh , history of ischemic heart disease , renal dysfunction , peripheral vascular disease , hyperlipidemia , and smoking ) as standard covariates and additional drugs ( diuretics , -blockers , and -blockers ) as time - varying covariates . fractional pulse pressure ( ppf ) , which is calculated as pulse pressure divided by mean arterial pressure , has recently been proposed as a new parameter of the pulsatile component of blood pressure ( 16 ) . ppf is thought to more directly reflect arterial stiffness than pulse pressure , because dividing by mean arterial pressure theoretically cancels out the influence of cardiac output and peripheral vascular resistance . we also evaluated the predictive value of this variable for new - onset diabetes by multiple cox regression analysis . because each blood pressure index is affected by aging ( 10 ) , we also conducted subgroup analyses stratified by age ( cutoff point : age 65 years ) , using the median age at baseline of all included patients . the test for interaction in the multiple cox model was evaluated with the interaction term . in addition , to clarify the significance of pulse pressure for new - onset diabetes , the associations of both sbp and dbp with the incidence of new - onset diabetes were examined by multiple cox regression analysis with sbp grouped into two categories ( sbp < 160 mmhg and 160 mmhg sbp ) and dbp plotted as a continuous variable . this model was plotted with the middle 80% of the distribution of dbp for each sbp group , and the hr of a dbp of 90 mmhg in the sbp < 160 mmhg category was assigned a reference value of 1.0 . all statistical tests were two - sided with an level of 0.05 and were performed using sas ( version 9.1 ; sas institute , cary , nc ) . during 3.3 0.8 years of follow - up , 97 patients ( 3.6% ) developed new - onset diabetes . baseline characteristics of patients with and without new - onset diabetes are shown in table 1 . patients developing diabetes were more likely to be male and obese , less likely to have been randomly assigned to a candesartan - based regimen , and more likely to have had lower dbp , higher pulse pressure , and lvh at baseline . at the time of randomization , 1,702 ( 65.8% ) patients without and 65 ( 67.0% ) patients with new - onset diabetes were under treatment with antihypertensive drugs ( ccb 40.1 vs. 34.0% , p = 0.229 ; ace inhibitor 13.3 vs. 16.5% , p = 0.363 ; arb 17.9 vs. 22.7% , p = 0.229 ; diuretic 3.1 vs. 5.2% , p = 0.255 ; -blocker 12.9 vs. 16.5% , p = 0.297 ; and -blocker 5.6 vs. 4.1% , p = 0.542 , respectively ) . * p < 0.05 , nod ( ) vs. nod ( + ) . stroke and transient ischemic attack . multiple cox regression analysis revealed that pulse pressure ( per 1 sd increase ) was an independent predictor of new - onset diabetes ( hr 1.44 [ 95% ci 1.151.79 ] , p = 0.001 ) ( table 2 ) . in addition , risk was also significantly associated with male sex , bmi , lvh , and concomitant use of diuretics . as reported previously , candesartan - based regimens significantly reduced the risk of new - onset diabetes compared with amlodipine - based regimens ( 15 ) . predictors of new - onset diabetes by multiple cox regression analysis data are hr ( 95% ci ) and are adjusted for each variable . * renal damage , proteinuria , and renal dysfunction . because pulse pressure was calculated as the difference between sbp and dbp , we conducted separate analyses for sbp and dbp and found that dbp ( per 1 sd decrease ) was also an independent predictor for new - onset diabetes , whereas sbp ( per 1 sd increase ) was not ( hr for sbp 1.13 [ 95% ci 0.901.41 ] , p = 0.284 ; and hr for dbp 1.45 [ 1.161.81 ] , p < 0.001 ) . subgroup analysis stratified by age ( cutoff point : age 65 years ) revealed that pulse pressure remained significantly associated with the risk of new - onset diabetes in both age - groups ( aged < 65 years : hr 1.72 [ 95% ci 1.182.49 ] , p = 0.004 ; aged 65 years : 1.34 [ 1.011.77 ] , p = 0.042 ; and pinteraction = 0.152 ) . however , dbp was significantly associated with risk only in the group aged < 65 years , whereas whole sbp was not associated in either age - group ( for sbp , aged < 65 years : 1.20 [ 0.861.67 ] , p = 0.284 ; aged 65 years : 1.16 [ 0.841.59 ] , p = 0.374 ; and pinteraction = 0.780 ; for dbp , aged < 65 years : 1.58 [ 1.102.28 ] , p = 0.014 ; aged 65 years : 1.32 [ 0.991.76 ] , p = 0.057 ; and pinteraction = 0.290 ) . because different combinations of sbp and dbp give the same pulse pressure value ( e.g. , blood pressures of 130/60 and 180/110 mmhg both give a pulse pressure of 70 mmhg ) , we evaluated the association of combinations of sbp and dbp with the risk of new - onset diabetes . as shown in fig . 1 , a strong association with risk was seen for higher pulse pressures arising mainly due to a lower dbp . from this result , we hypothesized that patients at high risk of new - onset diabetes had increased arterial stiffness . accordingly , we next examined the association between ppf and the risk of new - onset diabetes and found that ppf ( per 1 sd increase ) was an independent predictor of new - onset diabetes ( hr 1.49 [ 95% ci 1.211.84 ] , p < 0.001 ) . in subgroup analysis stratified by age , ppf ( per 1 sd increase ) was significantly associated with the risk of new - onset diabetes in both age - groups ( aged < 65 : 1.88 [ 1.292.73 ] , p < 0.001 ; aged 65 : 1.34 [ 1.031.74 ] , p = 0.027 ; and pinteraction = 0.057 ) . because fewer patients developed diabetes with candesartan- than amlodipine - based regimens , we examined the difference in this effect stratified by quartile of ppf . 2 , a trend to an increased incidence of new - onset diabetes with increasing ppf was seen in patients with amlodipine - based regimens , but not in those with candesartan - based regimens ( p = 0.0234 for interaction in the quadratic term ) . candesartan - based regimens significantly suppressed the incidence of new - onset diabetes in the highest quartile of ppf . this result was not changed after adjustment for baseline characteristics ( data not shown ) . hr of dbp of 90 mmhg in the sbp < 160 mmhg category was assigned a reference value of 1.0 . effect of candesartan and amlodipine on the incidence of new - onset diabetes stratified by quartile of ppf . ppf ( linear and quadratic terms ) , the allocated drugs , and their interaction terms were entered in multiple cox regression model . during 3.3 0.8 years of follow - up , 97 patients ( 3.6% ) developed new - onset diabetes . baseline characteristics of patients with and without new - onset diabetes are shown in table 1 . patients developing diabetes were more likely to be male and obese , less likely to have been randomly assigned to a candesartan - based regimen , and more likely to have had lower dbp , higher pulse pressure , and lvh at baseline . at the time of randomization , 1,702 ( 65.8% ) patients without and 65 ( 67.0% ) patients with new - onset diabetes were under treatment with antihypertensive drugs ( ccb 40.1 vs. 34.0% , p = 0.229 ; ace inhibitor 13.3 vs. 16.5% , p = 0.363 ; arb 17.9 vs. 22.7% , p = 0.229 ; diuretic 3.1 vs. 5.2% , p = 0.255 ; -blocker 12.9 vs. 16.5% , p = 0.297 ; and -blocker 5.6 vs. 4.1% , p = 0.542 , respectively ) . * p < 0.05 , nod ( ) vs. nod ( + ) . stroke and transient ischemic attack . multiple cox regression analysis revealed that pulse pressure ( per 1 sd increase ) was an independent predictor of new - onset diabetes ( hr 1.44 [ 95% ci 1.151.79 ] , p = 0.001 ) ( table 2 ) . in addition , risk was also significantly associated with male sex , bmi , lvh , and concomitant use of diuretics . as reported previously , candesartan - based regimens significantly reduced the risk of new - onset diabetes compared with amlodipine - based regimens ( 15 ) . predictors of new - onset diabetes by multiple cox regression analysis data are hr ( 95% ci ) and are adjusted for each variable . * renal damage , proteinuria , and renal dysfunction . because pulse pressure was calculated as the difference between sbp and dbp , we conducted separate analyses for sbp and dbp and found that dbp ( per 1 sd decrease ) was also an independent predictor for new - onset diabetes , whereas sbp ( per 1 sd increase ) was not ( hr for sbp 1.13 [ 95% ci 0.901.41 ] , p = 0.284 ; and hr for dbp 1.45 [ 1.161.81 ] , p < 0.001 ) . subgroup analysis stratified by age ( cutoff point : age 65 years ) revealed that pulse pressure remained significantly associated with the risk of new - onset diabetes in both age - groups ( aged < 65 years : hr 1.72 [ 95% ci 1.182.49 ] , p = 0.004 ; aged 65 years : 1.34 [ 1.011.77 ] , p = 0.042 ; and pinteraction = 0.152 ) . however , dbp was significantly associated with risk only in the group aged < 65 years , whereas whole sbp was not associated in either age - group ( for sbp , aged < 65 years : 1.20 [ 0.861.67 ] , p = 0.284 ; aged 65 years : 1.16 [ 0.841.59 ] , p = 0.374 ; and pinteraction = 0.780 ; for dbp , aged < 65 years : 1.58 [ 1.102.28 ] , p = 0.014 ; aged 65 years : 1.32 [ 0.991.76 ] , p = 0.057 ; and pinteraction = 0.290 ) . because different combinations of sbp and dbp give the same pulse pressure value ( e.g. , blood pressures of 130/60 and 180/110 mmhg both give a pulse pressure of 70 mmhg ) , we evaluated the association of combinations of sbp and dbp with the risk of new - onset diabetes . as shown in fig . 1 , a strong association with risk was seen for higher pulse pressures arising mainly due to a lower dbp . from this result , we hypothesized that patients at high risk of new - onset diabetes had increased arterial stiffness . accordingly , we next examined the association between ppf and the risk of new - onset diabetes and found that ppf ( per 1 sd increase ) was an independent predictor of new - onset diabetes ( hr 1.49 [ 95% ci 1.211.84 ] , p < 0.001 ) . in subgroup analysis stratified by age , ppf ( per 1 sd increase ) was significantly associated with the risk of new - onset diabetes in both age - groups ( aged < 65 : 1.88 [ 1.292.73 ] , p < 0.001 ; aged 65 : 1.34 [ 1.031.74 ] , p = 0.027 ; and pinteraction = 0.057 ) . because fewer patients developed diabetes with candesartan- than amlodipine - based regimens , we examined the difference in this effect stratified by quartile of ppf . 2 , a trend to an increased incidence of new - onset diabetes with increasing ppf was seen in patients with amlodipine - based regimens , but not in those with candesartan - based regimens ( p = 0.0234 for interaction in the quadratic term ) . candesartan - based regimens significantly suppressed the incidence of new - onset diabetes in the highest quartile of ppf . this result was not changed after adjustment for baseline characteristics ( data not shown ) . hr of dbp of 90 mmhg in the sbp < 160 mmhg category was assigned a reference value of 1.0 . effect of candesartan and amlodipine on the incidence of new - onset diabetes stratified by quartile of ppf . ppf ( linear and quadratic terms ) , the allocated drugs , and their interaction terms were entered in multiple cox regression model . in this study , we demonstrated that pulse pressure was a predictor of new - onset diabetes in high - risk hypertensive patients , independent of the effects of antihypertensive treatment and other possible risk factors for new - onset diabetes . further , a higher pulse pressure arising mainly due to a lower dbp , indicating that the increased pulse pressure resulted largely from increased arterial stiffness , was associated with a higher risk of new - onset diabetes . this finding suggests that increased arterial stiffness , reflected in an increased pulse pressure , may be related to the process of new - onset diabetes in high - risk hypertensive patients , albeit that the mechanism of this association remains to be elucidated . first , increased pulse pressure may be a surrogate marker for the risk of new - onset diabetes . supporting this suggestion , a higher pulse pressure , reflecting increased arterial stiffness , was observed in hypertensive patients with metabolic syndrome than in those without ( 17 ) . further , accumulating evidence supports the concept of increased arterial stiffness in patients with a metabolic disturbance , which is considered a potential mechanism linking metabolic disturbance to increased cvd risk ( 1113 ) . arterial properties are affected both functionally and structurally by many factors , including aging , blood pressure , sympathetic nervous system function , endothelial function , inflammation , bioactive peptides , and other cardiovascular risk factors . impaired glucose metabolism , including metabolic syndrome and insulin resistance , usually precedes the development of overt type 2 diabetes ( 18 ) . prolonged exposure to hyperglycemic conditions can lead to increased arterial stiffness via collagen cross - linking due to nonenzymatic glycation , endothelial dysfunction , inflammation , and local activation of the renin - angiotensin - aldosterone system in pre - diabetic as well as diabetic individuals ( 18 ) . indeed , ppf , represented as a parameter of the pulsatile component of blood pressure , was superior to pulse pressure in terms of the risk stratification of new - onset diabetes . recent findings have clarified that microvascular dysfunction may be a cause rather than a consequence of hypertension ( 19 ) . microvascular dysfunction may also contribute to impaired insulin - mediated changes in muscle perfusion and glucose metabolism , providing a novel pathophysiological framework for understanding the association among hypertension , obesity , and impaired insulin - mediated glucose disposal ( 19,20 ) . microvascular dysfunction is thus a potential mechanism explaining the clustering of hypertension and type 2 diabetes . interestingly , relations between microvascular function and both aortic stiffness and pressure pulsatility have been reported ( 21 ) . abnormalities in peripheral vascular resistance may have deleterious consequences for aortic stiffness , and microvascular dysfunction may in turn be further aggravated by increased transmission of the forward wave into the microcirculation . accordingly , increased pulse pressure , reflecting increased arterial stiffness , may be both a cause and a consequence of microvascular dysfunction , leading to a vicious cycle in impaired glucose metabolism as well as arteriosclerosis ( 9,19,20 ) . the present study also revealed that electrocardiographic or echocardiographic lvh at baseline was an independent predictor of new - onset diabetes . in their recent subanalysis of the losartan intervention for endpoint reduction in hypertension ( life ) study , oki et al . ( 22 ) reported that in - treatment resolution or continued absence of electrocardiographic lvh was associated with a lower incidence of diabetes . because pulse pressure was positively related to lvh ( 23 ) ( 24 ) found that treatment with the arb losartan was associated with less peripheral vascular hypertrophy / rarefaction and higher insulin sensitivity than that with atenolol , supporting the hypothesis that microvascular dysfunction in hypertension may induce insulin resistance . in the present study , the suppressive effect of the arb candesartan against new - onset diabetes tended to strengthen as ppf increased . these results suggest that arbs decrease the risk of new - onset diabetes partly via the improvement of microcirculation . although the prevalence of diabetes increases with age ( 25 ) , it remains unclear whether age is a risk factor for new - onset diabetes ( 68 ) . in the present study , age at baseline we assumed that high - risk elderly hypertensive patients who did not have diabetes at baseline were survivors who had avoided the development of diabetes and that their underlying risk of new - onset diabetes and ability to metabolize glucose may thus have differed from those of younger subjects . we also observed a strong association between pulse pressure and new - onset diabetes in patients aged < 65 years , possibly owing to the same mechanism . second , although we found an interesting association between pulse pressure and the risk of new - onset diabetes , the case - j trial was not designed to prospectively evaluate this association , and we were consequently unable to elucidate causality , because we did not directly measure parameters of arterial stiffness or collect the data to clarify the underlying mechanism . third , we were unable to include baseline data regarding glucose metabolism into the multiple cox regression analysis or information about a family history of diabetes , physical activity , or diet , which are well - known and important risk factors for new - onset diabetes . fourth , new - onset diabetes was prespecified as the end point just before the completion of the case - j trial . accordingly , there was a possibility of nonreporting bias , because the definition of new - onset diabetes was not in the original protocol and determination of whether new - onset diabetes had occurred depended on the participating investigators ' reports . nevertheless , the present study is the first to examine the association of pulse pressure with new - onset diabetes in hypertensive patients and may provide useful information in understanding the underlying mechanism between hypertension and new - onset diabetes . finally , because the study population consisted of japanese patients with high - risk hypertension , the generalizability of our findings to other ethnic groups or general populations may be limited . in summary , we found that pulse pressure is an independent predictor of new - onset diabetes in high - risk japanese hypertensive patients . the development of type 2 diabetes may involve increased arterial stiffness , suggesting the importance of the microvascular dysfunction theory in the underlying pathophysiological mechanism between hypertension and new - onset diabetes . to our knowledge , this study is the first to report the relation between pulse pressure and new - onset diabetes in hypertensive patients . further studies are required to elucidate the significance of pulse pressure in new - onset diabetes in hypertensive patients .

objectivehypertensive patients have an increased risk of developing diabetes . accumulating evidence suggests a close relation between metabolic disturbance and increased arterial stiffness . here , we examined the association between pulse pressure and the risk of new - onset diabetes in high - risk japanese hypertensive patients.research design and methodsthe candesartan antihypertensive survival evaluation in japan ( case - j ) trial examined the effects of candesartan and amlodipine on the incidence of cardiovascular events in 4,728 high - risk japanese hypertensive patients . in the present study , we analyzed the relationship between pulse pressure at baseline and new - onset diabetes in 2,685 patients without diabetes at baseline ( male 1,471 ; mean age 63.7 years ; mean bmi 24.8 kg / m2 ) as a subanalysis of the case - j trial.resultsduring 3.3 0.8 years of follow - up , 97 patients ( 3.6% ) developed diabetes . in multiple cox regression analysis , pulse pressure was an independent predictor for new - onset diabetes ( hazard ratio [ hr ] per 1 sd increase 1.44 [ 95% ci 1.151.79 ] ) as were male sex , bmi , and additional use of diuretics , whereas age and heart rate were not . plots of hrs for new - onset diabetes considering both systolic and diastolic blood pressure ( dbp ) revealed that a higher pulse pressure with a lower dbp , indicating that the increased pulse pressure was largely due to increased arterial stiffness , was strongly associated with the risk of new - onset diabetes.conclusionspulse pressure is an independent predictor of new - onset diabetes in high - risk japanese hypertensive patients . increased arterial stiffness may be involved in the development of diabetes .

RESEARCH DESIGN AND METHODS Outcome measurement Statistical analysis RESULTS Baseline characteristics Predictors of new-onset diabetes CONCLUSIONS

the case - j trial was a prospective , multicenter , randomized , open - label , active - controlled , two - arm parallel - group comparison with response - dependent dose titration and blinded assessment of end points conducted in high - risk japanese hypertensive patients . in brief , 4,728 high - risk japanese hypertensive patients aged 2084 years were randomly assigned to either candesartan- or amlodipine - based regimens . high - risk was defined as the presence of any one or more of the following : 1 ) severe hypertension ( sbp / dbp 180/110 mmhg ) ; 2 ) type 2 diabetes ( fasting blood glucose 126 mg / dl , casual blood glucose 200 mg / dl , a1c 6.5% , 2-h blood glucose on a 75-g oral glucose tolerance test 200 mg / dl , or current treatment with a hypoglycemic agent at baseline ) ; 3 ) a history of stroke or transient ischemic attack > 6 months before screening ; 4 ) left ventricular hypertrophy ( lvh ) , angina pectoris , or a history of myocardial infarction > 6 months before screening ; 5 ) proteinuria or renal dysfunction ( serum creatinine 1.3 mg / dl ) ; or 6 ) arteriosclerotic peripheral artery obstruction . of the 4,703 high - risk hypertensive patients analyzed in the case - j trial , 2,018 who had diabetes at baseline were excluded , leaving 2,685 patients for inclusion in the present study . new - onset diabetes was prespecified as the end point on 17 september 2005 , which was after the beginning but before the completion of the case - j trial ( 15 ) . a case of new - onset diabetes was defined as a patient reported as having developed diabetes on the adverse event form or a patient who had newly started antidiabetic agent therapy in the case report form . multiple cox regression analysis was used to examine the association between each blood pressure index ( sbp , dbp , and pulse pressure ) at baseline and the risk of new - onset diabetes with adjustment for baseline characteristics ( prior antihypertensive treatment , allocated drug , age , sex , bmi , heart rate , history of cerebrovascular events , lvh , history of ischemic heart disease , renal dysfunction , peripheral vascular disease , hyperlipidemia , and smoking ) as standard covariates and additional drugs ( diuretics , -blockers , and -blockers ) as time - varying covariates . fractional pulse pressure ( ppf ) , which is calculated as pulse pressure divided by mean arterial pressure , has recently been proposed as a new parameter of the pulsatile component of blood pressure ( 16 ) . we also evaluated the predictive value of this variable for new - onset diabetes by multiple cox regression analysis . in addition , to clarify the significance of pulse pressure for new - onset diabetes , the associations of both sbp and dbp with the incidence of new - onset diabetes were examined by multiple cox regression analysis with sbp grouped into two categories ( sbp < 160 mmhg and 160 mmhg sbp ) and dbp plotted as a continuous variable . this model was plotted with the middle 80% of the distribution of dbp for each sbp group , and the hr of a dbp of 90 mmhg in the sbp < 160 mmhg category was assigned a reference value of 1.0 . of the 4,703 high - risk hypertensive patients analyzed in the case - j trial , 2,018 who had diabetes at baseline were excluded , leaving 2,685 patients for inclusion in the present study . new - onset diabetes was prespecified as the end point on 17 september 2005 , which was after the beginning but before the completion of the case - j trial ( 15 ) . a case of new - onset diabetes was defined as a patient reported as having developed diabetes on the adverse event form or a patient who had newly started antidiabetic agent therapy in the case report form . multiple cox regression analysis was used to examine the association between each blood pressure index ( sbp , dbp , and pulse pressure ) at baseline and the risk of new - onset diabetes with adjustment for baseline characteristics ( prior antihypertensive treatment , allocated drug , age , sex , bmi , heart rate , history of cerebrovascular events , lvh , history of ischemic heart disease , renal dysfunction , peripheral vascular disease , hyperlipidemia , and smoking ) as standard covariates and additional drugs ( diuretics , -blockers , and -blockers ) as time - varying covariates . fractional pulse pressure ( ppf ) , which is calculated as pulse pressure divided by mean arterial pressure , has recently been proposed as a new parameter of the pulsatile component of blood pressure ( 16 ) . we also evaluated the predictive value of this variable for new - onset diabetes by multiple cox regression analysis . in addition , to clarify the significance of pulse pressure for new - onset diabetes , the associations of both sbp and dbp with the incidence of new - onset diabetes were examined by multiple cox regression analysis with sbp grouped into two categories ( sbp < 160 mmhg and 160 mmhg sbp ) and dbp plotted as a continuous variable . this model was plotted with the middle 80% of the distribution of dbp for each sbp group , and the hr of a dbp of 90 mmhg in the sbp < 160 mmhg category was assigned a reference value of 1.0 . during 3.3 0.8 years of follow - up , 97 patients ( 3.6% ) developed new - onset diabetes . patients developing diabetes were more likely to be male and obese , less likely to have been randomly assigned to a candesartan - based regimen , and more likely to have had lower dbp , higher pulse pressure , and lvh at baseline . multiple cox regression analysis revealed that pulse pressure ( per 1 sd increase ) was an independent predictor of new - onset diabetes ( hr 1.44 [ 95% ci 1.151.79 ] , p = 0.001 ) ( table 2 ) . in addition , risk was also significantly associated with male sex , bmi , lvh , and concomitant use of diuretics . as reported previously , candesartan - based regimens significantly reduced the risk of new - onset diabetes compared with amlodipine - based regimens ( 15 ) . predictors of new - onset diabetes by multiple cox regression analysis data are hr ( 95% ci ) and are adjusted for each variable . because pulse pressure was calculated as the difference between sbp and dbp , we conducted separate analyses for sbp and dbp and found that dbp ( per 1 sd decrease ) was also an independent predictor for new - onset diabetes , whereas sbp ( per 1 sd increase ) was not ( hr for sbp 1.13 [ 95% ci 0.901.41 ] , p = 0.284 ; and hr for dbp 1.45 [ 1.161.81 ] , p < 0.001 ) . subgroup analysis stratified by age ( cutoff point : age 65 years ) revealed that pulse pressure remained significantly associated with the risk of new - onset diabetes in both age - groups ( aged < 65 years : hr 1.72 [ 95% ci 1.182.49 ] , p = 0.004 ; aged 65 years : 1.34 [ 1.011.77 ] , p = 0.042 ; and pinteraction = 0.152 ) . , blood pressures of 130/60 and 180/110 mmhg both give a pulse pressure of 70 mmhg ) , we evaluated the association of combinations of sbp and dbp with the risk of new - onset diabetes . from this result , we hypothesized that patients at high risk of new - onset diabetes had increased arterial stiffness . accordingly , we next examined the association between ppf and the risk of new - onset diabetes and found that ppf ( per 1 sd increase ) was an independent predictor of new - onset diabetes ( hr 1.49 [ 95% ci 1.211.84 ] , p < 0.001 ) . in subgroup analysis stratified by age , ppf ( per 1 sd increase ) was significantly associated with the risk of new - onset diabetes in both age - groups ( aged < 65 : 1.88 [ 1.292.73 ] , p < 0.001 ; aged 65 : 1.34 [ 1.031.74 ] , p = 0.027 ; and pinteraction = 0.057 ) . because fewer patients developed diabetes with candesartan- than amlodipine - based regimens , we examined the difference in this effect stratified by quartile of ppf . 2 , a trend to an increased incidence of new - onset diabetes with increasing ppf was seen in patients with amlodipine - based regimens , but not in those with candesartan - based regimens ( p = 0.0234 for interaction in the quadratic term ) . candesartan - based regimens significantly suppressed the incidence of new - onset diabetes in the highest quartile of ppf . effect of candesartan and amlodipine on the incidence of new - onset diabetes stratified by quartile of ppf . during 3.3 0.8 years of follow - up , 97 patients ( 3.6% ) developed new - onset diabetes . patients developing diabetes were more likely to be male and obese , less likely to have been randomly assigned to a candesartan - based regimen , and more likely to have had lower dbp , higher pulse pressure , and lvh at baseline . at the time of randomization , 1,702 ( 65.8% ) patients without and 65 ( 67.0% ) patients with new - onset diabetes were under treatment with antihypertensive drugs ( ccb 40.1 vs. 34.0% , p = 0.229 ; ace inhibitor 13.3 vs. 16.5% , p = 0.363 ; arb 17.9 vs. 22.7% , p = 0.229 ; diuretic 3.1 vs. 5.2% , p = 0.255 ; -blocker 12.9 vs. 16.5% , p = 0.297 ; and -blocker 5.6 vs. 4.1% , p = 0.542 , respectively ) . multiple cox regression analysis revealed that pulse pressure ( per 1 sd increase ) was an independent predictor of new - onset diabetes ( hr 1.44 [ 95% ci 1.151.79 ] , p = 0.001 ) ( table 2 ) . in addition , risk was also significantly associated with male sex , bmi , lvh , and concomitant use of diuretics . as reported previously , candesartan - based regimens significantly reduced the risk of new - onset diabetes compared with amlodipine - based regimens ( 15 ) . predictors of new - onset diabetes by multiple cox regression analysis data are hr ( 95% ci ) and are adjusted for each variable . because pulse pressure was calculated as the difference between sbp and dbp , we conducted separate analyses for sbp and dbp and found that dbp ( per 1 sd decrease ) was also an independent predictor for new - onset diabetes , whereas sbp ( per 1 sd increase ) was not ( hr for sbp 1.13 [ 95% ci 0.901.41 ] , p = 0.284 ; and hr for dbp 1.45 [ 1.161.81 ] , p < 0.001 ) . subgroup analysis stratified by age ( cutoff point : age 65 years ) revealed that pulse pressure remained significantly associated with the risk of new - onset diabetes in both age - groups ( aged < 65 years : hr 1.72 [ 95% ci 1.182.49 ] , p = 0.004 ; aged 65 years : 1.34 [ 1.011.77 ] , p = 0.042 ; and pinteraction = 0.152 ) . however , dbp was significantly associated with risk only in the group aged < 65 years , whereas whole sbp was not associated in either age - group ( for sbp , aged < 65 years : 1.20 [ 0.861.67 ] , p = 0.284 ; aged 65 years : 1.16 [ 0.841.59 ] , p = 0.374 ; and pinteraction = 0.780 ; for dbp , aged < 65 years : 1.58 [ 1.102.28 ] , p = 0.014 ; aged 65 years : 1.32 [ 0.991.76 ] , p = 0.057 ; and pinteraction = 0.290 ) . , blood pressures of 130/60 and 180/110 mmhg both give a pulse pressure of 70 mmhg ) , we evaluated the association of combinations of sbp and dbp with the risk of new - onset diabetes . 1 , a strong association with risk was seen for higher pulse pressures arising mainly due to a lower dbp . from this result , we hypothesized that patients at high risk of new - onset diabetes had increased arterial stiffness . accordingly , we next examined the association between ppf and the risk of new - onset diabetes and found that ppf ( per 1 sd increase ) was an independent predictor of new - onset diabetes ( hr 1.49 [ 95% ci 1.211.84 ] , p < 0.001 ) . in subgroup analysis stratified by age , ppf ( per 1 sd increase ) was significantly associated with the risk of new - onset diabetes in both age - groups ( aged < 65 : 1.88 [ 1.292.73 ] , p < 0.001 ; aged 65 : 1.34 [ 1.031.74 ] , p = 0.027 ; and pinteraction = 0.057 ) . 2 , a trend to an increased incidence of new - onset diabetes with increasing ppf was seen in patients with amlodipine - based regimens , but not in those with candesartan - based regimens ( p = 0.0234 for interaction in the quadratic term ) . candesartan - based regimens significantly suppressed the incidence of new - onset diabetes in the highest quartile of ppf . effect of candesartan and amlodipine on the incidence of new - onset diabetes stratified by quartile of ppf . ppf ( linear and quadratic terms ) , the allocated drugs , and their interaction terms were entered in multiple cox regression model . in this study , we demonstrated that pulse pressure was a predictor of new - onset diabetes in high - risk hypertensive patients , independent of the effects of antihypertensive treatment and other possible risk factors for new - onset diabetes . further , a higher pulse pressure arising mainly due to a lower dbp , indicating that the increased pulse pressure resulted largely from increased arterial stiffness , was associated with a higher risk of new - onset diabetes . this finding suggests that increased arterial stiffness , reflected in an increased pulse pressure , may be related to the process of new - onset diabetes in high - risk hypertensive patients , albeit that the mechanism of this association remains to be elucidated . first , increased pulse pressure may be a surrogate marker for the risk of new - onset diabetes . supporting this suggestion , a higher pulse pressure , reflecting increased arterial stiffness , was observed in hypertensive patients with metabolic syndrome than in those without ( 17 ) . further , accumulating evidence supports the concept of increased arterial stiffness in patients with a metabolic disturbance , which is considered a potential mechanism linking metabolic disturbance to increased cvd risk ( 1113 ) . prolonged exposure to hyperglycemic conditions can lead to increased arterial stiffness via collagen cross - linking due to nonenzymatic glycation , endothelial dysfunction , inflammation , and local activation of the renin - angiotensin - aldosterone system in pre - diabetic as well as diabetic individuals ( 18 ) . indeed , ppf , represented as a parameter of the pulsatile component of blood pressure , was superior to pulse pressure in terms of the risk stratification of new - onset diabetes . accordingly , increased pulse pressure , reflecting increased arterial stiffness , may be both a cause and a consequence of microvascular dysfunction , leading to a vicious cycle in impaired glucose metabolism as well as arteriosclerosis ( 9,19,20 ) . the present study also revealed that electrocardiographic or echocardiographic lvh at baseline was an independent predictor of new - onset diabetes . ( 22 ) reported that in - treatment resolution or continued absence of electrocardiographic lvh was associated with a lower incidence of diabetes . because pulse pressure was positively related to lvh ( 23 ) ( 24 ) found that treatment with the arb losartan was associated with less peripheral vascular hypertrophy / rarefaction and higher insulin sensitivity than that with atenolol , supporting the hypothesis that microvascular dysfunction in hypertension may induce insulin resistance . in the present study , the suppressive effect of the arb candesartan against new - onset diabetes tended to strengthen as ppf increased . these results suggest that arbs decrease the risk of new - onset diabetes partly via the improvement of microcirculation . although the prevalence of diabetes increases with age ( 25 ) , it remains unclear whether age is a risk factor for new - onset diabetes ( 68 ) . in the present study , age at baseline we assumed that high - risk elderly hypertensive patients who did not have diabetes at baseline were survivors who had avoided the development of diabetes and that their underlying risk of new - onset diabetes and ability to metabolize glucose may thus have differed from those of younger subjects . we also observed a strong association between pulse pressure and new - onset diabetes in patients aged < 65 years , possibly owing to the same mechanism . second , although we found an interesting association between pulse pressure and the risk of new - onset diabetes , the case - j trial was not designed to prospectively evaluate this association , and we were consequently unable to elucidate causality , because we did not directly measure parameters of arterial stiffness or collect the data to clarify the underlying mechanism . third , we were unable to include baseline data regarding glucose metabolism into the multiple cox regression analysis or information about a family history of diabetes , physical activity , or diet , which are well - known and important risk factors for new - onset diabetes . fourth , new - onset diabetes was prespecified as the end point just before the completion of the case - j trial . accordingly , there was a possibility of nonreporting bias , because the definition of new - onset diabetes was not in the original protocol and determination of whether new - onset diabetes had occurred depended on the participating investigators ' reports . nevertheless , the present study is the first to examine the association of pulse pressure with new - onset diabetes in hypertensive patients and may provide useful information in understanding the underlying mechanism between hypertension and new - onset diabetes . in summary , we found that pulse pressure is an independent predictor of new - onset diabetes in high - risk japanese hypertensive patients . the development of type 2 diabetes may involve increased arterial stiffness , suggesting the importance of the microvascular dysfunction theory in the underlying pathophysiological mechanism between hypertension and new - onset diabetes . to our knowledge , this study is the first to report the relation between pulse pressure and new - onset diabetes in hypertensive patients . further studies are required to elucidate the significance of pulse pressure in new - onset diabetes in hypertensive patients .

victoria is the second most populous state in australia ; it has a temperate climate , and the annual influenza season usually occurs during may september . each season , on behalf of the victorian government department of health , the victorian infectious diseases reference laboratory conducts surveillance for influenza - like illness ( ili ; defined as history of fever , cough , and fatigue / malaise ) and laboratory - confirmed influenza . general practitioners within the network provide weekly reports on case - patients with ili as a proportion of total patients seen and send swabs from patients with ili to the laboratory for testing . in 2010 , a total of 87 practitioners participated in the program , which operated for 25 weeks , from may 3 ( week 19 ) through october 24 ( week 43 ) . practitioners were asked to collect nose and throat swabs from patients with an ili ( 20 ) within 4 days after onset of the patient 's symptoms . samples were collected by using copan dry swabs ( copan italia , brescia , italy ) and were placed in virus transport medium . practitioners were also asked to provide data on the patient 's age , sex , date of symptom onset , vaccination status , type of influenza vaccine ( monovalent or trivalent / seasonal ) received , and date of vaccination . type of vaccine and date of vaccination were ascertained from medical records and patient report . rna was extracted from clinical specimens by using a corbett extraction robot ( corbett robotics , brisbane , australia ) , followed by reverse transcription to cdna by using random hexamers . pcr amplification and detection selective for the type a influenza virus matrix gene was performed by using primers and a taqman probe on an abi-7500 fast real - time pcr system ( applied biosystems , foster city , ca , usa ) . samples determined to be positive by this assay were confirmed as positive or negative for pandemic ( h1n1 ) 2009 in a second real - time pcr that incorporated primers and probes specific for the hemagglutinin gene of the pandemic ( h1n1 ) 2009 virus . one practitioner chose to send samples to the state reference laboratory in south australia for testing with equivalent diagnostic assays . a case - patient was defined as a person with ili for whom test results for pandemic ( h1n1 ) 2009 were positive ; a control was defined as a person with negative test results for influenza virus . analysis of vaccine effectiveness against other influenza subtypes was not undertaken because of the almost exclusive circulation of pandemic ( h1n1 ) 2009 virus during the season ; therefore , patients with positive test results for other influenza viruses were excluded . a control could become a case - patient if another illness developed during the season , but a case - patient was no longer at risk and could not be included again . all analyses were conducted by using stata version 10.0 ( statacorp lp , college station , tx , usa ) . the test was used to compare proportions , and the mann - whitney u test was used to compare time from vaccination to time seen by practitioner ; p<0.05 was considered significant . patients were excluded from the vaccine effectiveness analysis if vaccination status was unknown , if the date of symptom onset was unknown , or if the interval between symptom onset and specimen collection was > 4 days ( because of decreased likelihood of a positive result after this time ) ( 21,22 ) . patients were considered not vaccinated if time between date of vaccination and symptom onset was < 14 days . if only the month of vaccination was reported , the date of vaccination was conservatively estimated to be the last day of the month . to avoid overestimation of vaccine effectiveness arising from recruitment of controls when influenza was not circulating in the population , analysis was restricted to case - patients and controls detected within the influenza season , defined as the period during which influenza - positive case - patients were detected ( weeks 2640 ) . vaccine effectiveness was defined as ( 1odds ratio ) 100% ; the odds ratio is the odds of laboratory - confirmed pandemic ( h1n1 ) 2009 case - patients having been vaccinated divided by the odds of controls having been vaccinated . in the test - negative case control design , the odds ratio estimates the incidence density ( rate ) ratio because controls are selected longitudinally throughout the course of the study ( i.e. , by density sampling ) ( 23,24 ) . control studies has also been shown to approximate the risk ratio under conditions of varying attack rates and test sensitivity and specificity ( 25 ) . logistic regression was used to calculate odds ratios and 95% confidence intervals ( cis ) for having laboratory - confirmed pandemic ( h1n1 ) 2009 , which were adjusted for the variables of age group and month of specimen collection against the following : seasonal vaccine , monovalent vaccine , both vaccines , and any ( either or both the seasonal and monovalent ) vaccine . sensitivity analyses were conducted to determine the effects of the following on vaccine effectiveness : not censoring for specimens collected from ili patients > 4 days after symptom onset , including controls recruited outside the defined influenza season , and assuming that patients with unspecified type a influenza had pandemic ( h1n1 ) 2009 . data in this study were collected , used and reported under the legislative authorization of the victorian public health and wellbeing act 2008 and public health and wellbeing regulations 2009 . victoria is the second most populous state in australia ; it has a temperate climate , and the annual influenza season usually occurs during may september . each season , on behalf of the victorian government department of health , the victorian infectious diseases reference laboratory conducts surveillance for influenza - like illness ( ili ; defined as history of fever , cough , and fatigue / malaise ) and laboratory - confirmed influenza . general practitioners within the network provide weekly reports on case - patients with ili as a proportion of total patients seen and send swabs from patients with ili to the laboratory for testing . in 2010 , a total of 87 practitioners participated in the program , which operated for 25 weeks , from may 3 ( week 19 ) through october 24 ( week 43 ) . practitioners were asked to collect nose and throat swabs from patients with an ili ( 20 ) within 4 days after onset of the patient 's symptoms . samples were collected by using copan dry swabs ( copan italia , brescia , italy ) and were placed in virus transport medium . practitioners were also asked to provide data on the patient 's age , sex , date of symptom onset , vaccination status , type of influenza vaccine ( monovalent or trivalent / seasonal ) received , and date of vaccination . type of vaccine and date of vaccination were ascertained from medical records and patient report . rna was extracted from clinical specimens by using a corbett extraction robot ( corbett robotics , brisbane , australia ) , followed by reverse transcription to cdna by using random hexamers . pcr amplification and detection selective for the type a influenza virus matrix gene was performed by using primers and a taqman probe on an abi-7500 fast real - time pcr system ( applied biosystems , foster city , ca , usa ) . samples determined to be positive by this assay were confirmed as positive or negative for pandemic ( h1n1 ) 2009 in a second real - time pcr that incorporated primers and probes specific for the hemagglutinin gene of the pandemic ( h1n1 ) 2009 virus . one practitioner chose to send samples to the state reference laboratory in south australia for testing with equivalent diagnostic assays . a case - patient was defined as a person with ili for whom test results for pandemic ( h1n1 ) 2009 were positive ; a control was defined as a person with negative test results for influenza virus . analysis of vaccine effectiveness against other influenza subtypes was not undertaken because of the almost exclusive circulation of pandemic ( h1n1 ) 2009 virus during the season ; therefore , patients with positive test results for other influenza viruses were excluded . a control could become a case - patient if another illness developed during the season , but a case - patient was no longer at risk and could not be included again . all analyses were conducted by using stata version 10.0 ( statacorp lp , college station , tx , usa ) . the test was used to compare proportions , and the mann - whitney u test was used to compare time from vaccination to time seen by practitioner ; p<0.05 was considered significant . patients were excluded from the vaccine effectiveness analysis if vaccination status was unknown , if the date of symptom onset was unknown , or if the interval between symptom onset and specimen collection was > 4 days ( because of decreased likelihood of a positive result after this time ) ( 21,22 ) . patients were considered not vaccinated if time between date of vaccination and symptom onset was < 14 days . if only the month of vaccination was reported , the date of vaccination was conservatively estimated to be the last day of the month . to avoid overestimation of vaccine effectiveness arising from recruitment of controls when influenza was not circulating in the population , analysis was restricted to case - patients and controls detected within the influenza season , defined as the period during which influenza - positive case - patients were detected ( weeks 2640 ) . vaccine effectiveness was defined as ( 1odds ratio ) 100% ; the odds ratio is the odds of laboratory - confirmed pandemic ( h1n1 ) 2009 case - patients having been vaccinated divided by the odds of controls having been vaccinated . in the test - negative case control design , the odds ratio estimates the incidence density ( rate ) ratio because controls are selected longitudinally throughout the course of the study ( i.e. , by density sampling ) ( 23,24 ) . control studies has also been shown to approximate the risk ratio under conditions of varying attack rates and test sensitivity and specificity ( 25 ) . logistic regression was used to calculate odds ratios and 95% confidence intervals ( cis ) for having laboratory - confirmed pandemic ( h1n1 ) 2009 , which were adjusted for the variables of age group and month of specimen collection against the following : seasonal vaccine , monovalent vaccine , both vaccines , and any ( either or both the seasonal and monovalent ) vaccine . sensitivity analyses were conducted to determine the effects of the following on vaccine effectiveness : not censoring for specimens collected from ili patients > 4 days after symptom onset , including controls recruited outside the defined influenza season , and assuming that patients with unspecified type a influenza had pandemic ( h1n1 ) 2009 . data in this study were collected , used and reported under the legislative authorization of the victorian public health and wellbeing act 2008 and public health and wellbeing regulations 2009 . a total of 172,411 patients were seen by participating practitioners during the study period , of whom 678 ( 0.4% ) had ili . after a nadir ili rate of 0.2% in week 21 , the rate gradually increased to 0.4% in week 31 before increasing more sharply to a peak of 0.9% in week 36 . swabs were collected from 478 ( 71% ) ili patients , among whom 170 ( 36% ) had positive influenza test results and the remainder were negative . influenza - positive patients were detected during weeks 2640 , which was defined as the influenza season ( figure ) . a total of 142 patients were excluded from further analysis because vaccination status was unknown ( n = 11 ) , symptom onset date was unknown ( n = 33 ) , time between symptom onset and specimen collection was > 4 days ( n = 43 ) , or the specimen was collected outside the influenza season ( n = 82 ) . a significantly higher proportion of influenza - negative patients ( 13% ) than influenza - positive patients ( 4% ) were excluded because > 4 days had elapsed between symptom onset and specimen collection ( p = 0.001 ) . no significant difference was found by age group for whether study participants had a specimen collected within 4 days after symptom onset ( p = 0.10 ) . influenza status of patients seen at sentinel general practices , victoria , australia , may 3 ( week 19 ) through october 24 ( week 43 ) , 2010 . of the remaining 336 patients , 156 ( 46% ) had positive influenza test results . most ( 89% ) influenza case - patients had pandemic ( h1n1 ) 2009 , 6% had unspecified type a influenza , 4% had influenza a ( h3n2 ) , and 1% had influenza type b ( figure ) . after exclusion of the other influenza patients , 139 pandemic ( h1n1 ) 2009 case - patients and 180 controls were included in the study analysis . most ( 57% ) participants were 2049 years of age , and case - patients were significantly younger than controls ( p = 0.001 ) ; no case - patient was > 65 years of age ( table 1 ) . no statistically significant difference was found between male and female study participants by case or control status ( p = 0.60 ) or by vaccination status ( p = 0.09 ) . the high proportion of case - patients detected in august resulted in a significant difference between case - patients and controls by month of swab collection ( p<0.001 ) . overall , 59 ( 18% ) study participants were reported as vaccinated with any vaccine , but the proportion was higher among controls ( 26% ) than among case - patients ( 9% ; p<0.001 ) . the proportion of controls , who were mostly older , who had received the trivalent seasonal vaccine was higher than the proportion of controls who had received the monovalent vaccine ( table 1 ) . similarly , controls who had received both vaccines were all > 20 years of age . only case - patients who were 519 and 2049 years of age influenza vaccine type was not specified for 1 case - patient and 1 control , each of whom was reported as vaccinated . reflecting the availability of each vaccine , the median period between vaccination and visit to a general practitioner was significantly shorter for those who received seasonal vaccine ( 114 days ) than for those who received monovalent vaccine ( 223 days ; p<0.0001 ) . no significant difference in the time from vaccination to practitioner visit was found between case - patients and controls for seasonal ( p = 0.70 ) or monovalent vaccine ( p = 0.95 ) . in general , point estimates of vaccine effectiveness adjusted for patient age and month of specimen collection differed little from crude estimates ( table 2 ) . a significant protective effect was observed for seasonal vaccine only ( adjusted vaccine effectiveness 79% ; 95% ci 33%93% ) and seasonal and monovalent vaccines ( adjusted vaccine effectiveness 81% ; 95% ci 7%96% ) . the adjusted vaccine effectiveness for receipt of any ( either or both the seasonal and monovalent ) vaccine was lower at 67% because of the 47% vaccine effectiveness for monovalent vaccine . the absence of vaccinated case - patients and controls meant vaccine effectiveness could not be estimated for several of the 5 age groups ( table 1 ) ; therefore , age was collapsed into 3 variables : children ( 019 years ) , working - age adults ( 2064 years ) , and elderly persons ( > 65 years ) . estimates of vaccine effectiveness for working adults were 0%14% higher than the overall adjusted estimates ; estimates for children were either undefined because no controls were vaccinated or were without a significant protective effect . vaccine effectiveness could not be calculated for elderly persons because there were no case - patients in this age group . sensitivity analyses to determine the effects of certain assumptions resulted in variations in the adjusted vaccine effectiveness point estimates of 0%3% and no changes to their relative significance . the effects considered were as follows : assumption that those patients with unspecified influenza type a had pandemic ( h1n1 ) 2009 , no exclusion of patients if > 4 days had elapsed between symptom onset and specimen collection , and no exclusion of patients if they were identified outside the defined influenza season . our results indicate that the 2010 seasonal trivalent influenza vaccine is > 80% effective against pandemic ( h1n1 ) 2009 virus , regardless whether given by itself or in addition to monovalent vaccine . groups in europe and canada have estimated the effectiveness of monovalent seasonal influenza vaccine against pandemic ( h1n1 ) 2009 virus to be 72%100% ( 1317 ) . however , the effectiveness of any vaccine ( monovalent , seasonal , or both ) against pandemic ( h1n1 ) 2009 virus was lower ( 67% , 95% ci 33%84% ) because effectiveness for monovalent vaccine only was 47% ( 95% ci 62% to 82% ) . the lower effectiveness of monovalent influenza vaccine against pandemic ( h1n1 ) 2009 virus compared with seasonal trivalent influenza vaccine is difficult to explain . both vaccines contain the same quantities ( 15 g ) of hemagglutinin ; and although the monovalent vaccine does not contain adjuvant and was available 6 months before the seasonal vaccine , it has been shown to be strongly immunogenic ( 3,9,10 ) . immunogenicity does not necessarily correlate directly with vaccine effectiveness , and we can not exclude waning immunity as an explanation for the lower effectiveness of monovalent vaccine in our study . waning immunity after receipt of monovalent vaccine has been suggested after an interim study from the united kingdom for the 201011 influenza season ( 26 ) . the finding could also be a product of the relatively small number of case - patients and controls who received only the monovalent vaccine , given that vaccine effectiveness estimates can change considerably by the inclusion or exclusion of 12 vaccinated study participants . when stratified by age , estimates of vaccine effectiveness for working - age adults were higher and more precise than those for children . we previously demonstrated that the sentinel practitioner surveillance program in victoria is well suited for estimating vaccine effectiveness among working - age adults , who account for most of the surveillance population ( 18 ) , and the 2010 results were consistent with this observation . the relatively few participants in the young ( childhood ) age groups meant the study had insufficient power to produce defined or significant estimates of vaccine effectiveness . at the other end of the age spectrum , 2% of study participants ( 5 controls and 0 case - patients ) in 2010 were > 65 years of age compared with an average of 7% in this age group during 200307 ( 18 ) . although the absence of pandemic ( h1n1 ) 2009 case - patients > 65 years of age is not surprising , given that older adults have been shown to have relatively higher levels of cross - reactive antibodies to pandemic ( h1n1 ) 2009 virus ( 2729 ) , the reason for the low proportion of controls in this age group remains unclear . among the several explanations are a true lower rate of ili in older persons during 2010 , a lower rate of visits to practitioners for ili by persons in this age group ( or treatment at other health services such as hospitals ) , or preferential sampling of younger persons by practitioners ( and perhaps awareness that pandemic [ h1n1 ] 2009 was the predominant circulating influenza virus subtype ) . in addition to having a sample size large enough to provide vaccine effectiveness estimates by age group and influenza type , several other considerations with regard to design of case control studies of influenza vaccine effectiveness have been proposed : 1 ) whether the control group best represents the vaccination coverage of the source population and 2 ) whether collection and confounding variables have been adjusted for , particularly underlying chronic conditions for which vaccine is recommended and previous influenza vaccination history ( 30 ) . a 2010 survey of pandemic vaccination suggests that monovalent vaccine coverage in the control group was generally consistent with that in the general population and that use of monovalent vaccine was 17% among those from victoria , compared with 13% among controls ( 31 ) . data about concurrent conditions of study participants that would indicate need for influenza vaccination were not collected during the 2010 influenza season ; thus , adjustment of the vaccine effectiveness estimates for this potentially confounding variable could not be conducted . such confounding by indication ( or negative confounding ) , in which persons at higher risk for influenza are more likely to be vaccinated , underestimates effectiveness of influenza vaccine but may be counteracted by healthy vaccinee bias ( or positive confounding ) , which overestimates effectiveness ( 30,32 ) . the extent to which these biases occur is likely to vary and may explain the positive and negative variation of crude influenza vaccine effectiveness estimates after adjustment for chronic conditions in several similar test - negative case speculation about the relative effects of these biases on how many received monovalent vaccine is also difficult ; vaccination was funded for the entire population of australia , but at the end of february 2010 , only 18% had been vaccinated ( 31 ) . similar methods using test - negative controls to assess seasonal and pandemic vaccine effectiveness against both seasonal and pandemic influenza viruses have been applied in north america and europe ( 13,16,17,3339 ) . observational studies provide a convenient and timely way to assess influenza vaccine effectiveness without the ethical , practical , and financial stringencies associated with clinical trials for vaccine efficacy , but they also have limitations . control design generally underestimates true vaccine effectiveness under most conditions of test sensitivity , specificity , and the ratio of influenza to noninfluenza attack rates ( 25 ) , although quantifying the extent of this effect in this study is difficult because the precise sensitivity and specificity of the test are not known . we attempted to limit ascertainment bias by censoring records that indicated specimen collection > 4 days after symptom onset and restricting the analysis to case - patients and controls tested within the influenza season only , although sensitivity analyses indicated little effect if these restrictions were relaxed . of note , these findings apply predominantly to working - age adults receiving medical care in the general practice setting ; the study did not include those who did not seek medical care for ili . thus , the study measured effectiveness of vaccine against illness severe enough to require a visit to a practitioner ; the results can not necessarily be generalized to other parts of the population , in particular young children and elderly persons . we were also unable to determine whether participants had previously been infected with pandemic ( h1n1 ) 2009 virus , which may result in overestimation of vaccine effectiveness . in conclusion control study design to an established sentinel surveillance system to estimate effectiveness of a trivalent seasonal influenza vaccine , which included an a / california/7/2009 ( h1n1)like virus , the pandemic ( h1n1 ) 2009 influenza virus strain . this strain is also a component of the trivalent influenza vaccine for the 201011 northern hemisphere influenza season ( 40 ) . the trivalent vaccine provided significant protection against laboratory - confirmed pandemic ( h1n1 ) 2009 virus infection .

to estimate effectiveness of seasonal trivalent and monovalent influenza vaccines against pandemic influenza a ( h1n1 ) 2009 virus , we conducted a test - negative case control study in victoria , australia , in 2010 . patients seen for influenza - like illness by general practitioners in a sentinel surveillance network during 2010 were tested for influenza ; vaccination status was recorded . case - patients had positive pcrs for pandemic ( h1n1 ) 2009 virus , and controls had negative influenza test results . of 319 eligible patients , test results for 139 ( 44% ) were pandemic ( h1n1 ) 2009 virus positive . adjusted effectiveness of seasonal vaccine against pandemic ( h1n1 ) 2009 virus was 79% ( 95% confidence interval 33%93% ) ; effectiveness of monovalent vaccine was 47% and not statistically significant . vaccine effectiveness was higher among adults . despite some limitations , this study indicates that the first seasonal trivalent influenza vaccine to include the pandemic ( h1n1 ) 2009 virus strain provided significant protection against laboratory - confirmed pandemic ( h1n1 ) 2009 infection .

Methods Sentinel Surveillance Laboratory Testing Ascertainment of Case-patients and Controls Data Analysis and Calculation of Vaccine Effectiveness Ethical Considerations Results Discussion

victoria is the second most populous state in australia ; it has a temperate climate , and the annual influenza season usually occurs during may september . each season , on behalf of the victorian government department of health , the victorian infectious diseases reference laboratory conducts surveillance for influenza - like illness ( ili ; defined as history of fever , cough , and fatigue / malaise ) and laboratory - confirmed influenza . general practitioners within the network provide weekly reports on case - patients with ili as a proportion of total patients seen and send swabs from patients with ili to the laboratory for testing . in 2010 , a total of 87 practitioners participated in the program , which operated for 25 weeks , from may 3 ( week 19 ) through october 24 ( week 43 ) . practitioners were also asked to provide data on the patient 's age , sex , date of symptom onset , vaccination status , type of influenza vaccine ( monovalent or trivalent / seasonal ) received , and date of vaccination . rna was extracted from clinical specimens by using a corbett extraction robot ( corbett robotics , brisbane , australia ) , followed by reverse transcription to cdna by using random hexamers . samples determined to be positive by this assay were confirmed as positive or negative for pandemic ( h1n1 ) 2009 in a second real - time pcr that incorporated primers and probes specific for the hemagglutinin gene of the pandemic ( h1n1 ) 2009 virus . a case - patient was defined as a person with ili for whom test results for pandemic ( h1n1 ) 2009 were positive ; a control was defined as a person with negative test results for influenza virus . analysis of vaccine effectiveness against other influenza subtypes was not undertaken because of the almost exclusive circulation of pandemic ( h1n1 ) 2009 virus during the season ; therefore , patients with positive test results for other influenza viruses were excluded . a control could become a case - patient if another illness developed during the season , but a case - patient was no longer at risk and could not be included again . the test was used to compare proportions , and the mann - whitney u test was used to compare time from vaccination to time seen by practitioner ; p<0.05 was considered significant . patients were excluded from the vaccine effectiveness analysis if vaccination status was unknown , if the date of symptom onset was unknown , or if the interval between symptom onset and specimen collection was > 4 days ( because of decreased likelihood of a positive result after this time ) ( 21,22 ) . patients were considered not vaccinated if time between date of vaccination and symptom onset was < 14 days . to avoid overestimation of vaccine effectiveness arising from recruitment of controls when influenza was not circulating in the population , analysis was restricted to case - patients and controls detected within the influenza season , defined as the period during which influenza - positive case - patients were detected ( weeks 2640 ) . vaccine effectiveness was defined as ( 1odds ratio ) 100% ; the odds ratio is the odds of laboratory - confirmed pandemic ( h1n1 ) 2009 case - patients having been vaccinated divided by the odds of controls having been vaccinated . in the test - negative case control design , the odds ratio estimates the incidence density ( rate ) ratio because controls are selected longitudinally throughout the course of the study ( i.e. , by density sampling ) ( 23,24 ) . logistic regression was used to calculate odds ratios and 95% confidence intervals ( cis ) for having laboratory - confirmed pandemic ( h1n1 ) 2009 , which were adjusted for the variables of age group and month of specimen collection against the following : seasonal vaccine , monovalent vaccine , both vaccines , and any ( either or both the seasonal and monovalent ) vaccine . sensitivity analyses were conducted to determine the effects of the following on vaccine effectiveness : not censoring for specimens collected from ili patients > 4 days after symptom onset , including controls recruited outside the defined influenza season , and assuming that patients with unspecified type a influenza had pandemic ( h1n1 ) 2009 . data in this study were collected , used and reported under the legislative authorization of the victorian public health and wellbeing act 2008 and public health and wellbeing regulations 2009 . victoria is the second most populous state in australia ; it has a temperate climate , and the annual influenza season usually occurs during may september . each season , on behalf of the victorian government department of health , the victorian infectious diseases reference laboratory conducts surveillance for influenza - like illness ( ili ; defined as history of fever , cough , and fatigue / malaise ) and laboratory - confirmed influenza . general practitioners within the network provide weekly reports on case - patients with ili as a proportion of total patients seen and send swabs from patients with ili to the laboratory for testing . in 2010 , a total of 87 practitioners participated in the program , which operated for 25 weeks , from may 3 ( week 19 ) through october 24 ( week 43 ) . practitioners were also asked to provide data on the patient 's age , sex , date of symptom onset , vaccination status , type of influenza vaccine ( monovalent or trivalent / seasonal ) received , and date of vaccination . rna was extracted from clinical specimens by using a corbett extraction robot ( corbett robotics , brisbane , australia ) , followed by reverse transcription to cdna by using random hexamers . samples determined to be positive by this assay were confirmed as positive or negative for pandemic ( h1n1 ) 2009 in a second real - time pcr that incorporated primers and probes specific for the hemagglutinin gene of the pandemic ( h1n1 ) 2009 virus . one practitioner chose to send samples to the state reference laboratory in south australia for testing with equivalent diagnostic assays . a case - patient was defined as a person with ili for whom test results for pandemic ( h1n1 ) 2009 were positive ; a control was defined as a person with negative test results for influenza virus . analysis of vaccine effectiveness against other influenza subtypes was not undertaken because of the almost exclusive circulation of pandemic ( h1n1 ) 2009 virus during the season ; therefore , patients with positive test results for other influenza viruses were excluded . a control could become a case - patient if another illness developed during the season , but a case - patient was no longer at risk and could not be included again . the test was used to compare proportions , and the mann - whitney u test was used to compare time from vaccination to time seen by practitioner ; p<0.05 was considered significant . patients were excluded from the vaccine effectiveness analysis if vaccination status was unknown , if the date of symptom onset was unknown , or if the interval between symptom onset and specimen collection was > 4 days ( because of decreased likelihood of a positive result after this time ) ( 21,22 ) . if only the month of vaccination was reported , the date of vaccination was conservatively estimated to be the last day of the month . to avoid overestimation of vaccine effectiveness arising from recruitment of controls when influenza was not circulating in the population , analysis was restricted to case - patients and controls detected within the influenza season , defined as the period during which influenza - positive case - patients were detected ( weeks 2640 ) . vaccine effectiveness was defined as ( 1odds ratio ) 100% ; the odds ratio is the odds of laboratory - confirmed pandemic ( h1n1 ) 2009 case - patients having been vaccinated divided by the odds of controls having been vaccinated . in the test - negative case control design , the odds ratio estimates the incidence density ( rate ) ratio because controls are selected longitudinally throughout the course of the study ( i.e. logistic regression was used to calculate odds ratios and 95% confidence intervals ( cis ) for having laboratory - confirmed pandemic ( h1n1 ) 2009 , which were adjusted for the variables of age group and month of specimen collection against the following : seasonal vaccine , monovalent vaccine , both vaccines , and any ( either or both the seasonal and monovalent ) vaccine . sensitivity analyses were conducted to determine the effects of the following on vaccine effectiveness : not censoring for specimens collected from ili patients > 4 days after symptom onset , including controls recruited outside the defined influenza season , and assuming that patients with unspecified type a influenza had pandemic ( h1n1 ) 2009 . data in this study were collected , used and reported under the legislative authorization of the victorian public health and wellbeing act 2008 and public health and wellbeing regulations 2009 . swabs were collected from 478 ( 71% ) ili patients , among whom 170 ( 36% ) had positive influenza test results and the remainder were negative . influenza - positive patients were detected during weeks 2640 , which was defined as the influenza season ( figure ) . a total of 142 patients were excluded from further analysis because vaccination status was unknown ( n = 11 ) , symptom onset date was unknown ( n = 33 ) , time between symptom onset and specimen collection was > 4 days ( n = 43 ) , or the specimen was collected outside the influenza season ( n = 82 ) . a significantly higher proportion of influenza - negative patients ( 13% ) than influenza - positive patients ( 4% ) were excluded because > 4 days had elapsed between symptom onset and specimen collection ( p = 0.001 ) . influenza status of patients seen at sentinel general practices , victoria , australia , may 3 ( week 19 ) through october 24 ( week 43 ) , 2010 . of the remaining 336 patients , 156 ( 46% ) had positive influenza test results . most ( 89% ) influenza case - patients had pandemic ( h1n1 ) 2009 , 6% had unspecified type a influenza , 4% had influenza a ( h3n2 ) , and 1% had influenza type b ( figure ) . after exclusion of the other influenza patients , 139 pandemic ( h1n1 ) 2009 case - patients and 180 controls were included in the study analysis . most ( 57% ) participants were 2049 years of age , and case - patients were significantly younger than controls ( p = 0.001 ) ; no case - patient was > 65 years of age ( table 1 ) . no statistically significant difference was found between male and female study participants by case or control status ( p = 0.60 ) or by vaccination status ( p = 0.09 ) . the high proportion of case - patients detected in august resulted in a significant difference between case - patients and controls by month of swab collection ( p<0.001 ) . overall , 59 ( 18% ) study participants were reported as vaccinated with any vaccine , but the proportion was higher among controls ( 26% ) than among case - patients ( 9% ; p<0.001 ) . the proportion of controls , who were mostly older , who had received the trivalent seasonal vaccine was higher than the proportion of controls who had received the monovalent vaccine ( table 1 ) . only case - patients who were 519 and 2049 years of age influenza vaccine type was not specified for 1 case - patient and 1 control , each of whom was reported as vaccinated . reflecting the availability of each vaccine , the median period between vaccination and visit to a general practitioner was significantly shorter for those who received seasonal vaccine ( 114 days ) than for those who received monovalent vaccine ( 223 days ; p<0.0001 ) . no significant difference in the time from vaccination to practitioner visit was found between case - patients and controls for seasonal ( p = 0.70 ) or monovalent vaccine ( p = 0.95 ) . in general , point estimates of vaccine effectiveness adjusted for patient age and month of specimen collection differed little from crude estimates ( table 2 ) . a significant protective effect was observed for seasonal vaccine only ( adjusted vaccine effectiveness 79% ; 95% ci 33%93% ) and seasonal and monovalent vaccines ( adjusted vaccine effectiveness 81% ; 95% ci 7%96% ) . the adjusted vaccine effectiveness for receipt of any ( either or both the seasonal and monovalent ) vaccine was lower at 67% because of the 47% vaccine effectiveness for monovalent vaccine . the absence of vaccinated case - patients and controls meant vaccine effectiveness could not be estimated for several of the 5 age groups ( table 1 ) ; therefore , age was collapsed into 3 variables : children ( 019 years ) , working - age adults ( 2064 years ) , and elderly persons ( > 65 years ) . estimates of vaccine effectiveness for working adults were 0%14% higher than the overall adjusted estimates ; estimates for children were either undefined because no controls were vaccinated or were without a significant protective effect . vaccine effectiveness could not be calculated for elderly persons because there were no case - patients in this age group . sensitivity analyses to determine the effects of certain assumptions resulted in variations in the adjusted vaccine effectiveness point estimates of 0%3% and no changes to their relative significance . the effects considered were as follows : assumption that those patients with unspecified influenza type a had pandemic ( h1n1 ) 2009 , no exclusion of patients if > 4 days had elapsed between symptom onset and specimen collection , and no exclusion of patients if they were identified outside the defined influenza season . our results indicate that the 2010 seasonal trivalent influenza vaccine is > 80% effective against pandemic ( h1n1 ) 2009 virus , regardless whether given by itself or in addition to monovalent vaccine . groups in europe and canada have estimated the effectiveness of monovalent seasonal influenza vaccine against pandemic ( h1n1 ) 2009 virus to be 72%100% ( 1317 ) . however , the effectiveness of any vaccine ( monovalent , seasonal , or both ) against pandemic ( h1n1 ) 2009 virus was lower ( 67% , 95% ci 33%84% ) because effectiveness for monovalent vaccine only was 47% ( 95% ci 62% to 82% ) . the lower effectiveness of monovalent influenza vaccine against pandemic ( h1n1 ) 2009 virus compared with seasonal trivalent influenza vaccine is difficult to explain . both vaccines contain the same quantities ( 15 g ) of hemagglutinin ; and although the monovalent vaccine does not contain adjuvant and was available 6 months before the seasonal vaccine , it has been shown to be strongly immunogenic ( 3,9,10 ) . immunogenicity does not necessarily correlate directly with vaccine effectiveness , and we can not exclude waning immunity as an explanation for the lower effectiveness of monovalent vaccine in our study . waning immunity after receipt of monovalent vaccine has been suggested after an interim study from the united kingdom for the 201011 influenza season ( 26 ) . the finding could also be a product of the relatively small number of case - patients and controls who received only the monovalent vaccine , given that vaccine effectiveness estimates can change considerably by the inclusion or exclusion of 12 vaccinated study participants . when stratified by age , estimates of vaccine effectiveness for working - age adults were higher and more precise than those for children . we previously demonstrated that the sentinel practitioner surveillance program in victoria is well suited for estimating vaccine effectiveness among working - age adults , who account for most of the surveillance population ( 18 ) , and the 2010 results were consistent with this observation . the relatively few participants in the young ( childhood ) age groups meant the study had insufficient power to produce defined or significant estimates of vaccine effectiveness . at the other end of the age spectrum , 2% of study participants ( 5 controls and 0 case - patients ) in 2010 were > 65 years of age compared with an average of 7% in this age group during 200307 ( 18 ) . although the absence of pandemic ( h1n1 ) 2009 case - patients > 65 years of age is not surprising , given that older adults have been shown to have relatively higher levels of cross - reactive antibodies to pandemic ( h1n1 ) 2009 virus ( 2729 ) , the reason for the low proportion of controls in this age group remains unclear . among the several explanations are a true lower rate of ili in older persons during 2010 , a lower rate of visits to practitioners for ili by persons in this age group ( or treatment at other health services such as hospitals ) , or preferential sampling of younger persons by practitioners ( and perhaps awareness that pandemic [ h1n1 ] 2009 was the predominant circulating influenza virus subtype ) . in addition to having a sample size large enough to provide vaccine effectiveness estimates by age group and influenza type , several other considerations with regard to design of case control studies of influenza vaccine effectiveness have been proposed : 1 ) whether the control group best represents the vaccination coverage of the source population and 2 ) whether collection and confounding variables have been adjusted for , particularly underlying chronic conditions for which vaccine is recommended and previous influenza vaccination history ( 30 ) . a 2010 survey of pandemic vaccination suggests that monovalent vaccine coverage in the control group was generally consistent with that in the general population and that use of monovalent vaccine was 17% among those from victoria , compared with 13% among controls ( 31 ) . data about concurrent conditions of study participants that would indicate need for influenza vaccination were not collected during the 2010 influenza season ; thus , adjustment of the vaccine effectiveness estimates for this potentially confounding variable could not be conducted . such confounding by indication ( or negative confounding ) , in which persons at higher risk for influenza are more likely to be vaccinated , underestimates effectiveness of influenza vaccine but may be counteracted by healthy vaccinee bias ( or positive confounding ) , which overestimates effectiveness ( 30,32 ) . the extent to which these biases occur is likely to vary and may explain the positive and negative variation of crude influenza vaccine effectiveness estimates after adjustment for chronic conditions in several similar test - negative case speculation about the relative effects of these biases on how many received monovalent vaccine is also difficult ; vaccination was funded for the entire population of australia , but at the end of february 2010 , only 18% had been vaccinated ( 31 ) . similar methods using test - negative controls to assess seasonal and pandemic vaccine effectiveness against both seasonal and pandemic influenza viruses have been applied in north america and europe ( 13,16,17,3339 ) . observational studies provide a convenient and timely way to assess influenza vaccine effectiveness without the ethical , practical , and financial stringencies associated with clinical trials for vaccine efficacy , but they also have limitations . control design generally underestimates true vaccine effectiveness under most conditions of test sensitivity , specificity , and the ratio of influenza to noninfluenza attack rates ( 25 ) , although quantifying the extent of this effect in this study is difficult because the precise sensitivity and specificity of the test are not known . we attempted to limit ascertainment bias by censoring records that indicated specimen collection > 4 days after symptom onset and restricting the analysis to case - patients and controls tested within the influenza season only , although sensitivity analyses indicated little effect if these restrictions were relaxed . thus , the study measured effectiveness of vaccine against illness severe enough to require a visit to a practitioner ; the results can not necessarily be generalized to other parts of the population , in particular young children and elderly persons . we were also unable to determine whether participants had previously been infected with pandemic ( h1n1 ) 2009 virus , which may result in overestimation of vaccine effectiveness . in conclusion control study design to an established sentinel surveillance system to estimate effectiveness of a trivalent seasonal influenza vaccine , which included an a / california/7/2009 ( h1n1)like virus , the pandemic ( h1n1 ) 2009 influenza virus strain . this strain is also a component of the trivalent influenza vaccine for the 201011 northern hemisphere influenza season ( 40 ) . the trivalent vaccine provided significant protection against laboratory - confirmed pandemic ( h1n1 ) 2009 virus infection .

staphylococcus aureus is the leading cause of both community and hospital - acquired infection in the united states . among s. aureus isolates observed in the united states , between 40% and 50% are methicillin - resistant ( mrsa ) , greatly reducing therapeutic options . mrsa are responsible for several serious infections , including endocarditis , pneumonia , catheter - associated bloodstream infections , osteomyelitis , and skin infections . vancomycin , a glycopeptide antibiotic derived in the 1950s from streptomyces orientalis , has been the mainstay in anti - mrsa therapy for more than 50 years . over this time , vancomycin has remained remarkably active against most gram - positive bacterial strains . a 2013 surveillance study of over 2000 mrsa bloodstream isolates found less than 0.1% possessed a vancomycin minimum inhibitory concentration ( mic ) of 4 g / ml , the mic that establishes mrsa as a vancomycin - intermediate susceptible staphylococcus aureus ( visa ) according to the clinical and laboratory standards institute ( clsi ) . however , isolates with reduced susceptibility are increasing in the literature after first being reported 20 years ago [ 4 , 5 ] . nephrotoxicity is often associated with vancomycin therapy , and its narrow therapeutic index makes it the only currently available antibiotic with a consensus guideline statement regarding its dosing . the difficulty of vancomycin administration , increase of mrsa with reduced susceptibility to vancomycin , and toxicities associated with vancomycin use have led to the recent development of several novel anti - mrsa antibiotics . dalbavancin is approved for the treatment of acute bacterial skin and skin structure infections ( absssi ) caused by susceptible , gram - positive isolates . it possesses a similar spectrum of activity to vancomycin , including activity against methicillin - susceptible s. aureus ( mssa ) and mrsa . owing to its extended half - life , dalbavancin is dosed once weekly , with only two doses required for the duration of therapy . this novel antibiotic possesses several qualities that make it an interesting addition to the anti - mrsa armamentarium . this review will serve to introduce dalbavancin , review pertinent in vitro and clinical data , and discuss possible future therapeutic uses for dalbavancin outside the currently fda - approved indication . dalbavancin is a semisynthetic lipoglycopeptide derived structurally from antibiotic a-40926 , a teicoplanin - like , natural antibiotic produced by nonomuria spp . . several structural alterations were made in an attempt to enhance activity against s. aureus as well as extend the half - life of dalbavancin . perhaps the most important addition to dalbavancin is the extended , lipophilic side chain not present in teicoplanin or a-40926 . this additional side chain allows dalbavancin to anchor to the bacterial cell membrane , enhancing its potency , prolonging its half - life , and allowing for extended dosing intervals . dalbavancin also possesses an amidated carboxyl side group that enhances the agent s anti - staphylococcal activity . 1chemical structure of dalbavancin chemical structure of dalbavancin like other agents in its class , dalbavancin exerts its antimicrobial activity through interaction with terminal d - alanyl - d - alanine residues of peptidoglycan precursors . the binding of dalbavancin to these terminal residues prevents both transpeptidase and transglycosylase enzymes from catalyzing peptidoglycan cross - linking and thereby destroying the integrity of the cell wall , ultimately causing cell death . recent data have demonstrated that the lipophilic side chain of dalbavancin allows dimerization of the molecule and anchors dalbavancin to lipid ii in the cellular membrane , strengthening adherence to the d - alanyl - d - alanine target site and allowing for enhanced activity compared to vancomycin and teicoplanin . dalbavancin possesses in vitro activity against several gram - positive pathogens , including s. aureus , streptococcus agalactiae , streptococcus pyogenes , streptococcus anginosus , enterococcus faecium , and enterococcus faecalis , although activity against enterococci has not been observed clinically [ 9 , 1518 ] . against staphylococci and streptococci , dalbavancin possesses 816-fold greater in vitro activity than vancomycin in broth microdilution mic testing [ 15 , 16 ] . currently , the fda has an mic breakpoint established for only s. aureus , s. pyogenes , s. agalactiae , and s. anginosus of 0.125 g / ml . population data of more than 1100 staphylococci and 300 -hemolytic streptococci isolates from us medical centers in 2012 report dalbavancin mic50/90 values of 0.06/0.06 and 0.06/0.12 g / ml , respectively [ 15 , 16 ] . only three staphylococcal isolates ( 0.3% ) and 14 streptococcal isolates ( 4% ) possessed dalbavancin mic values above the currently proposed fda breakpoint . each resistant streptococcal isolate was a member of the species s. agalactiae . against visa , vancomycin mic ( 48 g / ml ) and heteroresistant visa ( hvisa ) , dalbavancin maintains four- to eightfold greater potency than vancomycin , although the mic values largely fall above 0.12 g / ml . recently , jones and colleagues demonstrated that vancomycin susceptibility can be considered a surrogate for dalbavancin susceptibility , as greater than 99.9% of over 42,000 vancomycin - susceptible isolates were susceptible to dalbavancin . against enterococci , dalbavancin possesses higher mic values than it does against other gram - positive species . dalbavancin activity against enterococci is largely contingent upon vancomycin activity , as 100% of vancomycin - susceptible enterococcus faecalis and e. faecium are inhibited at 0.125 g / ml [ 15 , 16 ] . however , vancomycin - resistant isolates of each species possess dalbavancin mic90 values greater than 4 g / ml . resistance to dalbavancin among staphylococci is rare , being reported in less than 1% of isolates [ 15 , 16 ] . mechanistically , there is precedent for reduced vancomycin susceptibility predicting reduced susceptibility to dalbavancin . reduced susceptibility among hvisa and visa is mediated through thickening of the cell wall and increased d - alanine - d - alanine binding sites [ 1921 ] . these increased binding sites increase the amount of vancomycin bound to the cell wall , necessitating higher exposures for similar efficacy . although dalbavancin is more potent at the active site than vancomycin , it is still inhibited in the same fashion . it remains to be seen whether the increased potency of dalbavancin will give it appreciable activity against these phenotypes clinically . vancomycin - resistant s. aureus ( vrsa ) are similarly resistant to dalbavancin , although the mechanism for this resistance is different than for hvisa and visa isolates . vancomycin , and subsequently dalbavancin , resistance within vrsa is mediated through a plasmid gene vana or vanb carried over from enterococcal species . these genes are responsible for altering the d - alanyl - d - alanine target site to a d - alanyl - d - lactate , rendering glycopeptide antibiotics ineffective . interestingly , dalbavancin has in vitro activity against enterococci with vanb present , but not against those with vana . the vanb activity may be present due to dalbavancin being a derivative of teicoplanin , which also possesses activity against enterococci with vanb . although this activity is worth noting , the vast majority of vancomycin - resistant enterococci in the united states possess vana . a complete breakdown of currently available dalbavancin mic data against gram - positive organisms is available in table 1.table 1dalbavancin mics for several gram - positive organisms [ 1518 , 2527]number of isolatesmic50 ( g / ml)mic90 ( g / ml)range% susc . s. aureus [ 1518 , 25 , 27]64,8430.060.060.008 to 0.599.7mssa [ 1518 , 25 , 27]37,2220.060.060.008 to 0.599.7mrsa [ 1518 , 25 , 27]27,2610.060.060.008 to 0.599.6hvisa 100.250.50.12 to 0.520visa 80.5n / a0.5 to 20dns sa 370.060.120.03 to 0.591.9lr sa 190.060.120.03 to 0.5100cns [ 15 , 16 , 27]4730.030.060.03 to 0.2599.6ms cns [ 15 , 16 , 27]2810.030.060.03 to 1n / amr cns [ 15 , 16 , 27]1930.030.120.03 to 0.25n / a enterococcus spp . [ 15 , 16]1160.06>40.03 to > 456vse [ 15 , 16]630.030.120.03 to 0.2596.8vre [ 15 , 16]53>4>40.03 to > 47.5vana vre [ 15 , 16]49>4>40.25 to > 40vanb vre [ 15 , 16]40.030.120.03 to 0.12100 e. faecalis 250.06>40.03 to > 476vse faecalis 190.030.060.03 to 0.06100vre faecalis 6>4>4>40 e. faecium 311>40.03 to > 441.9vse faecium 110.060.120.03 to 0.12100vre faecium 20>4>40.03 to > 410-hemo strep [ 15 , 16 , 26]12420.030.030.03 to 0.2598.6viridans strep [ 15 , 16 , 26]7860.030.030.03 to 0.2599.7 s. anginosus 1900.030.030.03 to 0.06100 s. milleri 140.030.030.03 to 0.06100 s. bovis 470.030.060.03 to 0.12100 s. dysgalactiae 500.030.060.03 to 0.12100 s. mitis 3050.030.060.03 to 0.2599.7 s. mutans 200.030.060.03 to 0.12100 s. salivarius 490.030.060.03 to 0.2598gas [ 15 , 16 , 26]5060.030.030.03 to 0.12100gbs [ 15 , 16 , 26]2870.030.120.03 to 0.2594.4 s. pneumoniae 8930.030.030.03 to 0.12100pssp 7390.030.030.03 to 0.12100pisp 1200.030.030.03 to 0.12100prsp 340.030.030.03100 sa s. aureus , ms methicillin - susceptible , mr methicillin - resistant , dns daptomycin - nonsusceptible , lr linezolid - resistant , cns coagulase - negative staphylococci , vse vancomycin - susceptible enterococci , vre vancomycin - resistant enterococci , gas group a streptococci , gbs group b streptococci , sp s. pneumo , ps penicillin - susceptible , pi penicillin - intermediate , pr penicillin - resistant dalbavancin mics for several gram - positive organisms [ 1518 , 2527 ] sa s. aureus , ms methicillin - susceptible , mr methicillin - resistant , dns daptomycin - nonsusceptible , lr linezolid - resistant , cns coagulase - negative staphylococci , vse vancomycin - susceptible enterococci , vre vancomycin - resistant enterococci , gas group a streptococci , gbs group b streptococci , sp s. pneumo , ps penicillin - susceptible , pi penicillin - intermediate , pr penicillin - resistant dalbavancin is currently fda - approved at a dose of 1000 mg on day 1 followed by 500 mg on day 8 for a complete course of therapy for absssi . pharmacokinetic data from human studies demonstrate that dalbavancin possesses linear , dose - related pharmacokinetics with an extended elimination half - life of approximately 14.5 days ( 346 h ) , allowing for the extended interval between doses [ 2832 ] . the approved dosing regimen was based on a clinical study evaluating complicated skin and skin structure infections ( csssi ) in which dalbavancin dosed at 1000 mg initially followed by 500 mg on day 8 was numerically superior to a one - time dose of 1100 mg . nearly 33% of dalbavancin is excreted in the urine unchanged , suggesting that non - renal methods of elimination play an important role in the metabolism of dalbavancin . the extended half - life of dalbavancin is largely due to extensive , reversible binding to serum albumin , estimated to be roughly 95% . among both healthy subjects and those with varying degrees of renal dysfunction , the maximum concentration ( cmax ) of dalbavancin falls between 248 and 312 g / ml following the standard 1000 mg initial dose [ 28 , 30 ] . dalbavancin exhibits a large volume of distribution , with an initial average of 812 l and an ultimate volume of distribution of up to 15 l once the drug has distributed throughout the tissues . data from nine healthy male subjects given 1000 mg demonstrate that dalbavancin maintains concentrations in skin blister fluid well above current mic90 values through 7 days , suggesting that dalbavancin will be active for the duration of absssi treatment . in a pharmacokinetic study of dalbavancin in patients with varying renal and hepatic function , area under the curve ( auc ) values were markedly elevated over the treatment duration for patients with creatinine clearances ( crcl ) of 30 ml / min relative to patients with normal renal function . because of its reduced clearance , patients with renal impairment ( crcl < 30 ml / min ) should have dalbavancin doses adjusted to 750 mg initially followed by 375 mg on day 8 for a complete treatment course . interestingly , dalbavancin is not efficiently cleared by hemodialysis and levels are similar between patients on dialysis and those with normal renal function . therefore , although it seems contradictory to data in patients with crcl < 30 ml / min , patients on regularly scheduled hemodialysis do not require dosage adjustment . the same study found that hepatic function had no appreciable affect on dalbavancin clearance and likely does not need to be taken into account when dosing , although caution should be exercised in moderate to severe hepatic impairment , as clinical data are limited . the pharmacokinetic parameter associated with efficacy of dalbavancin both in vitro and in vivo is the ratio of auc to mic , as is generally the case with glycopeptides . in the initial pharmacokinetic studies , bactericidal concentrations of dalbavancin were present in serum samples up to 7 days following an initial , one - time dose of as little as 500 mg . with regard to the auc : mic target , population pharmacokinetic modeling of dalbavancin standard dosing from three clinical trials demonstrated a likelihood of target attainment of near 100% at dalbavancin mics up to 0.5 g / ml . similarly , a recent study with 10,000 monte carlo simulations of dalbavancin 1000 mg followed by 500 mg 1 week later also showed a 100% probability of target attainment for mrsa isolates with dalbavancin mics up to 0.12 g / ml . there are in vitro studies that have evaluated the activity of dalbavancin against gram - positive organisms , some of which examined isolates with reduced susceptibility to vancomycin . in one such study that evaluated several dalbavancin dose exposures against s. aureus , continuous concentrations of 3 g / ml were sufficient to provide bactericidal activity against both mssa and mrsa over 20 days . against visa , concentrations of 15 g dalbavancin was also studied in combination with several antibiotics against mssa , mrsa , visa , mrse , vancomycin - susceptible enterococci ( vse ) , s. pyogenes and s. pneumoniae using broth microdilution checkerboard techniques . oxacillin was synergistic with dalbavancin against mrsa , visa , mrse , and vse . among the other agents tested , including daptomycin , clindamycin , linezolid , levofloxacin , gentamicin , quinupristin / dalfopristin , rifampicin , and vancomycin , none were antagonistic when combined with dalbavancin against these bacteria . these in vitro data are limited but suggest dalbavancin may have activity , either alone or in combination , against problem organisms such as visa . when dalbavancin was evaluated against s. aureus and s. pneumoniae in neutropenic murine thigh and lung models , it was determined that auc : mic and cmax : mic were both correlated with dalbavancin activity . these data formed the dosing regimens used in human studies . in a study published by lefort et al . , dalbavancin possessed activity against vancomycin - susceptible s. aureus ( vssa ) and visa dependent upon dose in a rabbit model of endocarditis . in the authors in vitro time kill studies , dalbavancin possessed superior activity to teicoplanin and vancomycin against vssa and visa . however , dalbavancin was not bactericidal in the endocarditis model . the authors postulated that , due to the enhanced clearance of dalbavancin in rabbits compared to people , drug exposures were not appropriate for bactericidal activity , although they may be in humans . dalbavancin has also been studied in a foreign - body infection model in guinea pigs both alone and in combination with rifampin . dalbavancin alone was unable to eradicate adherent mrsa , but it was able to prevent resistance to rifampin . the combination of agents was effective and superior to either agent alone , suggesting that rifampin may play an important role in therapy targeting biofilm infections . in addition to the study that led to its approval for absssi , dalbavancin has been previously evaluated clinically in the setting of complicated skin and skin structure infections ( csssi ) and bloodstream infections . in a phase ii , multicenter , non - inferiority , randomized controlled trial , 2 weeks of twice daily vancomycin was compared to standard - dose dalbavancin against catheter - related bloodstream infections ( cr - bsi ) due to gram - positive pathogens . patients were 18 years old with signs of bacteremia and at least one of the following : intravascular catheter present at the start of infection , absence of any other likely source of infection , and either microbiologically documented gram - positive bacteremia or 2 signs of bacteremia . patients were excluded if they had impaired renal or hepatic function , recently received immunosuppressive therapy , prolonged neutropenia , received prior antibiotic active against gram - positive bacteria within 48 h of enrollment , s. aureus bacteremia within the previous 3 months , or an alternative source of infection . the primary outcome was overall efficacy at the follow - up visit defined by clinically and microbiologically documented responses among patients with a bacterial pathogen identified at study entry . clinical success was defined as resolution of signs and symptoms of cr - bsi such that no additional treatment was warranted . seventy - five patients were randomized to treatment , of which 67 were included in efficacy analyses . baseline characteristics were similar , although slightly more females were enrolled in the vancomycin group . coagulase - negative staphylococci and s. aureus were the primary pathogens isolated , with a higher proportion of s. aureus ( 32.1% ) reported as mrsa in the vancomycin group compared to the dalbavancin group ( 19.2% ) . the overall success rate was 87% for dalbavancin compared to 50% for vancomycin ( p < 0.05 ) , and both treatment groups were more successful with catheter removal . this effect carried across the microbiological intention to treat cohort at both end of therapy and test of cure visits , and the effect was also present among the evaluable population at both time points . adverse effects reported by study patients were generally mild , and no patients discontinued dalbavancin due to adverse effects . in the vancomycin group , three patients withdrew therapy due to adverse effects , with one patient perhaps experiencing renal toxicity due to vancomycin . the data from this study demonstrated the efficacy of dalbavancin 1000 mg on day 1 followed by 500 mg 1 week later against uncomplicated , gram - positive bacteremia . although not directly demonstrated by the study , the authors postulated that the increased peak levels and lipophilic tail of dalbavancin might have aided it in clearance of biofilm on infected catheters , perhaps explaining the enhanced efficacy . gram - positive pathogens also frequently cause skin infections , and they are a large source of healthcare expenditures with over 15 million infections annually in the united states . owing to the convenient dosing of dalbavancin and its ability to keep patients out of infusion centers , two published studies have evaluated dalbavancin for the treatment of absssi against active comparators [ 10 , 33 ] . in a phase ii , randomized controlled study , seltzer et al . evaluated dalbavancin given as either a single 1100 mg dose or 1000 mg followed by 500 mg 1 week later against several standard - of - care regimens for skin and soft - tissue infection ( ssti ) . sixty - two patients were enrolled in the study , and patients were 18 years old with ssti suspected or known to be caused by a gram - positive pathogen . infections were required to involve deep tissue or require surgical intervention , and patients were required to have 2 symptoms of ssti . of note , patients with creatinine clearance < 50 ml / min were excluded . failure was defined as persistence of 1 systemic sign or symptom of ssti that required further therapy . a higher proportion of 2-dose dalbavancin patients achieved cure in the intention - to - treat population ( 19/21 , 91% ) compared to 1-dose dalbavancin ( 12/20 , 60% ) or comparator regimen ( 16/21 , 76% ) . dalbavancin concentrations remained > 30 g / ml for 1 week after the 1100 mg dalbavancin dose , and concentrations remained > 20 g / ml for 20 days in patients who received 1000 mg followed by 500 mg . the data from this study established that dalbavancin is likely optimally dosed at 1000 mg with a supplementary 500 mg dose 1 week later for skin infections . were the first to evaluate dalbavancin at 1000 mg followed by 500 mg 1 week later in a phase iii trial for the treatment of csssi . the study was a multicenter , non - inferiority trial comparing dalbavancin to linezolid for 14 days . the primary end point was clinical success , defined as the lack of necessary further antibacterial therapy . the treatment groups were similar at baseline except for a higher proportion of vascular disease in the dalbavancin group . abscess ( 32% ) and cellulitis ( 28% ) were the primary types of infection . efficacy at test - of - cure for dalbavancin was 88.9% and was 91.2% for linezolid ( lower limit ci 7.28% ) , which achieved non - inferiority . the vast majority of cultured isolates were from abscesses , with 90% of those isolates being s. aureus , and dalbavancin and linezolid cured 91% and 89% of these isolates , respectively . adverse events were generally mild , and adverse events related to treatment were higher in the linezolid arm compared to dalbavancin ( 32.225.4% , respectively ) . boucher and colleagues evaluated dalbavancin dosed at 1000 mg followed by 500 mg in two phase iii , randomized controlled trials comparing dalbavancin to standard therapy against absssi , discover 1 and discover 2 . standard therapy consisted of at least 3 days of intravenous vancomycin followed by an optional switch to oral linezolid for the completion of 1014 days of therapy . the two trials were identically designed , double - dummy , international , non - inferiority studies , allowing for pooling of data . patients were included who were 18 years old and were diagnosed with absssi . the diagnosis of absssi was based on cellulitis , a major abscess , or wound infection with an area of at least 75 cm , which is about the area of a modern smart phone . patients were eligible if they were likely to require at least 3 days of intravenous antibiotics and possessed at least one systemic sign of infection . in the discover trials , enrollment was limited to a maximum of 30% of patients with abscess , owing to high cure rates in these patients with incision and drainage alone , and at least 25% of patients had fever , establishing a less healthy population than previous studies . per recent fda guidance , the primary endpoint was cessation of spread of erythema and a temperature of 37.6 c at 4872 h. more than 85% of patients enrolled possessed temperatures 38 c , and the median size of infection was 343 cm . the majority of patients were white ( 89.3% ) with a mean age of 49.5 years . greater than 50% of patients met sirs criteria , and < 26% of patients had abscesses . in the pooled dalbavancin group , 525 of 659 patients ( 79.7% ) had a successful outcome compared to 521 of 653 patients ( 79.8% ) in the standard therapy group ( 95% ci 4.5 to 4.2 ) , meeting the criteria for non - inferiority . missing temperature data was the primary reason for failure in both treatment arms and was the primary treatment - related reason for failure . the two arms were similarly successful in patients with bacteremia , as 23 of 23 patients ( 100% ) in the dalbavancin arm had negative cultures at follow - up and 12 of 14 patients ( 85.7% ) in the standard therapy arm had negative cultures . clinical success was similar between the arms according to infection type , severity of illness , and severity of infection . more patients in the standard therapy arm experienced an adverse event ( 645 vs. 540 , p = 0.05 ) compared to patients in the dalbavancin arm , and the total number of treatment - related adverse events was higher in the standard therapy arm as well ( 183 vs. 139 , p = 0.02 ) . the most common adverse events were nausea , diarrhea , and pruritus , and each occurred in < 3% of patients . only two patients in the study , both in the vancomycin - linezolid arm , experienced nephrotoxic adverse effects . the data from this study suggest that dalbavancin is similarly efficacious to standard therapy against absssi with regard to the newly established fda criteria . the patients in this study were quite sick , with > 85% possessing fever and more than half meeting sirs criteria , demonstrating that dalbavancin can be an effective alternative to therapy with vancomycin and linezolid , especially in the case of suspected mrsa infection . the incidence of serious adverse effects is low and has been similar to all comparator arms . currently , dalbavancin is being evaluated in a phase 3b study to compare the fda - approved dose against a one - time dose of 1500 mg for absssi ( clinicaltrials.gov nct02127970 ) . the results have yet to be published , but preliminary data presented in a press release suggest that the one - time dose of 1500 mg is similar to the two - dose regimen with regard to 20% reduction in absssi lesion size at 4872 h. although conclusions can not be made without the full breadth of data available , it does appear that dalbavancin may be efficacious and safe with a one - time dose , further enhancing its therapeutic ease of use . the safety of dalbavancin has been evaluated in several clinical and pre - clinical studies . combined phase ii and phase iii data thus far have revealed the most common adverse effects to be nausea ( 5.5% ) , headache ( 4.7% ) , diarrhea ( 4.4% ) , vomiting ( 2.8% ) , rash ( 2.7% ) , and pruritus ( 2.1% ) [ 10 , 2830 , 33 , 42 ] . in the phase ii study of cr - bsi , dalbavancin patients experienced more hypokalemia ( 6/33 , 18% ) and hypotension ( 7/33 , 21% ) than those treated with vancomycin , although these trends did not continue across other studies . additionally , dalbavancin has demonstrated a 0.8% rate of alt levels greater than three times the upper limit of normal , although these have been reversible with treatment cessation . in the phase iii clinical trials , treatment - related adverse events were fewer in the dalbavancin arms compared to treatment with vancomycin or linezolid [ 10 , 44 ] . in contrast to the nephrotoxic effects often associated with glycopeptides , dalbavancin has not been associated with nephrotoxicity to date . although increased nephrotoxicity was not demonstrated in the vancomycin arms of comparator trials , nephrotoxicity is a historically demonstrated adverse effect of vancomycin , and the lack of reduced renal function with dalbavancin therapy is encouraging . although further safety data are needed , it appears that dalbavancin has a favorable renal safety profile . due to potential concern for qtc prolongation with telavancin , another lipoglycopeptide antibiotic , dalbavancin has recently been studied at therapeutic and supratherapeutic doses , and dalbavancin caused no increase in qtc interval [ 45 , 46 ] . because dalbavancin has such an extended half - life , there has been some concern regarding the potential for long - term adverse effects . to date , no adverse effects have been demonstrated up to 28 days after treatment cessation . however , long - term data are still limited , and post - approval adverse effect reporting will shed more light on any long - term effects . several studies have evaluated dalbavancin pharmacokinetics in the presence of known cytochrome p450 inducers , substrates , and inhibitors to determine the presence of significant drug interactions [ 28 , 32 ] . unsurprisingly , dalbavancin was not affected by co - administration of other agents , as dalbavancin is not cleared by the p450 metabolic pathway . the available evidence suggests that dalbavancin can be administered without regard to agents known to affect the p450 pathway . dalbavancin possesses in vitro activity against several gram - positive pathogens , including s. aureus , streptococcus agalactiae , streptococcus pyogenes , streptococcus anginosus , enterococcus faecium , and enterococcus faecalis , although activity against enterococci has not been observed clinically [ 9 , 1518 ] . against staphylococci and streptococci , dalbavancin possesses 816-fold greater in vitro activity than vancomycin in broth microdilution mic testing [ 15 , 16 ] . currently , the fda has an mic breakpoint established for only s. aureus , s. pyogenes , s. agalactiae , and s. anginosus of 0.125 g / ml . population data of more than 1100 staphylococci and 300 -hemolytic streptococci isolates from us medical centers in 2012 report dalbavancin mic50/90 values of 0.06/0.06 and 0.06/0.12 g / ml , respectively [ 15 , 16 ] . only three staphylococcal isolates ( 0.3% ) and 14 streptococcal isolates ( 4% ) possessed dalbavancin mic values above the currently proposed fda breakpoint . each resistant streptococcal isolate was a member of the species s. agalactiae . against visa , vancomycin mic ( 48 g / ml ) and heteroresistant visa ( hvisa ) , dalbavancin maintains four- to eightfold greater potency than vancomycin , although the mic values largely fall above 0.12 g / ml . recently , jones and colleagues demonstrated that vancomycin susceptibility can be considered a surrogate for dalbavancin susceptibility , as greater than 99.9% of over 42,000 vancomycin - susceptible isolates were susceptible to dalbavancin . against enterococci , dalbavancin possesses higher mic values than it does against other gram - positive species . dalbavancin activity against enterococci is largely contingent upon vancomycin activity , as 100% of vancomycin - susceptible enterococcus faecalis and e. faecium are inhibited at 0.125 g / ml [ 15 , 16 ] . however , vancomycin - resistant isolates of each species possess dalbavancin mic90 values greater than 4 g / ml . resistance to dalbavancin among staphylococci is rare , being reported in less than 1% of isolates [ 15 , 16 ] . mechanistically , there is precedent for reduced vancomycin susceptibility predicting reduced susceptibility to dalbavancin . reduced susceptibility among hvisa and visa is mediated through thickening of the cell wall and increased d - alanine - d - alanine binding sites [ 1921 ] . these increased binding sites increase the amount of vancomycin bound to the cell wall , necessitating higher exposures for similar efficacy . although dalbavancin is more potent at the active site than vancomycin , it is still inhibited in the same fashion . it remains to be seen whether the increased potency of dalbavancin will give it appreciable activity against these phenotypes clinically . vancomycin - resistant s. aureus ( vrsa ) are similarly resistant to dalbavancin , although the mechanism for this resistance is different than for hvisa and visa isolates . vancomycin , and subsequently dalbavancin , resistance within vrsa is mediated through a plasmid gene vana or vanb carried over from enterococcal species . these genes are responsible for altering the d - alanyl - d - alanine target site to a d - alanyl - d - lactate , rendering glycopeptide antibiotics ineffective . interestingly , dalbavancin has in vitro activity against enterococci with vanb present , but not against those with vana . the vanb activity may be present due to dalbavancin being a derivative of teicoplanin , which also possesses activity against enterococci with vanb . although this activity is worth noting , the vast majority of vancomycin - resistant enterococci in the united states possess vana . a complete breakdown of currently available dalbavancin mic data against gram - positive organisms is available in table 1.table 1dalbavancin mics for several gram - positive organisms [ 1518 , 2527]number of isolatesmic50 ( g / ml)mic90 ( g / ml)range% susc . s. aureus [ 1518 , 25 , 27]64,8430.060.060.008 to 0.599.7mssa [ 1518 , 25 , 27]37,2220.060.060.008 to 0.599.7mrsa [ 1518 , 25 , 27]27,2610.060.060.008 to 0.599.6hvisa 100.250.50.12 to 0.520visa 80.5n / a0.5 to 20dns sa 370.060.120.03 to 0.591.9lr sa 190.060.120.03 to 0.5100cns [ 15 , 16 , 27]4730.030.060.03 to 0.2599.6ms cns [ 15 , 16 , 27]2810.030.060.03 to 1n / amr cns [ 15 , 16 , 27]1930.030.120.03 to 0.25n / a enterococcus spp . [ 15 , 16]1160.06>40.03 to > 456vse [ 15 , 16]630.030.120.03 to 0.2596.8vre [ 15 , 16]53>4>40.03 to > 47.5vana vre [ 15 , 16]49>4>40.25 to > 40vanb vre [ 15 , 16]40.030.120.03 to 0.12100 e. faecalis 250.06>40.03 to > 476vse faecalis 190.030.060.03 to 0.06100vre faecalis 6>4>4>40 e. faecium 311>40.03 to > 441.9vse faecium 110.060.120.03 to 0.12100vre faecium 20>4>40.03 to > 410-hemo strep [ 15 , 16 , 26]12420.030.030.03 to 0.2598.6viridans strep [ 15 , 16 , 26]7860.030.030.03 to 0.2599.7 s. anginosus 1900.030.030.03 to 0.06100 s. milleri 140.030.030.03 to 0.06100 s. bovis 470.030.060.03 to 0.12100 s. dysgalactiae 500.030.060.03 to 0.12100 s. mitis 3050.030.060.03 to 0.2599.7 s. mutans 200.030.060.03 to 0.12100 s. salivarius 490.030.060.03 to 0.2598gas [ 15 , 16 , 26]5060.030.030.03 to 0.12100gbs [ 15 , 16 , 26]2870.030.120.03 to 0.2594.4 s. pneumoniae 8930.030.030.03 to 0.12100pssp 7390.030.030.03 to 0.12100pisp 1200.030.030.03 to 0.12100prsp 340.030.030.03100 sa s. aureus , ms methicillin - susceptible , mr methicillin - resistant , dns daptomycin - nonsusceptible , lr linezolid - resistant , cns coagulase - negative staphylococci , vse vancomycin - susceptible enterococci , vre vancomycin - resistant enterococci , gas group a streptococci , gbs group b streptococci , sp s. pneumo , ps penicillin - susceptible , pi penicillin - intermediate , pr penicillin - resistant dalbavancin mics for several gram - positive organisms [ 1518 , 2527 ] sa s. aureus , ms methicillin - susceptible , mr methicillin - resistant , dns daptomycin - nonsusceptible , lr linezolid - resistant , cns coagulase - negative staphylococci , vse vancomycin - susceptible enterococci , vre vancomycin - resistant enterococci , gas group a streptococci , gbs group b streptococci , sp s. pneumo , ps penicillin - susceptible , pi penicillin - intermediate , pr penicillin - resistant dalbavancin is currently fda - approved at a dose of 1000 mg on day 1 followed by 500 mg on day 8 for a complete course of therapy for absssi . pharmacokinetic data from human studies demonstrate that dalbavancin possesses linear , dose - related pharmacokinetics with an extended elimination half - life of approximately 14.5 days ( 346 h ) , allowing for the extended interval between doses [ 2832 ] . the approved dosing regimen was based on a clinical study evaluating complicated skin and skin structure infections ( csssi ) in which dalbavancin dosed at 1000 mg initially followed by 500 mg on day 8 was numerically superior to a one - time dose of 1100 mg . nearly 33% of dalbavancin is excreted in the urine unchanged , suggesting that non - renal methods of elimination play an important role in the metabolism of dalbavancin . the extended half - life of dalbavancin is largely due to extensive , reversible binding to serum albumin , estimated to be roughly 95% . among both healthy subjects and those with varying degrees of renal dysfunction , the maximum concentration ( cmax ) of dalbavancin falls between 248 and 312 g / ml following the standard 1000 mg initial dose [ 28 , 30 ] . dalbavancin exhibits a large volume of distribution , with an initial average of 812 l and an ultimate volume of distribution of up to 15 l once the drug has distributed throughout the tissues . data from nine healthy male subjects given 1000 mg demonstrate that dalbavancin maintains concentrations in skin blister fluid well above current mic90 values through 7 days , suggesting that dalbavancin will be active for the duration of absssi treatment . in a pharmacokinetic study of dalbavancin in patients with varying renal and hepatic function , area under the curve ( auc ) values were markedly elevated over the treatment duration for patients with creatinine clearances ( crcl ) of 30 ml / min relative to patients with normal renal function . because of its reduced clearance , patients with renal impairment ( crcl < 30 ml / min ) should have dalbavancin doses adjusted to 750 mg initially followed by 375 mg on day 8 for a complete treatment course . interestingly , dalbavancin is not efficiently cleared by hemodialysis and levels are similar between patients on dialysis and those with normal renal function . therefore , although it seems contradictory to data in patients with crcl < 30 ml / min , patients on regularly scheduled hemodialysis do not require dosage adjustment . the same study found that hepatic function had no appreciable affect on dalbavancin clearance and likely does not need to be taken into account when dosing , although caution should be exercised in moderate to severe hepatic impairment , as clinical data are limited . the pharmacokinetic parameter associated with efficacy of dalbavancin both in vitro and in vivo is the ratio of auc to mic , as is generally the case with glycopeptides . in the initial pharmacokinetic studies , bactericidal concentrations of dalbavancin were present in serum samples up to 7 days following an initial , one - time dose of as little as 500 mg . with regard to the auc : mic target , population pharmacokinetic modeling of dalbavancin standard dosing from three clinical trials demonstrated a likelihood of target attainment of near 100% at dalbavancin mics up to 0.5 g / ml . similarly , a recent study with 10,000 monte carlo simulations of dalbavancin 1000 mg followed by 500 mg 1 week later also showed a 100% probability of target attainment for mrsa isolates with dalbavancin mics up to 0.12 g / ml . there are in vitro studies that have evaluated the activity of dalbavancin against gram - positive organisms , some of which examined isolates with reduced susceptibility to vancomycin . in one such study that evaluated several dalbavancin dose exposures against s. aureus , continuous concentrations of 3 g / ml were sufficient to provide bactericidal activity against both mssa and mrsa over 20 days . against visa , concentrations of 15 dalbavancin was also studied in combination with several antibiotics against mssa , mrsa , visa , mrse , vancomycin - susceptible enterococci ( vse ) , s. pyogenes and s. pneumoniae using broth microdilution checkerboard techniques . oxacillin was synergistic with dalbavancin against mrsa , visa , mrse , and vse . among the other agents tested , including daptomycin , clindamycin , linezolid , levofloxacin , gentamicin , quinupristin / dalfopristin , rifampicin , and vancomycin , none were antagonistic when combined with dalbavancin against these bacteria . these in vitro data are limited but suggest dalbavancin may have activity , either alone or in combination , against problem organisms such as visa . when dalbavancin was evaluated against s. aureus and s. pneumoniae in neutropenic murine thigh and lung models , it was determined that auc : mic and cmax : mic were both correlated with dalbavancin activity . these data formed the dosing regimens used in human studies . in a study published by lefort et al . , dalbavancin possessed activity against vancomycin - susceptible s. aureus ( vssa ) and visa dependent upon dose in a rabbit model of endocarditis . in the authors in vitro time kill studies , dalbavancin possessed superior activity to teicoplanin and vancomycin against vssa and visa . the authors postulated that , due to the enhanced clearance of dalbavancin in rabbits compared to people , drug exposures were not appropriate for bactericidal activity , although they may be in humans . dalbavancin has also been studied in a foreign - body infection model in guinea pigs both alone and in combination with rifampin . dalbavancin alone was unable to eradicate adherent mrsa , but it was able to prevent resistance to rifampin . the combination of agents was effective and superior to either agent alone , suggesting that rifampin may play an important role in therapy targeting biofilm infections . in addition to the study that led to its approval for absssi , dalbavancin has been previously evaluated clinically in the setting of complicated skin and skin structure infections ( csssi ) and bloodstream infections . in a phase ii , multicenter , non - inferiority , randomized controlled trial , 2 weeks of twice daily vancomycin was compared to standard - dose dalbavancin against catheter - related bloodstream infections ( cr - bsi ) due to gram - positive pathogens . patients were 18 years old with signs of bacteremia and at least one of the following : intravascular catheter present at the start of infection , absence of any other likely source of infection , and either microbiologically documented gram - positive bacteremia or 2 signs of bacteremia . patients were excluded if they had impaired renal or hepatic function , recently received immunosuppressive therapy , prolonged neutropenia , received prior antibiotic active against gram - positive bacteria within 48 h of enrollment , s. aureus bacteremia within the previous 3 months , or an alternative source of infection . the primary outcome was overall efficacy at the follow - up visit defined by clinically and microbiologically documented responses among patients with a bacterial pathogen identified at study entry . clinical success was defined as resolution of signs and symptoms of cr - bsi such that no additional treatment was warranted . seventy - five patients were randomized to treatment , of which 67 were included in efficacy analyses . baseline characteristics were similar , although slightly more females were enrolled in the vancomycin group . coagulase - negative staphylococci and s. aureus were the primary pathogens isolated , with a higher proportion of s. aureus ( 32.1% ) reported as mrsa in the vancomycin group compared to the dalbavancin group ( 19.2% ) . the overall success rate was 87% for dalbavancin compared to 50% for vancomycin ( p < 0.05 ) , and both treatment groups were more successful with catheter removal . this effect carried across the microbiological intention to treat cohort at both end of therapy and test of cure visits , and the effect was also present among the evaluable population at both time points . adverse effects reported by study patients were generally mild , and no patients discontinued dalbavancin due to adverse effects . in the vancomycin group , three patients withdrew therapy due to adverse effects , with one patient perhaps experiencing renal toxicity due to vancomycin . the data from this study demonstrated the efficacy of dalbavancin 1000 mg on day 1 followed by 500 mg 1 week later against uncomplicated , gram - positive bacteremia . although not directly demonstrated by the study , the authors postulated that the increased peak levels and lipophilic tail of dalbavancin might have aided it in clearance of biofilm on infected catheters , perhaps explaining the enhanced efficacy . gram - positive pathogens also frequently cause skin infections , and they are a large source of healthcare expenditures with over 15 million infections annually in the united states . owing to the convenient dosing of dalbavancin and its ability to keep patients out of infusion centers , two published studies have evaluated dalbavancin for the treatment of absssi against active comparators [ 10 , 33 ] . in a phase ii , randomized controlled study , seltzer et al . evaluated dalbavancin given as either a single 1100 mg dose or 1000 mg followed by 500 mg 1 week later against several standard - of - care regimens for skin and soft - tissue infection ( ssti ) . sixty - two patients were enrolled in the study , and patients were 18 years old with ssti suspected or known to be caused by a gram - positive pathogen . infections were required to involve deep tissue or require surgical intervention , and patients were required to have 2 symptoms of ssti . of note , patients with creatinine clearance < 50 ml / min were excluded . failure was defined as persistence of 1 systemic sign or symptom of ssti that required further therapy . at follow - up visit , a higher proportion of 2-dose dalbavancin patients achieved cure in the intention - to - treat population ( 19/21 , 91% ) compared to 1-dose dalbavancin ( 12/20 , 60% ) or comparator regimen ( 16/21 , 76% ) . dalbavancin concentrations remained > 30 g / ml for 1 week after the 1100 mg dalbavancin dose , and concentrations remained > 20 g / ml for 20 days in patients who received 1000 mg followed by 500 mg . the data from this study established that dalbavancin is likely optimally dosed at 1000 mg with a supplementary 500 mg dose 1 week later for skin infections . were the first to evaluate dalbavancin at 1000 mg followed by 500 mg 1 week later in a phase iii trial for the treatment of csssi . the study was a multicenter , non - inferiority trial comparing dalbavancin to linezolid for 14 days . the primary end point was clinical success , defined as the lack of necessary further antibacterial therapy . the treatment groups were similar at baseline except for a higher proportion of vascular disease in the dalbavancin group . abscess ( 32% ) and cellulitis ( 28% ) were the primary types of infection . efficacy at test - of - cure for dalbavancin was 88.9% and was 91.2% for linezolid ( lower limit ci 7.28% ) , which achieved non - inferiority . the vast majority of cultured isolates were from abscesses , with 90% of those isolates being s. aureus , and dalbavancin and linezolid cured 91% and 89% of these isolates , respectively . adverse events were generally mild , and adverse events related to treatment were higher in the linezolid arm compared to dalbavancin ( 32.225.4% , respectively ) . boucher and colleagues evaluated dalbavancin dosed at 1000 mg followed by 500 mg in two phase iii , randomized controlled trials comparing dalbavancin to standard therapy against absssi , discover 1 and discover 2 . standard therapy consisted of at least 3 days of intravenous vancomycin followed by an optional switch to oral linezolid for the completion of 1014 days of therapy . the two trials were identically designed , double - dummy , international , non - inferiority studies , allowing for pooling of data . the diagnosis of absssi was based on cellulitis , a major abscess , or wound infection with an area of at least 75 cm , which is about the area of a modern smart phone . patients were eligible if they were likely to require at least 3 days of intravenous antibiotics and possessed at least one systemic sign of infection . in the discover trials , enrollment was limited to a maximum of 30% of patients with abscess , owing to high cure rates in these patients with incision and drainage alone , and at least 25% of patients had fever , establishing a less healthy population than previous studies . per recent fda guidance , the primary endpoint was cessation of spread of erythema and a temperature of 37.6 c at 4872 h. more than 85% of patients enrolled possessed temperatures 38 c , and the median size of infection was 343 cm . the majority of patients were white ( 89.3% ) with a mean age of 49.5 years . greater than 50% of patients met sirs criteria , and < 26% of patients had abscesses . in the pooled dalbavancin group , 525 of 659 patients ( 79.7% ) had a successful outcome compared to 521 of 653 patients ( 79.8% ) in the standard therapy group ( 95% ci 4.5 to 4.2 ) , meeting the criteria for non - inferiority . missing temperature data was the primary reason for failure in both treatment arms and was the primary treatment - related reason for failure . the two arms were similarly successful in patients with bacteremia , as 23 of 23 patients ( 100% ) in the dalbavancin arm had negative cultures at follow - up and 12 of 14 patients ( 85.7% ) in the standard therapy arm had negative cultures . clinical success was similar between the arms according to infection type , severity of illness , and severity of infection . more patients in the standard therapy arm experienced an adverse event ( 645 vs. 540 , p = 0.05 ) compared to patients in the dalbavancin arm , and the total number of treatment - related adverse events was higher in the standard therapy arm as well ( 183 vs. 139 , p = 0.02 ) . the most common adverse events were nausea , diarrhea , and pruritus , and each occurred in < 3% of patients . only two patients in the study , both in the vancomycin - linezolid arm , experienced nephrotoxic adverse effects . the data from this study suggest that dalbavancin is similarly efficacious to standard therapy against absssi with regard to the newly established fda criteria . the patients in this study were quite sick , with > 85% possessing fever and more than half meeting sirs criteria , demonstrating that dalbavancin can be an effective alternative to therapy with vancomycin and linezolid , especially in the case of suspected mrsa infection . the incidence of serious adverse effects is low and has been similar to all comparator arms . currently , dalbavancin is being evaluated in a phase 3b study to compare the fda - approved dose against a one - time dose of 1500 mg for absssi ( clinicaltrials.gov nct02127970 ) . the results have yet to be published , but preliminary data presented in a press release suggest that the one - time dose of 1500 mg is similar to the two - dose regimen with regard to 20% reduction in absssi lesion size at 4872 h. although conclusions can not be made without the full breadth of data available , it does appear that dalbavancin may be efficacious and safe with a one - time dose , further enhancing its therapeutic ease of use . the safety of dalbavancin has been evaluated in several clinical and pre - clinical studies . combined phase ii and phase iii data thus far have revealed the most common adverse effects to be nausea ( 5.5% ) , headache ( 4.7% ) , diarrhea ( 4.4% ) , vomiting ( 2.8% ) , rash ( 2.7% ) , and pruritus ( 2.1% ) [ 10 , 2830 , 33 , 42 ] . in the phase ii study of cr - bsi , dalbavancin patients experienced more hypokalemia ( 6/33 , 18% ) and hypotension ( 7/33 , 21% ) than those treated with vancomycin , although these trends did not continue across other studies . additionally , dalbavancin has demonstrated a 0.8% rate of alt levels greater than three times the upper limit of normal , although these have been reversible with treatment cessation . in the phase iii clinical trials , treatment - related adverse events were fewer in the dalbavancin arms compared to treatment with vancomycin or linezolid [ 10 , 44 ] . in contrast to the nephrotoxic effects often associated with glycopeptides , dalbavancin has not been associated with nephrotoxicity to date . although increased nephrotoxicity was not demonstrated in the vancomycin arms of comparator trials , nephrotoxicity is a historically demonstrated adverse effect of vancomycin , and the lack of reduced renal function with dalbavancin therapy is encouraging . although further safety data are needed , it appears that dalbavancin has a favorable renal safety profile . due to potential concern for qtc prolongation with telavancin , another lipoglycopeptide antibiotic , dalbavancin has recently been studied at therapeutic and supratherapeutic doses , and dalbavancin caused no increase in qtc interval [ 45 , 46 ] . because dalbavancin has such an extended half - life , there has been some concern regarding the potential for long - term adverse effects . to date , no adverse effects have been demonstrated up to 28 days after treatment cessation . however , long - term data are still limited , and post - approval adverse effect reporting will shed more light on any long - term effects . several studies have evaluated dalbavancin pharmacokinetics in the presence of known cytochrome p450 inducers , substrates , and inhibitors to determine the presence of significant drug interactions [ 28 , 32 ] . unsurprisingly , dalbavancin was not affected by co - administration of other agents , as dalbavancin is not cleared by the p450 metabolic pathway . the available evidence suggests that dalbavancin can be administered without regard to agents known to affect the p450 pathway . with options for the treatment of resistant staphylococcal infections limited and vancomycin susceptibility demonstrating signs of decline , dalbavancin presents an exciting therapeutic alternative . it has evidenced an excellent safety and efficacy profile in the setting of absssi and has gained fda approval for this indication . absssi infections continue to be a large healthcare burden , and the availability of a two - dose therapeutic regimen has important implications for patient adherence , potential hospital avoidance , and cost savings versus inpatient therapy . emergency departments and observational care units stand to benefit especially , as patients will be able to be easily transitioned back into the community after being seen for only a limited amount of time . perhaps most exciting are the clinical opportunities for dalbavancin use outside of its current approved indication . dalbavancin has already demonstrated efficacy in cr - bsi , and further studies will be important to determine its place in deep - seated infections such as pneumonia , osteomyelitis , and endocarditis . preliminary data are promising , as dalbavancin achieves concentrations in bone tissue well above staphylococcal mic90 values 14 days after a 1000 mg dose . going forward , it will be imperative to determine optimal dosing strategies for infections that require weeks - long durations of therapy . owing to the activity dalbavancin possesses in vitro and in vivo against s. aureus with reduced vancomycin susceptibility , further study involving dalbavancin against these isolates is also intriguing and warranted . it remains to be seen whether antibiotic combinations with dalbavancin will display similar additive effects as they do with vancomycin and daptomycin against gram - positive pathogens . dalbavancin has the potential to be an effective agent to combat resistant gram - positive organisms , and expanded use of this agent post - approval will help determine its ultimate place in therapy .

dalbavancin is a lipoglycopeptide antibiotic recently approved by the united states food and drug administration ( fda ) for acute bacterial skin and skin structure infections ( absssis ) . it is active against gram - positive pathogens , including methicillin - resistant staphylococcus aureus ( mrsa ) , and minimum inhibitory concentrations ( mics ) are consistently < 0.125 g / ml , much lower than most other anti - mrsa agents . dalbavancin possesses an extended half - life of over 1 week , allowing an initial dose of 1000 mg followed by 500 mg 1 week later to complete a course of therapy for absssi . it is approximately 95% protein bound and is widely distributed throughout the body , achieving concentrations similar to plasma levels in numerous tissues . against mrsa , dalbavancin is 48 times more potent than vancomycin in vitro , and limited data suggest it possesses activity against mrsa with reduced susceptibility to vancomycin such as hvisa and visa . dalbavancin also possesses in vitro activity against streptococci and enterococci , although activity against vancomycin - resistant enterococci is lacking . in phase 3 absssi studies , dalbavancin demonstrated similar activity to vancomycin and provides a more convenient dosing regimen . limited phase 2 data suggest dalbavancin also possesses activity in catheter - related bloodstream infections . potential further therapeutic uses include conditions that require long - term treatment such as osteomyelitis and infective endocarditis , although data are currently lacking . the extended half - life of dalbavancin , along with its in vitro activity against gram - positive organisms with reduced susceptibility to other anti - mrsa antibiotics , suggest it could have an exciting clinical role going forward .

Introduction Structure and Mechanism of Action Microbiology and Resistance Dosing, Pharmacokinetics, and Pharmacodynamics In Vitro and Animal Data Clinical Trials Safety and Drug Interactions Conclusion

among s. aureus isolates observed in the united states , between 40% and 50% are methicillin - resistant ( mrsa ) , greatly reducing therapeutic options . mrsa are responsible for several serious infections , including endocarditis , pneumonia , catheter - associated bloodstream infections , osteomyelitis , and skin infections . a 2013 surveillance study of over 2000 mrsa bloodstream isolates found less than 0.1% possessed a vancomycin minimum inhibitory concentration ( mic ) of 4 g / ml , the mic that establishes mrsa as a vancomycin - intermediate susceptible staphylococcus aureus ( visa ) according to the clinical and laboratory standards institute ( clsi ) . the difficulty of vancomycin administration , increase of mrsa with reduced susceptibility to vancomycin , and toxicities associated with vancomycin use have led to the recent development of several novel anti - mrsa antibiotics . dalbavancin is approved for the treatment of acute bacterial skin and skin structure infections ( absssi ) caused by susceptible , gram - positive isolates . it possesses a similar spectrum of activity to vancomycin , including activity against methicillin - susceptible s. aureus ( mssa ) and mrsa . owing to its extended half - life , dalbavancin is dosed once weekly , with only two doses required for the duration of therapy . dalbavancin possesses in vitro activity against several gram - positive pathogens , including s. aureus , streptococcus agalactiae , streptococcus pyogenes , streptococcus anginosus , enterococcus faecium , and enterococcus faecalis , although activity against enterococci has not been observed clinically [ 9 , 1518 ] . against staphylococci and streptococci , dalbavancin possesses 816-fold greater in vitro activity than vancomycin in broth microdilution mic testing [ 15 , 16 ] . currently , the fda has an mic breakpoint established for only s. aureus , s. pyogenes , s. agalactiae , and s. anginosus of 0.125 g / ml . against visa , vancomycin mic ( 48 g / ml ) and heteroresistant visa ( hvisa ) , dalbavancin maintains four- to eightfold greater potency than vancomycin , although the mic values largely fall above 0.12 g / ml . dalbavancin activity against enterococci is largely contingent upon vancomycin activity , as 100% of vancomycin - susceptible enterococcus faecalis and e. faecium are inhibited at 0.125 g / ml [ 15 , 16 ] . vancomycin - resistant s. aureus ( vrsa ) are similarly resistant to dalbavancin , although the mechanism for this resistance is different than for hvisa and visa isolates . although this activity is worth noting , the vast majority of vancomycin - resistant enterococci in the united states possess vana . [ 15 , 16]1160.06>40.03 to > 456vse [ 15 , 16]630.030.120.03 to 0.2596.8vre [ 15 , 16]53>4>40.03 to > 47.5vana vre [ 15 , 16]49>4>40.25 to > 40vanb vre [ 15 , 16]40.030.120.03 to 0.12100 e. faecalis 250.06>40.03 to > 476vse faecalis 190.030.060.03 to 0.06100vre faecalis 6>4>4>40 e. faecium 311>40.03 to > 441.9vse faecium 110.060.120.03 to 0.12100vre faecium 20>4>40.03 to > 410-hemo strep [ 15 , 16 , 26]12420.030.030.03 to 0.2598.6viridans strep [ 15 , 16 , 26]7860.030.030.03 to 0.2599.7 s. anginosus 1900.030.030.03 to 0.06100 s. milleri 140.030.030.03 to 0.06100 s. bovis 470.030.060.03 to 0.12100 s. dysgalactiae 500.030.060.03 to 0.12100 s. mitis 3050.030.060.03 to 0.2599.7 s. mutans 200.030.060.03 to 0.12100 s. salivarius 490.030.060.03 to 0.2598gas [ 15 , 16 , 26]5060.030.030.03 to 0.12100gbs [ 15 , 16 , 26]2870.030.120.03 to 0.2594.4 s. pneumoniae 8930.030.030.03 to 0.12100pssp 7390.030.030.03 to 0.12100pisp 1200.030.030.03 to 0.12100prsp 340.030.030.03100 sa s. aureus , ms methicillin - susceptible , mr methicillin - resistant , dns daptomycin - nonsusceptible , lr linezolid - resistant , cns coagulase - negative staphylococci , vse vancomycin - susceptible enterococci , vre vancomycin - resistant enterococci , gas group a streptococci , gbs group b streptococci , sp s. pneumo , ps penicillin - susceptible , pi penicillin - intermediate , pr penicillin - resistant dalbavancin mics for several gram - positive organisms [ 1518 , 2527 ] sa s. aureus , ms methicillin - susceptible , mr methicillin - resistant , dns daptomycin - nonsusceptible , lr linezolid - resistant , cns coagulase - negative staphylococci , vse vancomycin - susceptible enterococci , vre vancomycin - resistant enterococci , gas group a streptococci , gbs group b streptococci , sp s. pneumo , ps penicillin - susceptible , pi penicillin - intermediate , pr penicillin - resistant dalbavancin is currently fda - approved at a dose of 1000 mg on day 1 followed by 500 mg on day 8 for a complete course of therapy for absssi . pharmacokinetic data from human studies demonstrate that dalbavancin possesses linear , dose - related pharmacokinetics with an extended elimination half - life of approximately 14.5 days ( 346 h ) , allowing for the extended interval between doses [ 2832 ] . the approved dosing regimen was based on a clinical study evaluating complicated skin and skin structure infections ( csssi ) in which dalbavancin dosed at 1000 mg initially followed by 500 mg on day 8 was numerically superior to a one - time dose of 1100 mg . the extended half - life of dalbavancin is largely due to extensive , reversible binding to serum albumin , estimated to be roughly 95% . among both healthy subjects and those with varying degrees of renal dysfunction , the maximum concentration ( cmax ) of dalbavancin falls between 248 and 312 g / ml following the standard 1000 mg initial dose [ 28 , 30 ] . in the initial pharmacokinetic studies , bactericidal concentrations of dalbavancin were present in serum samples up to 7 days following an initial , one - time dose of as little as 500 mg . similarly , a recent study with 10,000 monte carlo simulations of dalbavancin 1000 mg followed by 500 mg 1 week later also showed a 100% probability of target attainment for mrsa isolates with dalbavancin mics up to 0.12 g / ml . there are in vitro studies that have evaluated the activity of dalbavancin against gram - positive organisms , some of which examined isolates with reduced susceptibility to vancomycin . , dalbavancin possessed activity against vancomycin - susceptible s. aureus ( vssa ) and visa dependent upon dose in a rabbit model of endocarditis . in addition to the study that led to its approval for absssi , dalbavancin has been previously evaluated clinically in the setting of complicated skin and skin structure infections ( csssi ) and bloodstream infections . in a phase ii , multicenter , non - inferiority , randomized controlled trial , 2 weeks of twice daily vancomycin was compared to standard - dose dalbavancin against catheter - related bloodstream infections ( cr - bsi ) due to gram - positive pathogens . the data from this study demonstrated the efficacy of dalbavancin 1000 mg on day 1 followed by 500 mg 1 week later against uncomplicated , gram - positive bacteremia . gram - positive pathogens also frequently cause skin infections , and they are a large source of healthcare expenditures with over 15 million infections annually in the united states . evaluated dalbavancin given as either a single 1100 mg dose or 1000 mg followed by 500 mg 1 week later against several standard - of - care regimens for skin and soft - tissue infection ( ssti ) . dalbavancin concentrations remained > 30 g / ml for 1 week after the 1100 mg dalbavancin dose , and concentrations remained > 20 g / ml for 20 days in patients who received 1000 mg followed by 500 mg . were the first to evaluate dalbavancin at 1000 mg followed by 500 mg 1 week later in a phase iii trial for the treatment of csssi . because dalbavancin has such an extended half - life , there has been some concern regarding the potential for long - term adverse effects . dalbavancin possesses in vitro activity against several gram - positive pathogens , including s. aureus , streptococcus agalactiae , streptococcus pyogenes , streptococcus anginosus , enterococcus faecium , and enterococcus faecalis , although activity against enterococci has not been observed clinically [ 9 , 1518 ] . against staphylococci and streptococci , dalbavancin possesses 816-fold greater in vitro activity than vancomycin in broth microdilution mic testing [ 15 , 16 ] . currently , the fda has an mic breakpoint established for only s. aureus , s. pyogenes , s. agalactiae , and s. anginosus of 0.125 g / ml . against visa , vancomycin mic ( 48 g / ml ) and heteroresistant visa ( hvisa ) , dalbavancin maintains four- to eightfold greater potency than vancomycin , although the mic values largely fall above 0.12 g / ml . dalbavancin activity against enterococci is largely contingent upon vancomycin activity , as 100% of vancomycin - susceptible enterococcus faecalis and e. faecium are inhibited at 0.125 g / ml [ 15 , 16 ] . vancomycin - resistant s. aureus ( vrsa ) are similarly resistant to dalbavancin , although the mechanism for this resistance is different than for hvisa and visa isolates . [ 15 , 16]1160.06>40.03 to > 456vse [ 15 , 16]630.030.120.03 to 0.2596.8vre [ 15 , 16]53>4>40.03 to > 47.5vana vre [ 15 , 16]49>4>40.25 to > 40vanb vre [ 15 , 16]40.030.120.03 to 0.12100 e. faecalis 250.06>40.03 to > 476vse faecalis 190.030.060.03 to 0.06100vre faecalis 6>4>4>40 e. faecium 311>40.03 to > 441.9vse faecium 110.060.120.03 to 0.12100vre faecium 20>4>40.03 to > 410-hemo strep [ 15 , 16 , 26]12420.030.030.03 to 0.2598.6viridans strep [ 15 , 16 , 26]7860.030.030.03 to 0.2599.7 s. anginosus 1900.030.030.03 to 0.06100 s. milleri 140.030.030.03 to 0.06100 s. bovis 470.030.060.03 to 0.12100 s. dysgalactiae 500.030.060.03 to 0.12100 s. mitis 3050.030.060.03 to 0.2599.7 s. mutans 200.030.060.03 to 0.12100 s. salivarius 490.030.060.03 to 0.2598gas [ 15 , 16 , 26]5060.030.030.03 to 0.12100gbs [ 15 , 16 , 26]2870.030.120.03 to 0.2594.4 s. pneumoniae 8930.030.030.03 to 0.12100pssp 7390.030.030.03 to 0.12100pisp 1200.030.030.03 to 0.12100prsp 340.030.030.03100 sa s. aureus , ms methicillin - susceptible , mr methicillin - resistant , dns daptomycin - nonsusceptible , lr linezolid - resistant , cns coagulase - negative staphylococci , vse vancomycin - susceptible enterococci , vre vancomycin - resistant enterococci , gas group a streptococci , gbs group b streptococci , sp s. pneumo , ps penicillin - susceptible , pi penicillin - intermediate , pr penicillin - resistant dalbavancin mics for several gram - positive organisms [ 1518 , 2527 ] sa s. aureus , ms methicillin - susceptible , mr methicillin - resistant , dns daptomycin - nonsusceptible , lr linezolid - resistant , cns coagulase - negative staphylococci , vse vancomycin - susceptible enterococci , vre vancomycin - resistant enterococci , gas group a streptococci , gbs group b streptococci , sp s. pneumo , ps penicillin - susceptible , pi penicillin - intermediate , pr penicillin - resistant dalbavancin is currently fda - approved at a dose of 1000 mg on day 1 followed by 500 mg on day 8 for a complete course of therapy for absssi . pharmacokinetic data from human studies demonstrate that dalbavancin possesses linear , dose - related pharmacokinetics with an extended elimination half - life of approximately 14.5 days ( 346 h ) , allowing for the extended interval between doses [ 2832 ] . the approved dosing regimen was based on a clinical study evaluating complicated skin and skin structure infections ( csssi ) in which dalbavancin dosed at 1000 mg initially followed by 500 mg on day 8 was numerically superior to a one - time dose of 1100 mg . the extended half - life of dalbavancin is largely due to extensive , reversible binding to serum albumin , estimated to be roughly 95% . among both healthy subjects and those with varying degrees of renal dysfunction , the maximum concentration ( cmax ) of dalbavancin falls between 248 and 312 g / ml following the standard 1000 mg initial dose [ 28 , 30 ] . similarly , a recent study with 10,000 monte carlo simulations of dalbavancin 1000 mg followed by 500 mg 1 week later also showed a 100% probability of target attainment for mrsa isolates with dalbavancin mics up to 0.12 g / ml . there are in vitro studies that have evaluated the activity of dalbavancin against gram - positive organisms , some of which examined isolates with reduced susceptibility to vancomycin . , dalbavancin possessed activity against vancomycin - susceptible s. aureus ( vssa ) and visa dependent upon dose in a rabbit model of endocarditis . in addition to the study that led to its approval for absssi , dalbavancin has been previously evaluated clinically in the setting of complicated skin and skin structure infections ( csssi ) and bloodstream infections . in a phase ii , multicenter , non - inferiority , randomized controlled trial , 2 weeks of twice daily vancomycin was compared to standard - dose dalbavancin against catheter - related bloodstream infections ( cr - bsi ) due to gram - positive pathogens . the data from this study demonstrated the efficacy of dalbavancin 1000 mg on day 1 followed by 500 mg 1 week later against uncomplicated , gram - positive bacteremia . gram - positive pathogens also frequently cause skin infections , and they are a large source of healthcare expenditures with over 15 million infections annually in the united states . evaluated dalbavancin given as either a single 1100 mg dose or 1000 mg followed by 500 mg 1 week later against several standard - of - care regimens for skin and soft - tissue infection ( ssti ) . dalbavancin concentrations remained > 30 g / ml for 1 week after the 1100 mg dalbavancin dose , and concentrations remained > 20 g / ml for 20 days in patients who received 1000 mg followed by 500 mg . were the first to evaluate dalbavancin at 1000 mg followed by 500 mg 1 week later in a phase iii trial for the treatment of csssi . boucher and colleagues evaluated dalbavancin dosed at 1000 mg followed by 500 mg in two phase iii , randomized controlled trials comparing dalbavancin to standard therapy against absssi , discover 1 and discover 2 . because dalbavancin has such an extended half - life , there has been some concern regarding the potential for long - term adverse effects .

this is an investigator - initiated phase iii double - blind , randomized , placebo - controlled , two - arm crossover clinical study . the study adhered to the guidelines of the declaration of helsinki , and the protocol and consent form were approved by the local investigational review board , the national ethics committee , and the ministry of health . patients ( aged > 18 years ) with type 1 or type 2 diabetes and dme resulting in best corrected visual acuity ( bcva ) of 0.4 were eligible if they had at least two previous sessions of laser photocoagulation > 6 months before enrollment or if they had leaking microaneurysms within the foveal avascular zone , making laser photocoagulation unsafe for the central vision . in addition to standard inclusion and exclusion criteria for phase iii studies of infliximab , patients were excluded if they had 1 ) vitreoretinal traction , 2 ) retinal detachment , 3 ) proliferative diabetic retinopathy requiring immediate panretinal photocoagulation , 4 ) any previous eye surgery 6 months before the study , including any intravitreal infusions , 5 ) macular edema of the ischemic type or caused by retinal conditions other than diabetes , 6 ) cataract or media opacities of a degree that precluded accurate retinal photographs or optical coherence tomography ( oct ) measurement , 7 ) hard exudates under the fovea , or 8) uncontrolled arterial hypertension ( blood pressure > 180/110 mmhg ) , a major change in glycemic control ( e.g. , 2% change in a1c ) within the last 6 months , or a change in daily number of insulin injections . consenting patients were screened for the study within 2 weeks before random assignment with a medical history , physical examination , electrocardiogram , purified protein derivative test , chest x - ray , and laboratory tests including hemoglobin , a1c , platelet count , white blood cell count and differential , aspartate aminotransferase , alanine aminotransferase , -glutamyl transferase , alkaline phosphatase , total and conjugated bilirubin , lactate dehydrogenase , plasma lipids ( total cholesterol , hdl cholesterol , and triglycerides ) , creatinine phosphokinase , renal function ( urea and creatinine ) , sodium , potassium , calcium , phosphate , and serological tests for hepatitis and hiv infection . in addition , an experienced examiner obtained ophthalmic / dme history and performed , in both eyes , measurements of bcva , oct , stereoscopic fundus photographs ( seven fields ) , applanation tonometry , and fluorescein angiography . patients were randomly allocated 1:1 to receive placebo or infliximab in a two - armed crossover , double - blind design according to the permuted block randomization list generated in sas . patients received placebo at weeks 0 , 2 , 6 , and 14 , followed by infliximab at weeks 16 , 18 , 22 , and 30 ( group a ) , or vice versa ( group b ) in addition to standard therapy for diabetes , hypertension , and dyslipidemia , which remained unchanged during the study . all study drugs were administered via a 2-h intravenous infusion at a dose of 5 mg / kg body wt on the scheduled visits at weeks 0 , 2 , 6 , 14 , 16 , 18 , 22 , and 30 . blinding was maintained to week 32 , when the final clinical , laboratory , and ophthalmic evaluation was performed in all patients . finally , adverse event reporting and a complete physical examination were performed at week 56 ( long - term follow - up visit ) . physical examination and bcva measurements of the number of letters a patient was able to read from the early treatment diabetic retinopathy study ( etdrs ) charts with correction for individual refractive errors were performed at every visit . foveal thickness measurements by third - generation oct ( stratus oct iii ) , using the fast macular thickness scan , stereoscopic fundus photographs ( seven fields ) , and intraocular pressure measurements using a goldman applanation tonometer were performed at weeks 8 , 16 , 24 , and 32 . study physicians were blinded to the subject 's treatment ( infliximab or placebo ) as well as to the subject 's previous visual acuity assessments . the primary end point of the study was to assess the efficacy and safety of four infusions of infliximab on bcva , evaluated by a mixed - models approach for imbalanced crossover design using the percent difference between infliximab and placebo groups as an outcome variable . the secondary end points were 1 ) the effect of infliximab on the anatomic change of dme , assessed by oct and 2 ) the effect of infliximab on diabetic retinopathy , assessed by fundus photographs and fluoroangiographic studies . the treatment effect of infliximab versus placebo in macular thickness and fundus photographs results was also evaluated by a mixed - models approach for imbalanced crossover design using the percent difference as an outcome variable . the planned sample size of 26 patients was based on the expected reduction of bcva after treatment with infliximab ( 16 ) . it was estimated that 22 evaluable eyes ( 11 per study arm ) would provide 90% power to detect a mean difference in log minimum angle of resolution ( logmar ) bcva of 0.67 ( equivalent to 22 letters read in the edtrs chart ) at a 0.001 level of statistical significance . under the assumption of a 15% dropout rate , however , the study was terminated after enrollment of the first 12 patients because of inability to recruit additional patients who had not any intravitreal infusion within the prior 6 months ( exclusion criterion 4 , as described above ) . patients ( aged > 18 years ) with type 1 or type 2 diabetes and dme resulting in best corrected visual acuity ( bcva ) of 0.4 were eligible if they had at least two previous sessions of laser photocoagulation > 6 months before enrollment or if they had leaking microaneurysms within the foveal avascular zone , making laser photocoagulation unsafe for the central vision . in addition to standard inclusion and exclusion criteria for phase iii studies of infliximab , patients were excluded if they had 1 ) vitreoretinal traction , 2 ) retinal detachment , 3 ) proliferative diabetic retinopathy requiring immediate panretinal photocoagulation , 4 ) any previous eye surgery 6 months before the study , including any intravitreal infusions , 5 ) macular edema of the ischemic type or caused by retinal conditions other than diabetes , 6 ) cataract or media opacities of a degree that precluded accurate retinal photographs or optical coherence tomography ( oct ) measurement , 7 ) hard exudates under the fovea , or 8) uncontrolled arterial hypertension ( blood pressure > 180/110 mmhg ) , a major change in glycemic control ( e.g. , 2% change in a1c ) within the last 6 months , or a change in daily number of insulin injections . consenting patients were screened for the study within 2 weeks before random assignment with a medical history , physical examination , electrocardiogram , purified protein derivative test , chest x - ray , and laboratory tests including hemoglobin , a1c , platelet count , white blood cell count and differential , aspartate aminotransferase , alanine aminotransferase , -glutamyl transferase , alkaline phosphatase , total and conjugated bilirubin , lactate dehydrogenase , plasma lipids ( total cholesterol , hdl cholesterol , and triglycerides ) , creatinine phosphokinase , renal function ( urea and creatinine ) , sodium , potassium , calcium , phosphate , and serological tests for hepatitis and hiv infection . in addition , an experienced examiner obtained ophthalmic / dme history and performed , in both eyes , measurements of bcva , oct , stereoscopic fundus photographs ( seven fields ) , applanation tonometry , and fluorescein angiography . patients were randomly allocated 1:1 to receive placebo or infliximab in a two - armed crossover , double - blind design according to the permuted block randomization list generated in sas . patients received placebo at weeks 0 , 2 , 6 , and 14 , followed by infliximab at weeks 16 , 18 , 22 , and 30 ( group a ) , or vice versa ( group b ) in addition to standard therapy for diabetes , hypertension , and dyslipidemia , which remained unchanged during the study . all study drugs were administered via a 2-h intravenous infusion at a dose of 5 mg / kg body wt on the scheduled visits at weeks 0 , 2 , 6 , 14 , 16 , 18 , 22 , and 30 . blinding was maintained to week 32 , when the final clinical , laboratory , and ophthalmic evaluation was performed in all patients . finally , adverse event reporting and a complete physical examination were performed at week 56 ( long - term follow - up visit ) . physical examination and bcva measurements of the number of letters a patient was able to read from the early treatment diabetic retinopathy study ( etdrs ) charts with correction for individual refractive errors were performed at every visit . foveal thickness measurements by third - generation oct ( stratus oct iii ) , using the fast macular thickness scan , stereoscopic fundus photographs ( seven fields ) , and intraocular pressure measurements using a goldman applanation tonometer were performed at weeks 8 , 16 , 24 , and 32 . study physicians were blinded to the subject 's treatment ( infliximab or placebo ) as well as to the subject 's previous visual acuity assessments . the primary end point of the study was to assess the efficacy and safety of four infusions of infliximab on bcva , evaluated by a mixed - models approach for imbalanced crossover design using the percent difference between infliximab and placebo groups as an outcome variable . the secondary end points were 1 ) the effect of infliximab on the anatomic change of dme , assessed by oct and 2 ) the effect of infliximab on diabetic retinopathy , assessed by fundus photographs and fluoroangiographic studies . the treatment effect of infliximab versus placebo in macular thickness and fundus photographs results was also evaluated by a mixed - models approach for imbalanced crossover design using the percent difference as an outcome variable . the planned sample size of 26 patients was based on the expected reduction of bcva after treatment with infliximab ( 16 ) . it was estimated that 22 evaluable eyes ( 11 per study arm ) would provide 90% power to detect a mean difference in log minimum angle of resolution ( logmar ) bcva of 0.67 ( equivalent to 22 letters read in the edtrs chart ) at a 0.001 level of statistical significance . under the assumption of a 15% dropout rate , however , the study was terminated after enrollment of the first 12 patients because of inability to recruit additional patients who had not any intravitreal infusion within the prior 6 months ( exclusion criterion 4 , as described above ) . there were three women and eight men , aged between 40 and 73 years , with diabetes duration ranging between 3 and 20 years ( 1 patient with type 1 diabetes and 10 patients with type 2 diabetes ) . the additional enrolled patient , aged 72 , was randomly assigned to initially receive placebo ( group a ) , but had an acute myocardial infarction 2 days before the first scheduled injection and withdrew from the study . one patient from group a withdrew consent at week 18 after receiving four placebo injections and the first infliximab injection . in total 14 eyes were eligible for analysis ( 6 eyes in group a , including this patient 's response to placebo treatment , and 8 eyes in group b ) ( table 1 ) . demographic and disease characteristics of patients with dme and individual bcva values of eligible eyes at baseline ( week 2 ) , end of study treatment 1 ( week 16 ) , and end of study treatment 2 ( week 32 ) * a denotes placebo ; b denotes infliximab . study treatment 1 : from baseline to week 16 ; study treatment 2 : from week 16 to week 32 . individual values of bcva at baseline , week 16 ( end of the first study treatment ) , and week 32 ( final evaluation after the second study treatment ) are shown in table 1 . baseline bcva was not different between groups ( 31.6 5.1 vs. 23.5 10.3 letters read ; t = 1.7 ; p = 0.10 ) . 1a , bcva decreased from 31.6 5.1 at baseline to 28.8 11.6 letters read at week 16 in eyes treated initially with placebo and subsequently increased to 35.4 11.2 letters read at completion of infliximab treatment ( week 32 ) . on the other hand , bcva increased from 23.5 10.3 at baseline to 30.4 13.4 letters read at week 16 in eyes treated initially with infliximab and remained essentially unchanged at completion of placebo treatment ( 31.4 12.1 letters read , week 32 ) . changes in visual acuity ( va ) measured by the number of letters that a patient was able to read from the etdrs chart from baseline to study end . eyes of group a and group b were treated initially with placebo followed by infliximab or vice versa , respectively ( a ) . the improvement of visual acuity in infliximab - treated eyes is significantly greater by 24.3% compared with that of placebo - treated eyes , as evaluated by a mixed - models approach for imbalanced crossover design ( b ) . collectively , four infusions of infliximab resulted in an increase in bcva , from mean sd 25.5 10.7 ( range 640 ) to 32.3 12.4 ( range 947 ) letters read ( n = 13 ) . in contrast , bcva remained essentially unchanged in placebo - treated eyes ( n = 14 ) , from 31.5 10.5 ( range 945 ) to 31.1 11.3 ( range 1043 ) letters read . least squares means indicated that infliximab administration resulted in 28.6% and placebo resulted in 4.3% improvement in visual acuity . a possible carryover effect of infliximab in the second part of the study was tested in this model and was found to be nonsignificant . overall , the improvement in visual acuity in the infliximab - treated eyes was significantly greater by 24.3% compared with that in placebo - treated eyes ( 95% ci 4.843.7 ; p = 0.0167 ) ( fig . 1b ) . a similar analysis failed to reveal a significant effect of infliximab over placebo in the secondary end points of the study . least squares means indicated that central macular thickness assessed by oct decreased by 3.7% with infliximab and increased by 1.3% with placebo ( p > 0.5 ) . moreover , no significant difference between infliximab and placebo could be demonstrated in the scores of fundus photographs graded according to the etdrs protocol . as shown in table 2 , the following changes from baseline ( 2 week ) to the end of the study ( 32 weeks ) were evident in our 10 patients ( 13 eyes ) who , either in the first or second part of the study , received four infliximab infusions : bcva improved by at least one line ( 5 letters in the edtrs chart ) in 10 of 14 eyes ( 77% ) , whereas 5 eyes ( 38% ) gained two or more lines , 2 eyes remained stable , and 1 eye worsened by 5 letters . foveal thickness decreased by > 10% in 5 eyes ( 38% ) , remained stable in 5 eyes , and increased by > 10% in 3 eyes . finally , as documented by both fundus photographs and fluoroangiography , the status of diabetic retinopathy improved in 3 eyes , remained stable in 5 eyes , and deteriorated in 1 eye . changes from baseline to 32 weeks in bcva , dme , and retinopathy status after infliximab , given either during study treatment 1 ( eyes 0105 ) or study treatment 2 ( eyes 0714 ) * grading according to the etdrs protocol : 20 : macular edema only ; 35 , 43 , 47 , and 53 : mild , moderate , moderately severe , and severe nonproliferative diabetic retinopathy , respectively . infliximab was well tolerated and no safety issues emerged from hematologic monitoring , urinalysis , or ophthalmic assessments , including intraocular pressure or cataract formation during the study . moreover , no significant impact of placebo or infliximab on glycemic control was noted . one male patient ( aged 64 , with diabetes type 2 for 4 years ; table 1 ) had a diagnosis of breast cancer 5 months after the baseline evaluation . this condition was considered to be unrelated to infliximab treatment , because a slightly palpable mass leading to the final diagnosis was revealed in a physical examination at week 18 , only 14 days after the first infliximab injection . another male patient ( aged 73 , with diabetes type 2 for 18 years ; table 1 ) developed an upper respiratory tract infection that was treated successfully with antibiotics at week 29 while receiving placebo . finally , one male patient ( aged 71 , with diabetes type 2 for 11 years ; table 1 ) developed a neuro - ischemic foot ulcer at week 51 ( 18 weeks after receiving the eighth study injection of placebo ) . individual values of bcva at baseline , week 16 ( end of the first study treatment ) , and week 32 ( final evaluation after the second study treatment ) are shown in table 1 . baseline bcva was not different between groups ( 31.6 5.1 vs. 23.5 10.3 letters read ; t = 1.7 ; p = 0.10 ) . as shown in fig . 1a , bcva decreased from 31.6 5.1 at baseline to 28.8 11.6 letters read at week 16 in eyes treated initially with placebo and subsequently increased to 35.4 11.2 letters read at completion of infliximab treatment ( week 32 ) . on the other hand , bcva increased from 23.5 10.3 at baseline to 30.4 13.4 letters read at week 16 in eyes treated initially with infliximab and remained essentially unchanged at completion of placebo treatment ( 31.4 12.1 letters read , week 32 ) . changes in visual acuity ( va ) measured by the number of letters that a patient was able to read from the etdrs chart from baseline to study end . eyes of group a and group b were treated initially with placebo followed by infliximab or vice versa , respectively ( a ) . the improvement of visual acuity in infliximab - treated eyes is significantly greater by 24.3% compared with that of placebo - treated eyes , as evaluated by a mixed - models approach for imbalanced crossover design ( b ) . collectively , four infusions of infliximab resulted in an increase in bcva , from mean sd 25.5 10.7 ( range 640 ) to 32.3 12.4 ( range 947 ) letters read ( n = 13 ) . in contrast , bcva remained essentially unchanged in placebo - treated eyes ( n = 14 ) , from 31.5 10.5 ( range 945 ) to 31.1 11.3 ( range 1043 ) letters read . least squares means indicated that infliximab administration resulted in 28.6% and placebo resulted in 4.3% improvement in visual acuity . a possible carryover effect of infliximab in the second part of the study was tested in this model and was found to be nonsignificant . overall , the improvement in visual acuity in the infliximab - treated eyes was significantly greater by 24.3% compared with that in placebo - treated eyes ( 95% ci 4.843.7 ; p = 0.0167 ) ( fig . a similar analysis failed to reveal a significant effect of infliximab over placebo in the secondary end points of the study . least squares means indicated that central macular thickness assessed by oct decreased by 3.7% with infliximab and increased by 1.3% with placebo ( p > 0.5 ) . moreover , no significant difference between infliximab and placebo could be demonstrated in the scores of fundus photographs graded according to the etdrs protocol . as shown in table 2 , the following changes from baseline ( 2 week ) to the end of the study ( 32 weeks ) were evident in our 10 patients ( 13 eyes ) who , either in the first or second part of the study , received four infliximab infusions : bcva improved by at least one line ( 5 letters in the edtrs chart ) in 10 of 14 eyes ( 77% ) , whereas 5 eyes ( 38% ) gained two or more lines , 2 eyes remained stable , and 1 eye worsened by 5 letters . foveal thickness decreased by > 10% in 5 eyes ( 38% ) , remained stable in 5 eyes , and increased by > 10% in 3 eyes . finally , as documented by both fundus photographs and fluoroangiography , the status of diabetic retinopathy improved in 3 eyes , remained stable in 5 eyes , and deteriorated in 1 eye . changes from baseline to 32 weeks in bcva , dme , and retinopathy status after infliximab , given either during study treatment 1 ( eyes 0105 ) or study treatment 2 ( eyes 0714 ) * grading according to the etdrs protocol : 20 : macular edema only ; 35 , 43 , 47 , and 53 : mild , moderate , moderately severe , and severe nonproliferative diabetic retinopathy , respectively . infliximab was well tolerated and no safety issues emerged from hematologic monitoring , urinalysis , or ophthalmic assessments , including intraocular pressure or cataract formation during the study . moreover , no significant impact of placebo or infliximab on glycemic control was noted . one male patient ( aged 64 , with diabetes type 2 for 4 years ; table 1 ) had a diagnosis of breast cancer 5 months after the baseline evaluation . this condition was considered to be unrelated to infliximab treatment , because a slightly palpable mass leading to the final diagnosis was revealed in a physical examination at week 18 , only 14 days after the first infliximab injection . another male patient ( aged 73 , with diabetes type 2 for 18 years ; table 1 ) developed an upper respiratory tract infection that was treated successfully with antibiotics at week 29 while receiving placebo . finally , one male patient ( aged 71 , with diabetes type 2 for 11 years ; table 1 ) developed a neuro - ischemic foot ulcer at week 51 ( 18 weeks after receiving the eighth study injection of placebo ) . evidence suggests that altered local expression of tnf may play an important role in the pathogenesis of dme ( 17,18 ) and that low - grade subclinical inflammation is responsible for many of the signature vascular lesions of diabetic retinopathy ( 9 ) . moreover , studies in patients with arthritis have shown that anti - tnf therapy negatively affects vascular permeability and angiogenesis by decreasing vegf ( 19 ) , which has been implicated directly in the pathogenesis of dme and diabetic retinopathy ( 2,9,11 ) . although studies have shown the possible benefits of intravitreal corticosteroids and anti - vegf antibodies in the treatment of dme , focal / grid laser photocoagulation continues to be the only proven safe and effective treatment ( 2 ) . it is noteworthy that there are no previous randomized placebo - controlled phase iii studies for any treatment option in dme . the present study included patients with sight - threatening dme that was unmanageable by laser photocoagulation . of the 14 evaluable eyes , 12 had previously received at least two laser sessions ( maximum eight sessions , eye 12 ) ( table 1 ) . the two remaining eyes had leaking microaneurysms within the foveal avascular zone , making laser photocoagulation unsafe for the central vision . in view of our previously published encouraging preliminary results with infliximab ( 15 ) , the crossover design of this phase iii study was chosen to allow all participants with sight - threatening dme to receive infliximab and to enhance the statistical power of the study . because of the strict study exclusion criteria and because intravitreal administration of anti - vegf agents has been increasingly used over the past 2 years in greece , we were able to recruit only 12 patients . thus , the main limitation of the present study is the small sample size , limiting statistical analysis and not ensuring that randomization balanced all known and unknown risk factors between groups . however , the mean duration of diabetes , as well as the mean number of previous laser treatments and length of time since the last session were similar ( table 1 ) , whereas baseline bcva was also not different between groups . despite the small sample size , our short crossover trial of a conventional dose of infliximab demonstrated a significant improvement over placebo on the severely impaired visual acuity of these patients . infliximab , either as a first or second agent resulted in almost similar increases in bcva ( 6.9 and 6.6 mean letters read , respectively ) . thus , this infliximab - induced mean observed improvement of almost 7 letters read in the edtrs chart is comparable to the mean gain in bcva at 6 months in patients with dme treated with four intravitreal injections of the anti - vegf agent ranibizumab ( 11 ) . moreover , at the end of the study bcva improved by at least one line in 77% and by at least two lines in 38% of infliximab - treated eyes . these results are considered clinically important , given the fact that patients included in this study were unsuitable for all available approved treatment options . it seems that improvement of bcva was not correlated with the secondary anatomic and vision - related end points , because neither an anatomic improvement of dme by oct nor a decrease in fundus photographs grading by the etdrs protocol could be demonstrated . a recent study of 323 eyes from a randomized clinical trial of two methods of laser photocoagulation for dme found that the oct - based assessment of the extensiveness of dme neither explains additional variation in baseline visual acuity above that explained by other known important variables nor predicts changes in macular thickness or visual acuity after laser photocoagulation ( 20 ) . for example , local tnf neutralization by infliximab could have exerted a beneficial effect on photoreceptor function , explaining in part the improvement in bcva despite the persistence of macular edema in patients throughout our study . 2 , the photoreceptor inner / outer segment line , which was invisible at baseline or after the placebo treatment , partially reappeared after infliximab treatment . whether such changes underlie the infliximab - induced bcva improvement remains to be seen , because the photoreceptor inner / outer segment junction line was not identifiable by oct at any time in all other patients . sequential oct images at baseline ( a ) , at completion of placebo treatment ( b ) , and at completion of infliximab treatment ( c ) . the photoreceptor inner / outer segment junction line at the foveola is highly disrupted at week 2 ( a , arrow ) , becomes almost absent at week 16 ( b , arrow ) , and appears partially restored at week 32 ( c , arrow ) . the key safety considerations that emerged during the first years of clinical use of infliximab included infections , autoimmune disease , demyelinating disease , malignancies , and congestive heart failure ( 8) . overall rates of these conditions in randomized controlled trials were not significantly increased during treatment compared with placebo . postmarketing surveillance data in thousands of patients have clearly shown that the safety profile of infliximab is excellent , provided that it is not used to treat patients with active infection , malignancy , preexisting demyelinating conditions , and heart failure and that precautions are taken for reactivation of latent tuberculosis . no other particular safety signals in patients with diabetes have emerged ( 8) . overall , infliximab was well tolerated in our study . to summarize , a short - term treatment with infliximab significantly improved bcva in eyes with advanced - stage sight - threatening dme refractory to standard treatment , further suggesting an important role for tnf - mediated pathogenetic mechanisms in this condition . this positive result also suggests that larger and longer term placebo - controlled trials are warranted to assess the efficacy and safety of systemic tnf blockade and/or of local delivery of anti - tnf antibodies by intravitreal injection ( 2124 ) for the primary treatment of dme .

objectivebecause many patients with diabetic macular edema ( dme ) do not respond to focal / grid laser photocoagulation , the only currently approved treatment , alternatives are needed . based on encouraging preliminary findings , we aimed to assess efficacy and safety of the anti tumor necrosis factor ( tnf ) monoclonal antibody infliximab in this condition.research design and methodsthis was a single - center , double - blind , randomized , placebo - controlled , crossover study . eleven patients with sight - threatening dme persisting after two sessions of laser photocoagulation received infliximab ( 5 mg / kg ) intravenously at weeks 0 , 2 , 6 , and 14 , followed by placebo at weeks 16 , 18 , 22 , and 30 , or vice versa . blinding was maintained to week 32 , when the final assessments were performed . best corrected visual acuity evaluated by a mixed - models approach for imbalanced crossover design using the percentage difference as the outcome variable was the primary study end point . data were analyzed on an intention - to - treat basis.resultsearly treatment of diabetic retinopathy study ( etdrs ) scores dropped from 31.6 5.1 ( mean sd ) letters read at baseline to 28.8 11.6 letters read at week 16 in six placebo - treated eyes and improved to 35.4 11.2 letters read after infliximab . in contrast , visual acuity improved from 23.5 10.3 at baseline to 30.4 13.4 letters read at week 16 in eight infliximab - treated eyes and was sustained at completion of placebo treatment ( 31.4 12.1 letters read ) . the excess visual acuity in infliximab - treated eyes was greater by 24.3% compared with that in placebo - treated eyes ( 95% ci 4.843.7 ; p = 0.017 ) . infliximab treatment was well tolerated.conclusionsthe positive results of this small phase iii study suggest that larger and longer term trials should be conducted to assess the efficacy of systemic or intravitreal anti - tnf agent administration for primary treatment of dme .

RESEARCH DESIGN AND METHODS Patient eligibility and exclusion criteria Study protocol Outcome measures and statistical analysis RESULTS Primary study objective: changes in best corrected visual acuity Secondary anatomic and vision-related objectives Baseline versus 32-week evaluation measurements Safety issues CONCLUSIONS

this is an investigator - initiated phase iii double - blind , randomized , placebo - controlled , two - arm crossover clinical study . the study adhered to the guidelines of the declaration of helsinki , and the protocol and consent form were approved by the local investigational review board , the national ethics committee , and the ministry of health . patients ( aged > 18 years ) with type 1 or type 2 diabetes and dme resulting in best corrected visual acuity ( bcva ) of 0.4 were eligible if they had at least two previous sessions of laser photocoagulation > 6 months before enrollment or if they had leaking microaneurysms within the foveal avascular zone , making laser photocoagulation unsafe for the central vision . in addition to standard inclusion and exclusion criteria for phase iii studies of infliximab , patients were excluded if they had 1 ) vitreoretinal traction , 2 ) retinal detachment , 3 ) proliferative diabetic retinopathy requiring immediate panretinal photocoagulation , 4 ) any previous eye surgery 6 months before the study , including any intravitreal infusions , 5 ) macular edema of the ischemic type or caused by retinal conditions other than diabetes , 6 ) cataract or media opacities of a degree that precluded accurate retinal photographs or optical coherence tomography ( oct ) measurement , 7 ) hard exudates under the fovea , or 8) uncontrolled arterial hypertension ( blood pressure > 180/110 mmhg ) , a major change in glycemic control ( e.g. patients were randomly allocated 1:1 to receive placebo or infliximab in a two - armed crossover , double - blind design according to the permuted block randomization list generated in sas . patients received placebo at weeks 0 , 2 , 6 , and 14 , followed by infliximab at weeks 16 , 18 , 22 , and 30 ( group a ) , or vice versa ( group b ) in addition to standard therapy for diabetes , hypertension , and dyslipidemia , which remained unchanged during the study . all study drugs were administered via a 2-h intravenous infusion at a dose of 5 mg / kg body wt on the scheduled visits at weeks 0 , 2 , 6 , 14 , 16 , 18 , 22 , and 30 . blinding was maintained to week 32 , when the final clinical , laboratory , and ophthalmic evaluation was performed in all patients . physical examination and bcva measurements of the number of letters a patient was able to read from the early treatment diabetic retinopathy study ( etdrs ) charts with correction for individual refractive errors were performed at every visit . foveal thickness measurements by third - generation oct ( stratus oct iii ) , using the fast macular thickness scan , stereoscopic fundus photographs ( seven fields ) , and intraocular pressure measurements using a goldman applanation tonometer were performed at weeks 8 , 16 , 24 , and 32 . the primary end point of the study was to assess the efficacy and safety of four infusions of infliximab on bcva , evaluated by a mixed - models approach for imbalanced crossover design using the percent difference between infliximab and placebo groups as an outcome variable . the secondary end points were 1 ) the effect of infliximab on the anatomic change of dme , assessed by oct and 2 ) the effect of infliximab on diabetic retinopathy , assessed by fundus photographs and fluoroangiographic studies . the treatment effect of infliximab versus placebo in macular thickness and fundus photographs results was also evaluated by a mixed - models approach for imbalanced crossover design using the percent difference as an outcome variable . under the assumption of a 15% dropout rate , however , the study was terminated after enrollment of the first 12 patients because of inability to recruit additional patients who had not any intravitreal infusion within the prior 6 months ( exclusion criterion 4 , as described above ) . patients ( aged > 18 years ) with type 1 or type 2 diabetes and dme resulting in best corrected visual acuity ( bcva ) of 0.4 were eligible if they had at least two previous sessions of laser photocoagulation > 6 months before enrollment or if they had leaking microaneurysms within the foveal avascular zone , making laser photocoagulation unsafe for the central vision . in addition to standard inclusion and exclusion criteria for phase iii studies of infliximab , patients were excluded if they had 1 ) vitreoretinal traction , 2 ) retinal detachment , 3 ) proliferative diabetic retinopathy requiring immediate panretinal photocoagulation , 4 ) any previous eye surgery 6 months before the study , including any intravitreal infusions , 5 ) macular edema of the ischemic type or caused by retinal conditions other than diabetes , 6 ) cataract or media opacities of a degree that precluded accurate retinal photographs or optical coherence tomography ( oct ) measurement , 7 ) hard exudates under the fovea , or 8) uncontrolled arterial hypertension ( blood pressure > 180/110 mmhg ) , a major change in glycemic control ( e.g. patients were randomly allocated 1:1 to receive placebo or infliximab in a two - armed crossover , double - blind design according to the permuted block randomization list generated in sas . patients received placebo at weeks 0 , 2 , 6 , and 14 , followed by infliximab at weeks 16 , 18 , 22 , and 30 ( group a ) , or vice versa ( group b ) in addition to standard therapy for diabetes , hypertension , and dyslipidemia , which remained unchanged during the study . all study drugs were administered via a 2-h intravenous infusion at a dose of 5 mg / kg body wt on the scheduled visits at weeks 0 , 2 , 6 , 14 , 16 , 18 , 22 , and 30 . blinding was maintained to week 32 , when the final clinical , laboratory , and ophthalmic evaluation was performed in all patients . physical examination and bcva measurements of the number of letters a patient was able to read from the early treatment diabetic retinopathy study ( etdrs ) charts with correction for individual refractive errors were performed at every visit . foveal thickness measurements by third - generation oct ( stratus oct iii ) , using the fast macular thickness scan , stereoscopic fundus photographs ( seven fields ) , and intraocular pressure measurements using a goldman applanation tonometer were performed at weeks 8 , 16 , 24 , and 32 . the primary end point of the study was to assess the efficacy and safety of four infusions of infliximab on bcva , evaluated by a mixed - models approach for imbalanced crossover design using the percent difference between infliximab and placebo groups as an outcome variable . the secondary end points were 1 ) the effect of infliximab on the anatomic change of dme , assessed by oct and 2 ) the effect of infliximab on diabetic retinopathy , assessed by fundus photographs and fluoroangiographic studies . the treatment effect of infliximab versus placebo in macular thickness and fundus photographs results was also evaluated by a mixed - models approach for imbalanced crossover design using the percent difference as an outcome variable . it was estimated that 22 evaluable eyes ( 11 per study arm ) would provide 90% power to detect a mean difference in log minimum angle of resolution ( logmar ) bcva of 0.67 ( equivalent to 22 letters read in the edtrs chart ) at a 0.001 level of statistical significance . in total 14 eyes were eligible for analysis ( 6 eyes in group a , including this patient 's response to placebo treatment , and 8 eyes in group b ) ( table 1 ) . demographic and disease characteristics of patients with dme and individual bcva values of eligible eyes at baseline ( week 2 ) , end of study treatment 1 ( week 16 ) , and end of study treatment 2 ( week 32 ) * a denotes placebo ; b denotes infliximab . study treatment 1 : from baseline to week 16 ; study treatment 2 : from week 16 to week 32 . individual values of bcva at baseline , week 16 ( end of the first study treatment ) , and week 32 ( final evaluation after the second study treatment ) are shown in table 1 . baseline bcva was not different between groups ( 31.6 5.1 vs. 23.5 10.3 letters read ; t = 1.7 ; p = 0.10 ) . 1a , bcva decreased from 31.6 5.1 at baseline to 28.8 11.6 letters read at week 16 in eyes treated initially with placebo and subsequently increased to 35.4 11.2 letters read at completion of infliximab treatment ( week 32 ) . on the other hand , bcva increased from 23.5 10.3 at baseline to 30.4 13.4 letters read at week 16 in eyes treated initially with infliximab and remained essentially unchanged at completion of placebo treatment ( 31.4 12.1 letters read , week 32 ) . changes in visual acuity ( va ) measured by the number of letters that a patient was able to read from the etdrs chart from baseline to study end . eyes of group a and group b were treated initially with placebo followed by infliximab or vice versa , respectively ( a ) . the improvement of visual acuity in infliximab - treated eyes is significantly greater by 24.3% compared with that of placebo - treated eyes , as evaluated by a mixed - models approach for imbalanced crossover design ( b ) . collectively , four infusions of infliximab resulted in an increase in bcva , from mean sd 25.5 10.7 ( range 640 ) to 32.3 12.4 ( range 947 ) letters read ( n = 13 ) . in contrast , bcva remained essentially unchanged in placebo - treated eyes ( n = 14 ) , from 31.5 10.5 ( range 945 ) to 31.1 11.3 ( range 1043 ) letters read . a possible carryover effect of infliximab in the second part of the study was tested in this model and was found to be nonsignificant . overall , the improvement in visual acuity in the infliximab - treated eyes was significantly greater by 24.3% compared with that in placebo - treated eyes ( 95% ci 4.843.7 ; p = 0.0167 ) ( fig . as shown in table 2 , the following changes from baseline ( 2 week ) to the end of the study ( 32 weeks ) were evident in our 10 patients ( 13 eyes ) who , either in the first or second part of the study , received four infliximab infusions : bcva improved by at least one line ( 5 letters in the edtrs chart ) in 10 of 14 eyes ( 77% ) , whereas 5 eyes ( 38% ) gained two or more lines , 2 eyes remained stable , and 1 eye worsened by 5 letters . finally , as documented by both fundus photographs and fluoroangiography , the status of diabetic retinopathy improved in 3 eyes , remained stable in 5 eyes , and deteriorated in 1 eye . changes from baseline to 32 weeks in bcva , dme , and retinopathy status after infliximab , given either during study treatment 1 ( eyes 0105 ) or study treatment 2 ( eyes 0714 ) * grading according to the etdrs protocol : 20 : macular edema only ; 35 , 43 , 47 , and 53 : mild , moderate , moderately severe , and severe nonproliferative diabetic retinopathy , respectively . infliximab was well tolerated and no safety issues emerged from hematologic monitoring , urinalysis , or ophthalmic assessments , including intraocular pressure or cataract formation during the study . this condition was considered to be unrelated to infliximab treatment , because a slightly palpable mass leading to the final diagnosis was revealed in a physical examination at week 18 , only 14 days after the first infliximab injection . individual values of bcva at baseline , week 16 ( end of the first study treatment ) , and week 32 ( final evaluation after the second study treatment ) are shown in table 1 . baseline bcva was not different between groups ( 31.6 5.1 vs. 23.5 10.3 letters read ; t = 1.7 ; p = 0.10 ) . 1a , bcva decreased from 31.6 5.1 at baseline to 28.8 11.6 letters read at week 16 in eyes treated initially with placebo and subsequently increased to 35.4 11.2 letters read at completion of infliximab treatment ( week 32 ) . on the other hand , bcva increased from 23.5 10.3 at baseline to 30.4 13.4 letters read at week 16 in eyes treated initially with infliximab and remained essentially unchanged at completion of placebo treatment ( 31.4 12.1 letters read , week 32 ) . changes in visual acuity ( va ) measured by the number of letters that a patient was able to read from the etdrs chart from baseline to study end . eyes of group a and group b were treated initially with placebo followed by infliximab or vice versa , respectively ( a ) . the improvement of visual acuity in infliximab - treated eyes is significantly greater by 24.3% compared with that of placebo - treated eyes , as evaluated by a mixed - models approach for imbalanced crossover design ( b ) . collectively , four infusions of infliximab resulted in an increase in bcva , from mean sd 25.5 10.7 ( range 640 ) to 32.3 12.4 ( range 947 ) letters read ( n = 13 ) . in contrast , bcva remained essentially unchanged in placebo - treated eyes ( n = 14 ) , from 31.5 10.5 ( range 945 ) to 31.1 11.3 ( range 1043 ) letters read . a possible carryover effect of infliximab in the second part of the study was tested in this model and was found to be nonsignificant . overall , the improvement in visual acuity in the infliximab - treated eyes was significantly greater by 24.3% compared with that in placebo - treated eyes ( 95% ci 4.843.7 ; p = 0.0167 ) ( fig . as shown in table 2 , the following changes from baseline ( 2 week ) to the end of the study ( 32 weeks ) were evident in our 10 patients ( 13 eyes ) who , either in the first or second part of the study , received four infliximab infusions : bcva improved by at least one line ( 5 letters in the edtrs chart ) in 10 of 14 eyes ( 77% ) , whereas 5 eyes ( 38% ) gained two or more lines , 2 eyes remained stable , and 1 eye worsened by 5 letters . finally , as documented by both fundus photographs and fluoroangiography , the status of diabetic retinopathy improved in 3 eyes , remained stable in 5 eyes , and deteriorated in 1 eye . changes from baseline to 32 weeks in bcva , dme , and retinopathy status after infliximab , given either during study treatment 1 ( eyes 0105 ) or study treatment 2 ( eyes 0714 ) * grading according to the etdrs protocol : 20 : macular edema only ; 35 , 43 , 47 , and 53 : mild , moderate , moderately severe , and severe nonproliferative diabetic retinopathy , respectively . infliximab was well tolerated and no safety issues emerged from hematologic monitoring , urinalysis , or ophthalmic assessments , including intraocular pressure or cataract formation during the study . this condition was considered to be unrelated to infliximab treatment , because a slightly palpable mass leading to the final diagnosis was revealed in a physical examination at week 18 , only 14 days after the first infliximab injection . evidence suggests that altered local expression of tnf may play an important role in the pathogenesis of dme ( 17,18 ) and that low - grade subclinical inflammation is responsible for many of the signature vascular lesions of diabetic retinopathy ( 9 ) . moreover , studies in patients with arthritis have shown that anti - tnf therapy negatively affects vascular permeability and angiogenesis by decreasing vegf ( 19 ) , which has been implicated directly in the pathogenesis of dme and diabetic retinopathy ( 2,9,11 ) . although studies have shown the possible benefits of intravitreal corticosteroids and anti - vegf antibodies in the treatment of dme , focal / grid laser photocoagulation continues to be the only proven safe and effective treatment ( 2 ) . it is noteworthy that there are no previous randomized placebo - controlled phase iii studies for any treatment option in dme . the present study included patients with sight - threatening dme that was unmanageable by laser photocoagulation . in view of our previously published encouraging preliminary results with infliximab ( 15 ) , the crossover design of this phase iii study was chosen to allow all participants with sight - threatening dme to receive infliximab and to enhance the statistical power of the study . because of the strict study exclusion criteria and because intravitreal administration of anti - vegf agents has been increasingly used over the past 2 years in greece , we were able to recruit only 12 patients . however , the mean duration of diabetes , as well as the mean number of previous laser treatments and length of time since the last session were similar ( table 1 ) , whereas baseline bcva was also not different between groups . thus , this infliximab - induced mean observed improvement of almost 7 letters read in the edtrs chart is comparable to the mean gain in bcva at 6 months in patients with dme treated with four intravitreal injections of the anti - vegf agent ranibizumab ( 11 ) . moreover , at the end of the study bcva improved by at least one line in 77% and by at least two lines in 38% of infliximab - treated eyes . a recent study of 323 eyes from a randomized clinical trial of two methods of laser photocoagulation for dme found that the oct - based assessment of the extensiveness of dme neither explains additional variation in baseline visual acuity above that explained by other known important variables nor predicts changes in macular thickness or visual acuity after laser photocoagulation ( 20 ) . 2 , the photoreceptor inner / outer segment line , which was invisible at baseline or after the placebo treatment , partially reappeared after infliximab treatment . sequential oct images at baseline ( a ) , at completion of placebo treatment ( b ) , and at completion of infliximab treatment ( c ) . the photoreceptor inner / outer segment junction line at the foveola is highly disrupted at week 2 ( a , arrow ) , becomes almost absent at week 16 ( b , arrow ) , and appears partially restored at week 32 ( c , arrow ) . to summarize , a short - term treatment with infliximab significantly improved bcva in eyes with advanced - stage sight - threatening dme refractory to standard treatment , further suggesting an important role for tnf - mediated pathogenetic mechanisms in this condition . this positive result also suggests that larger and longer term placebo - controlled trials are warranted to assess the efficacy and safety of systemic tnf blockade and/or of local delivery of anti - tnf antibodies by intravitreal injection ( 2124 ) for the primary treatment of dme .

since 1994 , bacterial artificial chromosome ( bac ) libraries have become an invaluable resource tool to initiate genomics research in theareas of genome sequencing , physical mapping , positional cloning , complex analysis of targeted genomic regions , and analysis of gene structure and function [ 18 ] . more recently , physical map tiles of bacs have been shown to be suitable for next generation sequencing of chromosomal regions or whole genomes . bac - end sequencing ( bes ) is a powerful tool that enhances the value of bac libraries as a genomic resource by providing partial sequence information that can be used to understand genome content and architecture and develop genetic markers [ 10 , 11 ] . physical maps constructed from fingerprinted bac clones , together with associated bac - end sequence information , can be used to : construct bac fingerprint-/bes- based physical maps [ 1 , 12 ] which can be aligned to reference genome sequences ; sequence genomes by walking from one clone to the next ; anchor whole genome shotgun sequence data ; integrate genetic linkage maps with physical maps . about 3.9 million tons of tea is produced ( http://faostat.fao.org/site/567/desktopdefault.aspx?pageid=567#ancor ) and consumed yearly with an estimated world value of $ 9.04 billion ( $ 2.39 per kg ; http://www.fao.org/docrep/meeting/019/k8336e.pdf ) . throughout history , it has become well known that tea is one of the most beneficial beverages to drink due to its health attributes . tea polyphenols ( catechins ) and antioxidants , saponin , polysaccharides , l - theanine , pigments , and tea water extracts are the most important components that contribute to tea 's medicinal qualities [ 1619 ] . there are different varieties or types of tea , that is , green tea , black tea , oolong tea , white tea , and so on , ( see also http://jingtea.com/tea-knowledge/tea-varieties ) . oolong tea is a special variety of tea and has been studied for its effects on diabetes , eczema , allergies , bacterial infections , dental caries ( cavities ) , obesity , cardiovascular disease , cancer , and its antioxidant properties [ 2126 ] . it has been shown that consumption of oolong tea stimulates both energy expenditure and fat oxidation in normal weight men . oolong tea is semifermented which occurs when the leaves are gently rolled during processing , and this gives oolong tea a unique appearance and flavor . chin - shin oolong is one of the most widely cultivated oolong tea varieties worldwide . it has been suggested that functional genomic research should be a major emphasis of tea genetics and breeding in the future . although rapid progress of gene identification and isolation from tea plants has been made in the past several years , the study of the tea plant genome lags far behind other crop species due to the lack of good genomic research tools . this is most likely due to the difficulties of preparing high - quality tea dna and due to the distinctness of tea plant from other taxa ( i.e. , perennial nature , high inbreeding depression , unavailability of distinct mutants of different biotic and abiotic stress , and large genome size of 4 gb ) . one key genomic tool that is completely lacking for tea is the availability of a high - quality , deep - coverage , bac library . in this paper , we report the construction of a high - quality , publicly available bac library of tea plant from the variety chin - shin oolong . we generated and analyzed a limited data set of bac - end sequences from this library which provided an early glimpse into the sequence composition of the tea genome . sixty 6-year - old plants , derived from a clonally propagated single mother plant of a camellia sinensis cultivar chin - shin oolong , were selected as the plant germplasm source . healthy young shoot tips and the uppermost two leaves were collected ( similar to harvesting top - quality tea ) , then washed quickly to remove debris , and immediately frozen by submersion in liquid nitrogen followed by short - term storage at 80c . all plant growth and tea genetic resources have lagged behind other important plants due to the complex and difficult nature of adequate extraction of quality nuclear dna . however , a detailed manuscript describing the experiments that led to the successful isolation of high - quality , high molecular weight , nuclear dna , suitable for bac library construction , was recently made available . the method for tea plant bac library construction utilized the standard arizona genomics institute ( agi ) protocol , the method of luo and wing , and ammiraju et al . . modifications necessary to achieve successful construction of a tea plant bac library are described as follows . twenty grams of frozen tissue was homogenized and transferred to a flask containing 200 ml of prechilled extraction buffer ( 10 mm tris - hcl , ph 8.0 , 10 mm edta , ph 8.0 , 100 mm kcl , 0.5 m sucrose , 4 mm spermidine , 1 mm spermine , 0.10% w / v l - ascorbic acid , 2.00% w / v pvp-40 , 0.13% w / v sodium diethyldithiocarbamate trihydrate ; pvp-40 was only about 50 percent dissolved ) and 400 l -mercaptoethanol . the homogenate was filtered into a flask that contained 200 ml of the same above prechilled extraction buffer and 400 l -mercaptoethanol . the homogenate was filtered a second time into a fresh flask that contained 45 ml of prechilled extraction buffer with 10.00% v / v triton x-100 . the resulting pellet was washed several times with the same buffer and resuspended in 1 ml of prechilled extraction buffer , incubated in a 45c water bath for 5 minutes , and gently mixed with one - third volume of 1.00% low melting temperature agarose ( in extraction buffer ) at 45c . twenty - four plugs were transferred into a 50 ml - falcon tube , containing 40 ml of standard proteinase k solution ( 1.00% w / v n - lauroylsarcosine ( sodium salt ) , 0.1 mg / ml proteinase k , dissolved in 0.5 m edta , ph 9.4 ) , and incubated in a hybridization oven at 50c with a gentle rotation for 24 h. after the plugs were rewashed with fresh proteinase k solution for an additional 24 h , they were rewashed in 2 additional solutions . first , two times with 40 ml t10e10 containing 1 mm pmsf ( phenylmethanesulfonyl fluoride ) and then twice with 40 ml te , each time for about 1 h at room temperature with gentle shaking . the plugs were stored in 70.00% ethanol at 20c ( for long - term storage ) . two and a half dna plugs were used to establish optimal hindiii partial digestion conditions . formal partial digestion , using 5 dna plugs , was performed using 5u of hindiii restriction enzyme added to each sample ( per half plug ) . digested samples were loaded to 1.00% agarose gel and subjected to pulsed - field gel electrophoresis ( pfge ) . dna was visualized , and agarose fragments , containing specific dna sizes , were cut from the gel slabs . a second- and third - pfge run of the fragments was performed to further purify the dna and remove small dna fragments . after finishing the third size selection , the gel fractions containing different sized fragments ( b2 , b1 , a2 ) were recovered and stored at 20c in 70.00% ethanol ( for long - term storage ) . for each size - selected fraction , the high molecular weight genomic dna was electroeluted from the agarose at 4c . pipet tips ( with cutoff tips ) were used when manipulating high molecular weight genomic dna to avoid mechanical shearing . the dna concentrations were estimated , and 120 ng200 ng dna was used to ligate to the linearized ( hindiii site ) and dephosphorylated vector pindigobac536 swai , commonly known as pagibac1 . separately , ligations were mixed well by tapping and then incubated in a water bath at 16c for 19 hours . ligation samples were transferred into 0.1 m glucose/1.00% agarose cones to desalt for 1.5 h on ice . ligations were transferred into fresh microcentrifuge tubes and stored at 4c until transformation tests were completed . the methods for test transformations , bac dna isolations , bac insert size analyses , bulk transformations , colony arrays , and library characterizations followed exactly the agi protocols ( http://www.genome.arizona.edu/information/publications/wingpub/construct.pdf and http://www2.genome.arizona.edu/pdfs/publications/meizhong_metmb.pdf ) . from each hmw fraction ( b2 , b1 , a2 ) , 2.3 l of each ligation was used to transform 20 l of dh10b t1 phage - resistant e. coli cells ( invitrogen ) by electroporation using the cell porator and voltage booster electroporation system ( life technologies ) . electroporation was performed on ice at 327 dc v with fast charge rate at a low resistance ( 4 k ) and a capacitance of 330 f . the cells were transferred into 3 ml of soc media , and incubated at 37c for 1 h with shaking at 250 rpm , followed by the addition of an equal volume of sterile glycerol and gentle shaking for 3 minutes . these mixtures were immediately frozen by submersion into liquid nitrogen followed by long - term storage at 80c . to evaluate these transformation tests , 300 l of each ( containing cells , soc , and glycerol ) were spread on petri dishes ( containing lb x - gal iptg agar with 12.5 g / ml of chloramphenicol , 80 g / ml x - gal , and 100 g / ml iptg ) and incubated at 37c overnight . five - hundred - seventy - six white recombinants ( positive for insert ) , 192 from each of the three sublibraries ( b2 , b1 , a2 ) , were randomly selected and grown overnight at 37c in 1.2 ml lb broth ( with chloramphenicol 12.5 g / ml ) with shaking at 220 rpm . digestions were separated by pfge at 6 v / cm , switch time from 5 to 15 s , angle 120 , and run for 16 h followed by staining , destaining , and visualization . transformed e. coli from the b2 ligation , selected to contain the largest insert sizes , were prepared for array to 384-well microtiter dishes . five ml of the mixture from the b2 ligation were spread on lb x - gal iptg - cm agar q - trays ( 22.5 22.5 cm ) and incubated at 37c overnight . the white ( insert positive recombinant ) clones were robotically picked ( using a genetix q - bot ; genetix ltd . ) into forty - eight 384-well microtiter plates containing freezing media and stored at 80c . hybridization screening filters were printed from library copies to facilitate future experiments . to further evaluate this sub library ( b2 ligation ) , 576 bacs were randomly selected from these forty - eight plates and grown overnight at 37c with shaking at 220 rpm in 1.2 ml lb supplemented with chloramphenicol ( 12.5 g / ml ) . the digested clones were separated by pfge and analyzed . additionally , 192 bac clones were subjected to bac clone end sequencing ( bes ) using the method of kim et al . , and the resultant sequences were analyzed for chloroplast and mitochondrial genome contaminations and for repeat content using blastn searches with settings that required 98% identity over lengths of at least 51 bp . the successful c. sinensis bac library , composed of e. coli transformants from each of the three ligations , was named csbcba . camellia sinensis cultivar chin - shin oolong was chosen to construct the bac library since it is one of the most widely cultivated oolong tea varieties . to avoid contamination with small , trapped dna fragments and improve the size and uniformity of the inserts , the high molecular weight ( hmw ) genomic dna was partially digested with hindiii and three separate size fractionations were collected . following ligations into the hindiii site of the pagibac1 vector , the three size fractionations were transformed , and the new tea bac library , consisting of 401,280 bac clones , was named csbcba ; see table 1 . the overall average insert size of the total library was calculated to be 135 kb with inserts ranging from 8 to 314 kb , and the total library coverage was estimated to equal 13.54 haploid genome equivalents based upon mathematical calculations that utilize a genome size of 4,000 mb . sub library 1 ( ligation b2 ) was composed of 40,320 clones with an average insert size 140 kb ; sub library 2 ( ligation b1 ) was composed of 160,896 clones with an average insert size 140 kb ; sub library 3 ( ligation a2 ) was composed of 200,064 clones with an average insert size 130 kb . five - hundred - seventy - six bacs , 192 from each of the three sublibraries , were randomly selected and analyzed by noti restriction enzyme digestion in order to evaluate the library ( figure 1 ) . based on this sample size were shown to carry dna inserts greater than 130 kb , with a reasonable fraction ( 22.30% ) carrying inserts larger than 170 kb ( figure 2 ) . bac clones from sub library 1 , ligation b2 , were spread on agar dishes , clones ( 18,432 ) were robotically picked and arrayed to 48- 384well plates , and hybridization screening filters were printed to facilitate future experiments . to examine the quality of the library and to obtain a preliminary view of the major repetitive element content of the c. sinensis genome , unidirectional end sequencing was performed on 192 random bac clones which produced 182 reads with an average length of 581 high - quality bases ; the bess are available at the url : http://www2.genome.arizona.edu/files/camellia_bes.txt . of the 182 random bess , the total number of nucleotides was 111,695 bp and the gc level was 40.35% ( table 2 ) . likewise , the 182 random bac - end sequences were evaluated using blast searches and queries to the transposable element databases of arabidopsis ( arabidopsis thaliana ) at genetic information research institute and of rice ( oryza sativa ) at the arizona genomics institute . this preliminary analysis of repetitive sequences from pilot bac - end sequences indicated that 97 to 98 percent of the bess contained sequences related to transposable elements . ltr retrotransposons were the predominant class of repeat elements in c. sinensis ( arabidopsis 86.93% and rice 87.24% ; table 2 ) . the second major class of transposable elements belongs to dna retrotransposon ( arabidopsis 11.16% and rice 11.69% ) . line and non - ltr elements were also observed though at much lower amounts . blastn searches against the chloroplast and mitochondrial genomes of arabidopsis ( arabidopsis thaliana ) and rice ( oryza sativa ) using the 182 random bess revealed no significant homology found , thus indicating that the bac library contained very low levels of chloroplast or mitochondrial dna contamination . visualization of the inserts from the random 576 bacs from pulsed - field electrophoresis also indicated no patterns typical of organelle contamination ( data not shown ) . further repeat analysis of the 182 random bess revealed 25 different microsatellite repeats ( table 3 ) . the 25 simple sequence repeats ( ssrs ) were found from 17 different bac clones ( from the forward and reverse reads ) , thus suggesting that eight bacs represented genomic regions that contained high repeat content . interestingly , one bes read , > _ camellia_g1 - 12 - 3 - 07_d02_.g1 , contained two different dimer , and one pentamer , ssrs . generally , the world 's agriculture and food systems come from the tremendous biological diversity encompassed in over 200 independently ( and perhaps convergent ) domesticated species of angiosperms . paradoxically , much of the potential genomic research with these taxa has been centered on a few model species , or taxonomic families , that represent only a very small amount of this diversity . in the past decade , these include molecular / genetic maps , transcript databases , large - insert libraries , five complete genome sequences ( rice , arabidopsis , poplar , grape , and maize ) , and a suite of several others scheduled for completion . the ultimate goals of these projects are to continue to discover new ways to meet future world agricultural and food needs while simultaneously providing an understanding of functional systems biology . however , the expected dramatic advances in theoretical and applied plant biology for other taxa , following these innovations , have been critically slow due to two major obstacles : lack of adequate genomic tools and resources to efficiently and effectively transfer existing genomic knowledge to other economically and diverse agriculturally important species and lack of representation of novel genes and regulatory networks that underlie key traits of agriculture and ecology in the sequenced model species . despite the significant economic impact of tea and other similar commodities ( i.e. , coffee , cocoa ) , a lack of adequate public genomic resources , especially access to large - insert libraries , has contributed to a lack of advanced genetic knowledge useable for modern breeding and improvement . recent published tea plant works involving molecular tools have shown an aflp and rapd marker - based linkage map , identified cdnas involved in secondary metabolism , described sequence analysis from 4320 tea ests , and used intersimple sequence repeats to analyze genetic variability of somaclonal embryo - derived tea plants . a recent review described the use of molecular resources for tea cultivar classification , the identification of parentage of mulberry scale resistance , and more advanced linkage maps with ssr markers . while these and other reports provide important insight to the understanding of the tea plant , the availability and utilization of bac resources are absent . here , we report the construction and public availability of a high - quality , deep - coverage , large - insert , bacterial artificial chromosome ( bac ) library of cultivated tea plant ( camellia sinensis ) variety chin - shin oolong . this bac library resource is publicly available in the form of whole libraries , filters , and individual clones , through the agi bac / est resource center ( http://www.genome.arizona.edu/orders ) , and we expect it to be extensively used worldwide for the analysis of genome evolution and organization , positional cloning , and eventual gap closure of a c. sinensis reference sequence . this tea bac library was made possible after significant methodological improvements were accomplished in the preparation of purified high molecular weight ( hmw ) dna and subsequent partial enzymatic digestions and ligations . we showed that , despite expected difficulties in obtaining hmw dna of reliable quality from tissues known to contain compounds deleterious to established extraction techniques , our results yielded ligations that produced a bac library with low organellar contamination , such as chloroplast and mitochondria , yet with high transformation efficiencies and large - insert sizes . though the genome size of tea is quite large , 4 gb , the described bac library has an average insert size of 135 kb and provides over 13x genome coverage to allow for adequate utility . it was divided into three sublibraries : sub library 1 ( ligation b2 ) composed of 40,320 clones with average insert sizes of 140 kb , sub library 2 ( ligation b1 ) composed of 160,896 clones with average insert sizes of 140 kb , and sub library 3 ( ligation a2 ) composed of 200,064 clones with average insert sizes of 130 kb . fresh tea leaves are rich in both volatile secondary compounds such as tea polyphenols and carbohydrate matrices such as tea polysaccharides . it has been shown that tea leaves contain 2033% dry weight of polyphenols , and polysaccharides have been shown to be present at 7.02% dry weight in oolong tea . these types of compounds contribute negatively toward the handling of high molecular weight ( hmw ) dna necessary for construction of large - insert genomic clone libraries . tea polyphenols must be prevented from interacting with the nuclear dna , and tea polysaccharides must be prevented from trapping nuclei in the process of tissue homogenization . in this study , to prepare high - quality hmw dna , the frozen tissue was homogenized with a buffer ( composed of combining ingredients from two methods but requiring the pvp-40 to be partially undissolved ) in double volumes followed by additional filtration . double volumes of buffer were found to dilute the polyphenols and polysaccharides , as evidenced by the absence of sticky nuclear pellets , thus allowing for increased removal during the centrifugation steps . the combining of the nuclei with the low melting temperature agarose at a lower ratio of 1 : 3 ( instead of 1 : 1 ) was required to concentrate the nuclei but was performed in the presence of the undissolved pvp-40 . to lower the organellar dna contamination and to further eliminate tea polysaccharides and tea polyphenols in the process of preparing tea plant hmw dna plugs , removing small restriction fragments is vital for construction of a high - quality bac library [ 46 , 47 ] . tea plant genomic plugs prepared by the method used in this paper contained abundant hmw dna and produced satisfactory restriction fragments when digested with hindiii . to avoid contamination with small - trapped dna fragments and improve the size and uniformity of the inserts usually two size selections are sufficient , but visual examination of the dna fragments after the second size selection revealed that an additional selection was required ( data not shown ) . a detailed manuscript describing the experiments for the appropriate isolation of high - quality , high molecular weight dna that led to the successful construction of this tea plant bac library ( tea plant nuclei isolation , buffer compositions , hmw dna , large - insert ligations , etc . ) was recently published , and this method will also yield sufficient quality tea dna for other purposes , such as next generation sequencing ( ngs ) . pulsed - field gel electrophoresis analysis of 576 random bac clone plasmids showed that the majority of c. sinensis cloned dna inserts were present as single noti fragments . this indicated that the c. sinensis genome apparently contained few noti sites , a feature commonly observed with the genomes of other plant dicot species and contrary to the results obtained with monocot species . long terminal repeats ( ltrs ) , a component of genome repeat analysis [ 4951 ] , are sequences of dna that are repeated hundreds or thousands of times in the genome . they are often found in retrotransposons and flanking functional genes [ 51 , 52 ] . ltr retrotransposons constitute a significant portion of most eukaryote genomes and in plants have been suggested to be causative for the dramatic differences of genome sizes ( and polyploidization ) and for disruptions of genome organization and structure [ 2 , 51 , 53 , 54 ] . while insertions of dna have been attributed to amplifications of retrotransposons [ 51 , 5457 ] , deletions have been suggested to involve all dna sequence class types and may be the result of homologous recombination and/or illegitimate recombination . recent analysis of bes from different oryza species found good correlation with flow cytometric genome sizing and repeat content . our preliminary analysis of tea repetitive sequences from pilot bac - end sequences indicated that over 98 percent of the tea genome could be repetitive . we found that ltr retrotransposons are the predominant class of repeat elements in c. sinensis followed by dna retroelements . these results support the correlation of large genome size with proliferation of repeat content since the tea genome is quite large , 4,000 mb , which is 1.6x maize and 0.8x barley whose genomes are approximately 8095 percent repetitive . therefore , it is not surprising that the majority of tea bess contained sequences highly enriched with transposable elements . a high - quality , deep - coverage , hindiii bac library of tea plant ( camellia sinensis ) has been constructed . the library , named csbcba , is publicly available from the arizona genomics institute resource center ( http://www.genome.arizona.edu/orders/ ) . the average insert size of the library was 135 kb , it contained very low organellar contamination , and it provides 13.54x genome equivalent coverage from a total of 401,280 clones . analysis of bac clone end sequences revealed that the repetitive fraction of the tea genome was highly enriched for ltrs and dna tes . this public resource will provide a useful platform for genomics research , such as genome sequencing , dna fingerprinting and physical mapping , gene identification , isolation and regulation , as well as complex analysis of targeted genomic regions . research of genomic information and gene expression patterns of tea plant have advanced slowly probably owing to the lack of an available high - quality bac library as one key genomic tool . since this bac library has been made available to the public , we expect that the most advanced work with dna tools , which has been initiated by other researchers , will move forward for deepening genomics research of tea plant . the specific genes of tea plant , such as the genes involved in the oxidative pathways of catechins by polyphenol oxidase , which are involved in the pathways of the powerful antioxidant epigallocatechin-3-gallate ( egcg ) , may be revealed by use of this resource . previous work on tea plant genetics , such as linkage mapping , genetic integrity of somaclonal variants [ 38 , 41 , 58 , 59 ] , and tea plant functional genes and biopathways [ 39 , 40 , 60 , 61 ] , may be integrated and/or expanded with this library to help unravel the tea plant genome .

we describe the construction and characterization of a publicly available bac library for the tea plant , camellia sinensis . using modified methods , the library was constructed with the aim of developing public molecular resources to advance tea plant genomics research . the library consists of a total of 401,280 clones with an average insert size of 135 kb , providing an approximate coverage of 13.5 haploid genome equivalents . no empty vector clones were observed in a random sampling of 576 bac clones . further analysis of 182 bac - end sequences from randomly selected clones revealed a gc content of 40.35% and low chloroplast and mitochondrial contamination . repetitive sequence analyses indicated that ltr retrotransposons were the most predominant sequence class ( 86.93%87.24% ) , followed by dna retrotransposons ( 11.16%11.69% ) . additionally , we found 25 simple sequence repeats ( ssrs ) that could potentially be used as genetic markers .

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

since 1994 , bacterial artificial chromosome ( bac ) libraries have become an invaluable resource tool to initiate genomics research in theareas of genome sequencing , physical mapping , positional cloning , complex analysis of targeted genomic regions , and analysis of gene structure and function [ 18 ] . bac - end sequencing ( bes ) is a powerful tool that enhances the value of bac libraries as a genomic resource by providing partial sequence information that can be used to understand genome content and architecture and develop genetic markers [ 10 , 11 ] . physical maps constructed from fingerprinted bac clones , together with associated bac - end sequence information , can be used to : construct bac fingerprint-/bes- based physical maps [ 1 , 12 ] which can be aligned to reference genome sequences ; sequence genomes by walking from one clone to the next ; anchor whole genome shotgun sequence data ; integrate genetic linkage maps with physical maps . about 3.9 million tons of tea is produced ( http://faostat.fao.org/site/567/desktopdefault.aspx?pageid=567#ancor ) and consumed yearly with an estimated world value of $ 9.04 billion ( $ 2.39 per kg ; http://www.fao.org/docrep/meeting/019/k8336e.pdf ) . throughout history , it has become well known that tea is one of the most beneficial beverages to drink due to its health attributes . tea polyphenols ( catechins ) and antioxidants , saponin , polysaccharides , l - theanine , pigments , and tea water extracts are the most important components that contribute to tea 's medicinal qualities [ 1619 ] . oolong tea is a special variety of tea and has been studied for its effects on diabetes , eczema , allergies , bacterial infections , dental caries ( cavities ) , obesity , cardiovascular disease , cancer , and its antioxidant properties [ 2126 ] . although rapid progress of gene identification and isolation from tea plants has been made in the past several years , the study of the tea plant genome lags far behind other crop species due to the lack of good genomic research tools . this is most likely due to the difficulties of preparing high - quality tea dna and due to the distinctness of tea plant from other taxa ( i.e. , perennial nature , high inbreeding depression , unavailability of distinct mutants of different biotic and abiotic stress , and large genome size of 4 gb ) . one key genomic tool that is completely lacking for tea is the availability of a high - quality , deep - coverage , bac library . in this paper , we report the construction of a high - quality , publicly available bac library of tea plant from the variety chin - shin oolong . we generated and analyzed a limited data set of bac - end sequences from this library which provided an early glimpse into the sequence composition of the tea genome . sixty 6-year - old plants , derived from a clonally propagated single mother plant of a camellia sinensis cultivar chin - shin oolong , were selected as the plant germplasm source . healthy young shoot tips and the uppermost two leaves were collected ( similar to harvesting top - quality tea ) , then washed quickly to remove debris , and immediately frozen by submersion in liquid nitrogen followed by short - term storage at 80c . however , a detailed manuscript describing the experiments that led to the successful isolation of high - quality , high molecular weight , nuclear dna , suitable for bac library construction , was recently made available . the method for tea plant bac library construction utilized the standard arizona genomics institute ( agi ) protocol , the method of luo and wing , and ammiraju et al . modifications necessary to achieve successful construction of a tea plant bac library are described as follows . the resulting pellet was washed several times with the same buffer and resuspended in 1 ml of prechilled extraction buffer , incubated in a 45c water bath for 5 minutes , and gently mixed with one - third volume of 1.00% low melting temperature agarose ( in extraction buffer ) at 45c . twenty - four plugs were transferred into a 50 ml - falcon tube , containing 40 ml of standard proteinase k solution ( 1.00% w / v n - lauroylsarcosine ( sodium salt ) , 0.1 mg / ml proteinase k , dissolved in 0.5 m edta , ph 9.4 ) , and incubated in a hybridization oven at 50c with a gentle rotation for 24 h. after the plugs were rewashed with fresh proteinase k solution for an additional 24 h , they were rewashed in 2 additional solutions . for each size - selected fraction , the high molecular weight genomic dna was electroeluted from the agarose at 4c . separately , ligations were mixed well by tapping and then incubated in a water bath at 16c for 19 hours . the methods for test transformations , bac dna isolations , bac insert size analyses , bulk transformations , colony arrays , and library characterizations followed exactly the agi protocols ( http://www.genome.arizona.edu/information/publications/wingpub/construct.pdf and http://www2.genome.arizona.edu/pdfs/publications/meizhong_metmb.pdf ) . from each hmw fraction ( b2 , b1 , a2 ) , 2.3 l of each ligation was used to transform 20 l of dh10b t1 phage - resistant e. coli cells ( invitrogen ) by electroporation using the cell porator and voltage booster electroporation system ( life technologies ) . the cells were transferred into 3 ml of soc media , and incubated at 37c for 1 h with shaking at 250 rpm , followed by the addition of an equal volume of sterile glycerol and gentle shaking for 3 minutes . these mixtures were immediately frozen by submersion into liquid nitrogen followed by long - term storage at 80c . five - hundred - seventy - six white recombinants ( positive for insert ) , 192 from each of the three sublibraries ( b2 , b1 , a2 ) , were randomly selected and grown overnight at 37c in 1.2 ml lb broth ( with chloramphenicol 12.5 g / ml ) with shaking at 220 rpm . the white ( insert positive recombinant ) clones were robotically picked ( using a genetix q - bot ; genetix ltd . ) to further evaluate this sub library ( b2 ligation ) , 576 bacs were randomly selected from these forty - eight plates and grown overnight at 37c with shaking at 220 rpm in 1.2 ml lb supplemented with chloramphenicol ( 12.5 g / ml ) . the digested clones were separated by pfge and analyzed . additionally , 192 bac clones were subjected to bac clone end sequencing ( bes ) using the method of kim et al . , and the resultant sequences were analyzed for chloroplast and mitochondrial genome contaminations and for repeat content using blastn searches with settings that required 98% identity over lengths of at least 51 bp . the successful c. sinensis bac library , composed of e. coli transformants from each of the three ligations , was named csbcba . camellia sinensis cultivar chin - shin oolong was chosen to construct the bac library since it is one of the most widely cultivated oolong tea varieties . to avoid contamination with small , trapped dna fragments and improve the size and uniformity of the inserts , the high molecular weight ( hmw ) genomic dna was partially digested with hindiii and three separate size fractionations were collected . following ligations into the hindiii site of the pagibac1 vector , the three size fractionations were transformed , and the new tea bac library , consisting of 401,280 bac clones , was named csbcba ; see table 1 . the overall average insert size of the total library was calculated to be 135 kb with inserts ranging from 8 to 314 kb , and the total library coverage was estimated to equal 13.54 haploid genome equivalents based upon mathematical calculations that utilize a genome size of 4,000 mb . sub library 1 ( ligation b2 ) was composed of 40,320 clones with an average insert size 140 kb ; sub library 2 ( ligation b1 ) was composed of 160,896 clones with an average insert size 140 kb ; sub library 3 ( ligation a2 ) was composed of 200,064 clones with an average insert size 130 kb . five - hundred - seventy - six bacs , 192 from each of the three sublibraries , were randomly selected and analyzed by noti restriction enzyme digestion in order to evaluate the library ( figure 1 ) . based on this sample size were shown to carry dna inserts greater than 130 kb , with a reasonable fraction ( 22.30% ) carrying inserts larger than 170 kb ( figure 2 ) . bac clones from sub library 1 , ligation b2 , were spread on agar dishes , clones ( 18,432 ) were robotically picked and arrayed to 48- 384well plates , and hybridization screening filters were printed to facilitate future experiments . to examine the quality of the library and to obtain a preliminary view of the major repetitive element content of the c. sinensis genome , unidirectional end sequencing was performed on 192 random bac clones which produced 182 reads with an average length of 581 high - quality bases ; the bess are available at the url : http://www2.genome.arizona.edu/files/camellia_bes.txt . of the 182 random bess , the total number of nucleotides was 111,695 bp and the gc level was 40.35% ( table 2 ) . likewise , the 182 random bac - end sequences were evaluated using blast searches and queries to the transposable element databases of arabidopsis ( arabidopsis thaliana ) at genetic information research institute and of rice ( oryza sativa ) at the arizona genomics institute . this preliminary analysis of repetitive sequences from pilot bac - end sequences indicated that 97 to 98 percent of the bess contained sequences related to transposable elements . ltr retrotransposons were the predominant class of repeat elements in c. sinensis ( arabidopsis 86.93% and rice 87.24% ; table 2 ) . the second major class of transposable elements belongs to dna retrotransposon ( arabidopsis 11.16% and rice 11.69% ) . blastn searches against the chloroplast and mitochondrial genomes of arabidopsis ( arabidopsis thaliana ) and rice ( oryza sativa ) using the 182 random bess revealed no significant homology found , thus indicating that the bac library contained very low levels of chloroplast or mitochondrial dna contamination . further repeat analysis of the 182 random bess revealed 25 different microsatellite repeats ( table 3 ) . the 25 simple sequence repeats ( ssrs ) were found from 17 different bac clones ( from the forward and reverse reads ) , thus suggesting that eight bacs represented genomic regions that contained high repeat content . the ultimate goals of these projects are to continue to discover new ways to meet future world agricultural and food needs while simultaneously providing an understanding of functional systems biology . however , the expected dramatic advances in theoretical and applied plant biology for other taxa , following these innovations , have been critically slow due to two major obstacles : lack of adequate genomic tools and resources to efficiently and effectively transfer existing genomic knowledge to other economically and diverse agriculturally important species and lack of representation of novel genes and regulatory networks that underlie key traits of agriculture and ecology in the sequenced model species . , coffee , cocoa ) , a lack of adequate public genomic resources , especially access to large - insert libraries , has contributed to a lack of advanced genetic knowledge useable for modern breeding and improvement . recent published tea plant works involving molecular tools have shown an aflp and rapd marker - based linkage map , identified cdnas involved in secondary metabolism , described sequence analysis from 4320 tea ests , and used intersimple sequence repeats to analyze genetic variability of somaclonal embryo - derived tea plants . a recent review described the use of molecular resources for tea cultivar classification , the identification of parentage of mulberry scale resistance , and more advanced linkage maps with ssr markers . while these and other reports provide important insight to the understanding of the tea plant , the availability and utilization of bac resources are absent . here , we report the construction and public availability of a high - quality , deep - coverage , large - insert , bacterial artificial chromosome ( bac ) library of cultivated tea plant ( camellia sinensis ) variety chin - shin oolong . this bac library resource is publicly available in the form of whole libraries , filters , and individual clones , through the agi bac / est resource center ( http://www.genome.arizona.edu/orders ) , and we expect it to be extensively used worldwide for the analysis of genome evolution and organization , positional cloning , and eventual gap closure of a c. sinensis reference sequence . this tea bac library was made possible after significant methodological improvements were accomplished in the preparation of purified high molecular weight ( hmw ) dna and subsequent partial enzymatic digestions and ligations . we showed that , despite expected difficulties in obtaining hmw dna of reliable quality from tissues known to contain compounds deleterious to established extraction techniques , our results yielded ligations that produced a bac library with low organellar contamination , such as chloroplast and mitochondria , yet with high transformation efficiencies and large - insert sizes . though the genome size of tea is quite large , 4 gb , the described bac library has an average insert size of 135 kb and provides over 13x genome coverage to allow for adequate utility . it was divided into three sublibraries : sub library 1 ( ligation b2 ) composed of 40,320 clones with average insert sizes of 140 kb , sub library 2 ( ligation b1 ) composed of 160,896 clones with average insert sizes of 140 kb , and sub library 3 ( ligation a2 ) composed of 200,064 clones with average insert sizes of 130 kb . tea polyphenols must be prevented from interacting with the nuclear dna , and tea polysaccharides must be prevented from trapping nuclei in the process of tissue homogenization . in this study , to prepare high - quality hmw dna , the frozen tissue was homogenized with a buffer ( composed of combining ingredients from two methods but requiring the pvp-40 to be partially undissolved ) in double volumes followed by additional filtration . the combining of the nuclei with the low melting temperature agarose at a lower ratio of 1 : 3 ( instead of 1 : 1 ) was required to concentrate the nuclei but was performed in the presence of the undissolved pvp-40 . to lower the organellar dna contamination and to further eliminate tea polysaccharides and tea polyphenols in the process of preparing tea plant hmw dna plugs , removing small restriction fragments is vital for construction of a high - quality bac library [ 46 , 47 ] . tea plant genomic plugs prepared by the method used in this paper contained abundant hmw dna and produced satisfactory restriction fragments when digested with hindiii . a detailed manuscript describing the experiments for the appropriate isolation of high - quality , high molecular weight dna that led to the successful construction of this tea plant bac library ( tea plant nuclei isolation , buffer compositions , hmw dna , large - insert ligations , etc . ) pulsed - field gel electrophoresis analysis of 576 random bac clone plasmids showed that the majority of c. sinensis cloned dna inserts were present as single noti fragments . this indicated that the c. sinensis genome apparently contained few noti sites , a feature commonly observed with the genomes of other plant dicot species and contrary to the results obtained with monocot species . long terminal repeats ( ltrs ) , a component of genome repeat analysis [ 4951 ] , are sequences of dna that are repeated hundreds or thousands of times in the genome . ltr retrotransposons constitute a significant portion of most eukaryote genomes and in plants have been suggested to be causative for the dramatic differences of genome sizes ( and polyploidization ) and for disruptions of genome organization and structure [ 2 , 51 , 53 , 54 ] . while insertions of dna have been attributed to amplifications of retrotransposons [ 51 , 5457 ] , deletions have been suggested to involve all dna sequence class types and may be the result of homologous recombination and/or illegitimate recombination . recent analysis of bes from different oryza species found good correlation with flow cytometric genome sizing and repeat content . our preliminary analysis of tea repetitive sequences from pilot bac - end sequences indicated that over 98 percent of the tea genome could be repetitive . we found that ltr retrotransposons are the predominant class of repeat elements in c. sinensis followed by dna retroelements . these results support the correlation of large genome size with proliferation of repeat content since the tea genome is quite large , 4,000 mb , which is 1.6x maize and 0.8x barley whose genomes are approximately 8095 percent repetitive . a high - quality , deep - coverage , hindiii bac library of tea plant ( camellia sinensis ) has been constructed . the library , named csbcba , is publicly available from the arizona genomics institute resource center ( http://www.genome.arizona.edu/orders/ ) . the average insert size of the library was 135 kb , it contained very low organellar contamination , and it provides 13.54x genome equivalent coverage from a total of 401,280 clones . analysis of bac clone end sequences revealed that the repetitive fraction of the tea genome was highly enriched for ltrs and dna tes . this public resource will provide a useful platform for genomics research , such as genome sequencing , dna fingerprinting and physical mapping , gene identification , isolation and regulation , as well as complex analysis of targeted genomic regions . research of genomic information and gene expression patterns of tea plant have advanced slowly probably owing to the lack of an available high - quality bac library as one key genomic tool . since this bac library has been made available to the public , we expect that the most advanced work with dna tools , which has been initiated by other researchers , will move forward for deepening genomics research of tea plant . the specific genes of tea plant , such as the genes involved in the oxidative pathways of catechins by polyphenol oxidase , which are involved in the pathways of the powerful antioxidant epigallocatechin-3-gallate ( egcg ) , may be revealed by use of this resource . previous work on tea plant genetics , such as linkage mapping , genetic integrity of somaclonal variants [ 38 , 41 , 58 , 59 ] , and tea plant functional genes and biopathways [ 39 , 40 , 60 , 61 ] , may be integrated and/or expanded with this library to help unravel the tea plant genome .

the visual cortex has been traditionally considered as a stimulus - driven , unimodal system with a hierarchical organization , in which the early visual areas ( v1 , v2 ) tune to general features while the higher - tier ones ( v3a , v4v , v7 , hmt+ , and v8 ) respond selectively to the specific features of a visual stimulus [ 15 ] . two parallel visual streams have been proposed to generalize the hierarchical organization of the visual processing [ 68 ] . the dorsal stream or where pathway serves to analyze visual spatial information about object location , motion , and visuomotor planning . in this pathway , visual signals are conveyed to the posterior parietal cortex through the dorsal part of the visual cortex ( such as the v3d , v3a , v7 , and hmt+ ) and finally reach the prefrontal cortex . the ventral stream or pathway has been associated with the processing of form , object identity , and color . this pathway conveys visual signals along the ventral part of visual cortex ( such as vp , v4 , and v8 ) , the inferior temporal ( it ) areas , and finally to the prefrontal cortex . the structural and functional organization of the visual areas is supposed to develop through a combination of genetic instruction [ 911 ] and experience - dependent refinement [ 12 , 13 ] . the role of visual experience in the development of the visual areas is supported by a large number of neuroimaging studies revealing that the visual areas of congenitally blind ( cb ) and early blind ( eb ) subjects have increased cortical thickness [ 1417 ] , local brain spontaneous activity , metabolism , and blood flow [ 1922 ] and decreased regional volume [ 2325 ] , white matter integrity [ 26 , 27 ] , anatomical network efficiency [ 28 , 29 ] , and altered resting - state functional connectivity ( rsfc ) [ 30 , 31 ] . moreover , converging evidence suggests that both the early and higher - tier visual areas in cb subjects are recruited during performing a variety of tasks given through nonvisual sensory modalities , as detailed in previous reviews [ 3236 ] . however , the notion of the visual cortex as a unimodal system molded only by visual experience has recently been challenged because the visual cortex of both the sighted controls ( sc ) and the cb responded to a variety of nonvisual perceptive stimuli , including tactile , auditory , and olfactory . furthermore , the visual cortex of the cb was also involved in cognitive processes , such as linguistic processing , working memory , and attention . although the extent and magnitude of the activation in the visual areas depend on the tasks and subjects ' characteristics [ 37 , 38 ] , the coactivation of several visual areas by nonvisual tasks in sc and cb highly indicates that the development of the functional organization of these visual areas does not require visual experience . the main topic of this review is to elucidate how the nonvisual signals recruit the visual cortex . we firstly provide evidence if the visual cortex is supramodal in nature , and then we reviewed how the nonvisual signals reach the visual cortex . finally , we also discussed about the nature ( stimulus - driven or top - down ) of nonvisual signals . unimodal theory supposes that the visual cortex is specifically allocated to process visual stimuli in sighted people ; however , this hypothesis has recently been challenged . using the positron emission tomography ( pet ) , sathian et al . first reported that the extrastriate area close to the parietooccipital fissure ( v6 ) was activated during discrimination of grating orientation compared with discrimination of grating groove width , suggesting this visual area is recruited in the processing spatial information of tactile signals . to further confirm that the occipital area is functionally involved in nonvisual processing , the transcranial magnetic stimulation ( tms ) technique was used to transiently disrupt the functioning of this area . the authors found impaired tactile discrimination of grating orientation after exerting tms on this occipital area and concluded that this occipital area is really functionally involved in tactile spatial perception . since then , many studies have reported the involvement of visual areas in a series of tactile processing , including the hmt+ complex for tactile motion perception [ 37 , 41 ] and the ventral visual pathway for tactile object discrimination [ 4249 ] . it is interesting to note that the hmt+ is capable of processing motion - related information even when the stimulus is delivered to the tongue . furthermore , several studies using both visual and tactile stimuli showed that these two modalities drive the same visual areas for motion and object processing , supporting the cross - modal involvement of visual areas in the abstract representation of the concepts of objects , space , and motion [ 38 , 42 ] . the cross - modal recruitment of the visual cortex has also been reported in auditory domain . as early as 1972 , morrell found that up to 41% of recorded neurons in extrastriate cortex of adult cats were modulated by both visual and auditory stimuli and that the receptive fields for both responses typically overlapped in space . recent studies in humans have also provided evidence of occipital activation in auditory processing [ 52 , 53 ] . for example , the hmt+ complex was activated in sighted subjects while listening to auditory motion stimuli [ 52 , 54 , 55 ] . in accordance with the neuroimaging findings , several tms studies showed that transient disruption of the specific occipital regions can impair the auditory perception in sighted subjects , such as inhibition of the extrastriate cortex induced a systematic error in auditory spatial perception , and disruption of the dorsal extrastriate cortex impaired the sound localization [ 57 , 58 ] . the tms evidence supports that the visual cortex is involved in spatial hearing in sighted subjects . it is also interesting to note that the visual cortex in sighted subjects can not only percept the sound itself but also response to abstract auditory information such as action sounds . relative to the sighted subjects , cross - modal processing of nonvisual signals in the visual cortex has been more extensively reported in the cb and eb subjects when they perform nonvisual perception and high - order cognitive tasks . numerous studies reported that the visual cortex was recruited during diverse tactile tasks , such as the early and higher visual areas were activated in vibrotactile frequency discrimination , the hmt+ in tactile motion perception [ 37 , 50 , 61 ] , and the ventral visual pathway in tactile object perception . the visual cortex is also involved in tactile perception of the tongue [ 42 , 62 , 63 ] . using the tongue display device ( tdu ) , a tactile - to - vision sensory substitution device that translates a visual image into electrotactile stimulation , several studies have shown that the specific visual areas were recruited to process different types of tongue tactile stimuli , such as the ventral stream for tactile - form recognition , the hmt+ complex for tactile motion discrimination , and the ventral lateral occipitotemporal cortex for virtual route recognition . moreover , rtms inhibition of the human hmt+ impaired the tactile speed discrimination , indicating that the recruitment of hmt+ is necessary for tactile motion processing . interestingly , tms stimulation of the visual cortex can induce subjective tongue - tactile sensations in the cb who is proficient at the use of the tdu , which indicates that the perceptual correlate of activity in the visual cortex reflects the characteristics of its novel sensory input source . similar with the tactile perception , the activation of the visual cortex by auditory perception was also frequently reported in cb subjects [ 54 , 65 , 66 ] . in an early pet study , weeks et al . reported that the right dorsal visual cortex was activated by auditory perception task in the cb but not in the sc . this region was also activated in eb subjects by an auditory spatial processing task using a sensory substitution prosthesis translating visual information into sounds . the activation pattern in eb was also confirmed by recent studies [ 66 , 68 , 69 ] . accordingly , the visual areas previously considered to be involved in visual motion processing ( such as the hmt+ ) were specifically recruited in the eb by motion stimuli presented through the auditory modality [ 50 , 54 , 55 , 70 , 71 ] . the ventral pathway can be recruited to process auditory object recognition [ 72 , 73 ] in the cb . in a recent study , striem - amit et al . showed that the dorsal stream processed the location information , whereas the ventral stream responded to shape information via the vision - to - sound substitutes in cb subjects . the dorsal and ventral pathway recruited by auditory perception was further confirmed by electrophysiological and tms evidence . using erp , several groups showed greater amplitude of the n1 component at the visual region in sound localization , suggesting the involvement of the visual cortex in early auditory processing in the eb [ 7577 ] . tms inhibition of the visual area can impair specific auditory performance in the eb [ 57 , 78 , 79 ] . for example , rtms delivered to right dorsal extrastriate cortex disrupted the spatial processing of sounds in the eb [ 57 , 78 ] , and rtms over the loc can impair a eb subject 's ability to identify objects . in the cb , besides auditory and tactile perception , the visual cortex was also recruited in olfactory processing . in this study , a simple odor detection task , the authors found that cb subjects not only showed strong activation in the olfactory cortex , but also showed widespread activation in the visual cortex . combining earlier studies reported that superior olfactory perception in the cb [ 81 , 82 ] ; the recruitment of the visual cortex during odor detection suggests a preferential access of olfactory stimuli to this area in the cb . the visual cortex in eb subjects is not only involved in nonvisual perception , but also takes part in the process of higher - level cognitive tasks , such as language , attention , and working memory . converging evidence supports the involvement of the visual cortex in language processing in the cb or eb subjects . the medial visual cortex was recruited during braille reading [ 8385 ] and the occipitotemporal visual areas were activated during covert verb generation in the eb [ 86 , 87 ] . further studies reveal that the visual cortex is preferentially recruited by semantic relative to phonological processing , and the magnitude of fmri activation is associated with both semantic and syntactic complexity . a recent study showed that the visual word form area ( vwfa ) , a component of the ventral stream that develops expertise for visual reading , can also process braille reading in the eb . further evidence for the involvement of the visual cortex in language processing in the eb has been provided by a combination of task activation and functional connectivity analyses . the authors found that ( 1 ) the responses of the visual regions and classic language regions across conditions were similar ; ( 2 ) language sensitivity was restricted to the left visual cortex ; ( 3 ) the left visual regions that responded to language had increased functional connectivity with classic language regions . besides the neuroimaging findings , in the cb , disruption of the visual cortex by tms or lesions impairs the performance of braille reading and verb generation [ 84 , 9295 ] . besides the language processing , the visual cortex that normally subserves vision is activated in the cb subjects when performing nonvisual attention - demanding tasks , such as spatial attention discrimination [ 32 , 35 , 36 ] . more importantly , the amplitude of the occipital activation in the cb was correlated with the spatial attention performance [ 66 , 96 , 97 ] . these findings suggest that the occipital activation is associated with the enhanced nonvisual attention abilities in the cb . the visual cortex can also be activated by memory task with nonvisual stimuli or without any sensory input [ 98101 ] . the posterior occipital region ( including v1 ) was recruited during a verbal memory task even without real sensory stimulation in the cb , and the activation magnitude of this region was correlated with verbal memory performance . reported that tactile spatial working memory task activated the dorsal extrastriate areas in the cb individuals . moreover , using three different kinds of working memory tasks ( verbal , tactile , and auditory ) , a recent study showed that the visual cortex of the eb subjects responded to all types of stimuli . two neural mechanisms have been proposed to explain the involvement of the visual cortex in the processing of nonvisual stimuli . one hypothesis holds that the visual cortex is supramodal in nature , which means that an occipital area relies on a common , abstract representation of the perceived stimuli irrespective of the sensory modality . another hypothesis is the cross - modal plasticity . in the cb or eb , the visual cortex that normally serves to process visual input shifts to cross - modal process nonvisual information via plastic reorganization of the inner structure and function . however , the two mechanisms are not mutually exclusive , and they might coexist in the eb . as discussed above , many pieces of evidence that showed the involvement of the visual areas in processing nonvisual inputs in both the sc and eb may support the supramodal hypothesis ( see sections 2.1 and 2.2 ) . the cross - modal involvement of the visual cortex in processing nonvisual stimuli means that the functional specialization of the visual areas is task - dependent rather than sensory modality - dependent . furthermore , this pattern can not be fully explained by visual imagery [ 102108 ] because the cb subjects who never have visual experience also show occipital response to the nonvisual stimuli [ 69 , 80 ] . the supramodal hypothesis can explain that visual experience is not necessary to develop the normal functional organization of the visual areas , which has been confirmed in a variety of previous studies on the cb [ 74 , 90 , 109 , 110 ] , because the development of the functional organization may be driven by inputs from other sensory modalities . the cb subjects commonly showed superior performance during auditory or tactile perception than normal sighted subjects [ 97 , 101 , 111115 ] . the superior performance has also been associated with the occipital activation in the cb [ 96 , 97 , 101 ] , suggesting the hypothesis of the cross - modal plasticity . this mechanism can also be applied to explain the involvement of the visual cortex in the higher - level cognitive tasks in the cb but not in the sc [ 84 , 9295 ] . furthermore , the plasticity mechanism may also partly explain the increased cortical thickness [ 1417 ] , local brain spontaneous activity , metabolism and blood flow [ 1922 ] , and rsfc [ 30 , 31 ] in the eb . studies on the functional characteristics of the hmt+ have well described the coexistence of the two mechanisms in the eb . in sighted subjects , the hmt+ complex is segregated into an anterior part ( supramodal region ) , that is , involved in processing both visual and tactile motion , and a posterior part ( unimodal region ) , which is only involved in processing visual information . in the eb , however , the entire hmt+ is involved in the representation of tactile motion , suggesting the coexistence of the supramodal ( anterior part ) and plastic ( posterior part ) mechanisms in this region . these results represent competitive interactions between visual and nonvisual inputs in the reshape of hmt+ complex [ 37 , 116 ] . it should be noted that the improved nonvisual perception performance in the early blind subjects can also be the consequences of the experience - dependent plasticity of their auditory or somatosensory related cortices . for example , the mice that are binocularly enucleated from birth demonstrate remarkable expansion of their barrel cortex , which may be interpreted by increased usage of the whiskers after visual deprivation . cats that were deprived of vision from birth also show expanded primary somatosensory and auditory areas ; furthermore , the neurons in the anterior ectosylvian visual area ( aev ) that normally respond to visual stimuli are replaced by neurons of neighboring auditory ectosylvian area ( aea ) , which is accompanied by the improvement of auditory spatial tuning of this region than sighted controls [ 118 , 119 ] . as a result , the improved auditory / tactile perceptive performance might both be caused by the experience - dependent plasticity of the classic auditory / tactile regions with expanded cortical area , and by classic visual regions that turn to subserve the nonvisual information . caution should be paid that some cortical regions such as the aev in the congenitally blind subjects are actually replaced by expanded auditory cortices and their activation by auditory stimuli can not be interpreted as cross - modal involvement of visual areas any more . as discussed above , much evidence supports the cross - modal processing of nonvisual signals in the visual cortex . it is then important to understand how the nonvisual sensory information reaches the occipital areas , especially in the cb subjects who have no visual experience about the external world . the candidate pathways include the thalamooccipital and corticooccipital pathway , which is categorized in figures 1 and 2 . the thalamus is an important relay that receives afferents from different sensory organs and sends efferents to the primary sensory cortex . under normal condition , the thalamic nuclei are relay stations via which sensory information from the peripheral sensory receptors can reach the primary sensory cortex . for example , the lateral geniculate nucleus ( lgn ) , the visual thalamic relay , mainly transfers visual signals to the primary visual cortex ( v1 ) ; the medial geniculate nucleus ( mgn ) is the auditory thalamic relay that connects the inferior colliculus ( ic ) and the primary auditory cortex ( a1 ) ; and the ventral posterior nuclei ( vp ) is the somatosensory thalamic relay that receives tactile signals and transmits them to the primary somatosensory cortex ( s1 ) [ 120 , 121 ] . furthermore , the thalamus can also receive feedback signals from the sensory cortex [ 122125 ] and the associate cortex [ 126132 ] to modulate the input signals . it is hypothesized that nonvisual signals may bypass the traditional sensory pathway and rewire into the visual thalamus and project to the v1 . this hypothesis is supported by the findings that the lgn receives rewired auditory projections from the inferior colliculus [ 133139 ] and the mgn , receives rewired somatosensory projections from the vp [ 140 , 141 ] , and then projects efferent fibers to the visual areas in enucleated animals since birth . recent findings have shown that the lgn and its output projections to the v1 are atrophied in early blind subjects [ 24 , 26 , 27 ] , which seems to contradict with this hypothesis . one putative explanation is that the atrophy of the lgn and the retinofugal pathway is the consequence of the interaction between disused neurodegeneration of the visual part of the pathway and cross - modal plasticity of the rewired nonvisual part of the pathway . in fact , pervious animal experiments have shown that the visual projections to the dorsal lateral geniculate nucleus are dramatically reduced in blind animals , while the auditory projections to the same region are strengthened [ 133 , 142 ] . another possibility is that the nonvisual signals bypass traditional retinofugal pathway ( from the lgn to the v1 ) and pass through the pulvinar - occipital pathway [ 143146 ] , or from the lgn to the higher visual areas ( such as the hmt+ and v4 ) [ 147150 ] . in agreement with this interpretation , a recent fmri study in monkeys demonstrates that direct lgn projections to the extrastriate cortex have a critical functional contribution to blindsight with v1 lesions ; a human study shows a direct anatomical connection between the thalamus and the hmt+ complex , that would directly convey motion information to the hmt+ , thereby bypassing the v1 . an alternative pathway is that the visual areas receive nonvisual sensory information through corticooccipital connections between these sensory modalities [ 140 , 152 , 153 ] . these corticooccipital connections can be further subdivided into direct corticooccipital connections and indirect polysynaptic corticooccipital connections . the former has been found between the a1 and visual cortex in adult mongolian gerbils , cats , primates [ 153 , 156 , 157 ] , humans , and congenitally blind opossums , and between s1 and v1 in enucleated opossums . the latter has been indicated by studies showing multisensory processing in the association cortices , such as the posterior parietal area ( ppa ) [ 160 , 161 ] , superior temporal sulcus ( sts ) [ 162164 ] , ventral lateral prefrontal cortex ( vlpfc ) [ 165167 ] , and extrastriate areas [ 155 , 168 , 169 ] . the dense anatomical connections between multisensory areas and both the visual and nonvisual sensory cortices have been identified in both animal and human studies [ 155 , 159 , 163 , 170 , 171 ] . the corticooccipital pathway hypothesis is also supported by task - based fmri studies [ 165 , 172 ] and a resting - state fmri study in sighted subjects , an effective connectivity study , and tms studies in sighted and eb subjects [ 23 , 78 , 175 , 176 ] . in a recent study by our group , we found the rsfc between the early visual areas and s1 was dramatically decreased , while those between the higher - tier visual areas and s1 , and between the early and higher - tier visual areas , were relatively preserved or even strengthened in the cb . our findings support the hypothesis of the indirect corticooccipital pathway mediating nonvisual sensory information to the early visual areas via the relay of higher - tier ones . it should be noted that these two pathways can not be absolutely segregated in the brain network . they are interacted with each other by feed forward and feed back projections . for example , the auditory signals can first feed forward to the a1 for initial processing , and then feed back to the mgn and multimodal thalamic nuclei ( e.g. , the pulvinar ) , and finally project to the v1 ( cortico - thalamooccipital pathway ) . the multisensory areas such as the ppa and vlpfc also project efferents to the multimodal thalamus ( the pulvinar and medial dorsal nucleus ) [ 127131 ] , so they can convey the modulated nonvisual signals to the occipital area via the thalamus ( figure 1 ) . the existing thalamo - cortical and corticooccipital pathways found in the normal adult animals and humans provide anatomical evidence of the supramodal nature of the visual cortex . auditory and tactile information this anatomical connections pattern can explain the cross - modal involvement of visual cortex by nonvisual sensory tasks ; furthermore , it can explain the development of a portion of the visual areas does not depend on the visual experience because inputs from other sensory modalities are sufficient to support the development of these functional patterns . two competing hypotheses have been proposed to explain the neural mechanisms of cross - modal plasticity after early visual deprivation . according to the rewiring hypothesis , cross - modal brain responses are mediated by the formation of new pathways in the sensory deprived brain . for example , after experimental destruction of the superior colliculi and the visual cortex in neonatal hamsters , the authors observed a strong projection from the retina to the a1 , which can perceive the visual information [ 178 , 179 ] . studies in animals have also shown that when the brain is deprived of peripheral visual input at an early age , auditory inputs are re - routed to the visual cortex via the thalamooccipital pathway [ 133 , 139141 ] . however , this subcortical rewired pathway is questioned because of the lack of in vivo evidence . in contrast , there are considerable studies showed that the whole segments of retinofugal pathway , including the optic tract , the lgn , and optic radiation , suffered atrophy and loss of integrity in humans after early visual deprivation [ 16 , 23 , 27 ] . the unmasking hypothesis proposed that the loss of a sensory input induces unmasking and/or strengthening of the existing neural pathways . as discussed in sections 3.1 and 3.2 , the tactile and auditory inputs can be conveyed to the visual cortex via the existing thalamooccipital pathway or corticooccipital pathway that have been confirmed in normal adult animals and humans . generally , these nonvisual signals can modulate the processing of visual information in sighted subjects ; however , they can not induce subjective nonvisual sensations and occipital activation due to being masked by the dominant visual input [ 40 , 64 , 176 ] . the occasional findings of occipital processing nonvisual signals might be task - dependent that dramatically reduced the masking effects of the visual input [ 40 , 52 , 55 ] . however , after early visual deprivation , nonvisual processing in the visual cortex is strengthened or unmasked because of the lack of visual input . the unmasking hypothesis is also supported by the cross - modal responses after short - term visual deprivation ( blindfolding ) . several hours to days blindfolding resulted in rapid , reversible improvement in task performance and recruitment of the visual cortex in nonvisual processing , such as tactile discrimination [ 181 , 182 ] , braille reading , and sound localization [ 184 , 185 ] . rapid cross - modal responses exclude the possibility that these are mediated by the establishment of new anatomical connections . this claim was also supported by sensory substitution devices ( ssd ) studies that the visual cortex was also involved in processing nonvisual tasks after a short period of ssd training in sighted subject [ 50 , 72 , 186 ] . it is possible that a short period of blindfolding and ssd training unmasks and strengthens pre - existing connections between the nonvisual and the occipital cortices . as shown in figure 1 , nonvisual signals can reach the visual cortex via thalamooccipital pathway , corticooccipital pathway , or combinations of these two pathways . an important but unsolved question is the nature of these nonvisual signals : stimulus - driven or top - down . the stimulus - driven signals refer to those from sensory organs or early sensory cortex , whereas the top - down signals are refer to those who came from the higher - level cortical regions . clarifying this question can help us to understand the neural mechanisms underlying cross - modal recruitment of the visual cortex and to design appropriate interventions to improve the adaptive capacity of blind subjects to the external environments . the following evidence supports stimulus - driven nature of the nonvisual signals that reach the visual cortex . these nonvisual signals can be conveyed from sensory organs to the visual cortex via rewired thalamic - cortical pathway [ 133139 ] and those from the nonvisual primary sensory cortices via the direct corticooccipital connections ( such as from a1 to v1 ) [ 153 , 156 , 157 ] , which bypass the higher - tier cognitive cortex , so the inputted nonvisual signals may not be modulated and reflect the pure stimulus - driven information . the involvement of the visual cortex in nonvisual perception in both sighted [ 45 , 46 , 72 ] and eb [ 37 , 42 , 50 , 6163 , 66 , 68 , 69 , 80 ] subjects also suggests the stimulus - driven nature of these nonvisual signals because the top - down effects such as visual imagery and attention were well controlled in these studies . the event - related potentials ( erps ) studies demonstrated that the n1 component of occipital response following nonvisual stimulation was as early as the typical component of the visual perception in eb subjects [ 76 , 77 , 187 ] . for example , a recent report showed that the shape - selective activity in the loc was present as early as 150 ms following the onset of tactile stimulation , which support the stimulus - driven somatosensory input to the loc . according to the top - down mechanism , the peripheral auditory / tactile signals are firstly modulated and refined by the higher - level cortical regions , such as the multisensory associate areas ( vlpfc , ppa , and sts ) , and then feed back to the visual cortex through the indirect corticooccipital pathway and cortico - thalamooccipital pathway ( figure 1 ) . the existing anatomical feedback projections from higher - level cortical regions to the thalamus and visual cortex support this top - down mechanism [ 132 , 155 , 159 , 163 , 170 , 171 ] . furthermore , many task - evoked and lesion studies have demonstrated the modulation effects of the fronto - parietal network on the thalamus and visual cortex [ 165 , 188191 ] . it seems that in cb subjects , the top - down attention modulation of the occipital activity was strengthened [ 75 , 96 , 192195 ] . visual imagery , a complex mental process , can also activate the specific visual areas that are usually recruited by certain visual properties ( shape , space , and color ) , which supports the top - down controlling the visual activities [ 102108 ] . it should be noted that the earlier erp response can not exclude the effects of top - down modulation , because top - down attention can also module the early components of erp , for example , the n1 negativity or even earlier peak [ 196 , 197 ] . furthermore , a erp study demonstrated that the top - down attention modulation of the occipital activity was significantly strengthened in eb subjects . the following paragraphs state the two popular cognitive processes ( visual imagery and attention ) that might contribute to the top - down recruitment of the visual cortex . visual imagery mechanism supports the hypothesis of top - down recruitment of the visual cortex because the mental progress can recruit the visual cortex [ 102108 ] . for example , kosslyn et al . showed that the visual areas 17 and 18/19 were activated by a stripe imagery task , and rtms delivered to area 17 did disrupt both visual perception and imagery performance . this study indicates that the early visual areas are involved in at least some forms of visual imagery as well as in visual perception . it is interesting to noted that not only the early visual area , but also the higher - tier ones can specifically respond to the visual imagery task , including the ventral stream for shape imagery [ 47 , 198200 ] , and the dorsal stream for motion and spatial imagery [ 107 , 108 , 201203 ] , which highly corresponded with the hierarchy representation of the visual perception . de volder showed that in both sighted and eb subjects , auditory triggered mental imagery of shape can also activate the ventral occipitotemporal and visual association areas . top - down attention may be the most extensively studied cognitive process that can modulate the activation of the visual cortex [ 204208 ] . it is proposed that visual attention modulates visual processing even at an early stage ; it not only modulates the gain on incoming visual information , but also adds a pure top - down signal that increases baseline activity in the visual cortex ; moreover , attentional modulation can exert on different aspects of visual perception , such as locations , features , objects , or a combination . furthermore , not only the visual attention , but also the nonvisual attention can also activate the visual cortex [ 207 , 209211 ] . in the cb subjects , electrophysiological or neuroimaging studies have revealed that the top - down attention modulation was strengthened when they performing tactile / auditory attention - demanding tasks [ 75 , 111 , 112 , 212215 ] . additionally , the occipital cortical areas were activated in the cb subjects by attention tasks through nonvisual modalities [ 60 , 96 , 194 , 216 , 217 ] . in combination with evidence of attention modulation on visual perception in the sc [ 218220 ] , these findings indicate increased top - down attention modulation of occipital activity in the cb . in summary , the nature of the nonvisual signals to the visual cortex may be either stimulus - driven or top - down , or both . indeed , effective connectivity analysis offers evidence for the coexistence of both bottom - up and top - down information flows during nonvisual perception [ 221223 ] . the cross - modal processing of nonvisual signals in the occipital areas in both sighted and eb subjects suggests that the functional organization of the visual cortex is supramodal in nature . the cross - modal plasticity can also account for parts of the findings in the eb subjects . the normally existing thalamo - cortical and corticooccipital pathways provide anatomical evidence for the supramodal nature of the visual cortex . the cross - modal plasticity in the eb might be driven by two neural mechanisms : rewiring and unmasking , although there is lack of in vivo evidence for the former . further studies with more advanced in vivo imaging techniques should be implemented to clarify this issue . finally , the nature of the nonvisual signals to the visual cortex may be either stimulus - driven or top - down , or both . further understanding the issue may help us to design appropriate interventions to improve the adaptive capacity of blind subjects to the external environments .

the visual cortex has been traditionally considered as a stimulus - driven , unimodal system with a hierarchical organization . however , recent animal and human studies have shown that the visual cortex responds to non - visual stimuli , especially in individuals with visual deprivation congenitally , indicating the supramodal nature of the functional representation in the visual cortex . to understand the neural substrates of the cross - modal processing of the non - visual signals in the visual cortex , we firstly showed the supramodal nature of the visual cortex . we then reviewed how the nonvisual signals reach the visual cortex . moreover , we discussed if these non - visual pathways are reshaped by early visual deprivation . finally , the open question about the nature ( stimulus - driven or top - down ) of non - visual signals is also discussed .

1. Introduction 2. The Supramodal Nature of Visual Cortex 3. The Candidate Pathways That Nonvisual Signals Reach the Visual Cortex 4. Rewiring versus Unmasking of the Nonvisual Pathway after Visual Deprivation 5. Stimulus-Driven or Top-Down Control of the Nonvisual Processing in the Visual Cortex 6. Conclusions

the visual cortex has been traditionally considered as a stimulus - driven , unimodal system with a hierarchical organization , in which the early visual areas ( v1 , v2 ) tune to general features while the higher - tier ones ( v3a , v4v , v7 , hmt+ , and v8 ) respond selectively to the specific features of a visual stimulus [ 15 ] . two parallel visual streams have been proposed to generalize the hierarchical organization of the visual processing [ 68 ] . in this pathway , visual signals are conveyed to the posterior parietal cortex through the dorsal part of the visual cortex ( such as the v3d , v3a , v7 , and hmt+ ) and finally reach the prefrontal cortex . this pathway conveys visual signals along the ventral part of visual cortex ( such as vp , v4 , and v8 ) , the inferior temporal ( it ) areas , and finally to the prefrontal cortex . the role of visual experience in the development of the visual areas is supported by a large number of neuroimaging studies revealing that the visual areas of congenitally blind ( cb ) and early blind ( eb ) subjects have increased cortical thickness [ 1417 ] , local brain spontaneous activity , metabolism , and blood flow [ 1922 ] and decreased regional volume [ 2325 ] , white matter integrity [ 26 , 27 ] , anatomical network efficiency [ 28 , 29 ] , and altered resting - state functional connectivity ( rsfc ) [ 30 , 31 ] . however , the notion of the visual cortex as a unimodal system molded only by visual experience has recently been challenged because the visual cortex of both the sighted controls ( sc ) and the cb responded to a variety of nonvisual perceptive stimuli , including tactile , auditory , and olfactory . furthermore , the visual cortex of the cb was also involved in cognitive processes , such as linguistic processing , working memory , and attention . although the extent and magnitude of the activation in the visual areas depend on the tasks and subjects ' characteristics [ 37 , 38 ] , the coactivation of several visual areas by nonvisual tasks in sc and cb highly indicates that the development of the functional organization of these visual areas does not require visual experience . the main topic of this review is to elucidate how the nonvisual signals recruit the visual cortex . we firstly provide evidence if the visual cortex is supramodal in nature , and then we reviewed how the nonvisual signals reach the visual cortex . finally , we also discussed about the nature ( stimulus - driven or top - down ) of nonvisual signals . unimodal theory supposes that the visual cortex is specifically allocated to process visual stimuli in sighted people ; however , this hypothesis has recently been challenged . furthermore , several studies using both visual and tactile stimuli showed that these two modalities drive the same visual areas for motion and object processing , supporting the cross - modal involvement of visual areas in the abstract representation of the concepts of objects , space , and motion [ 38 , 42 ] . the cross - modal recruitment of the visual cortex has also been reported in auditory domain . it is also interesting to note that the visual cortex in sighted subjects can not only percept the sound itself but also response to abstract auditory information such as action sounds . relative to the sighted subjects , cross - modal processing of nonvisual signals in the visual cortex has been more extensively reported in the cb and eb subjects when they perform nonvisual perception and high - order cognitive tasks . numerous studies reported that the visual cortex was recruited during diverse tactile tasks , such as the early and higher visual areas were activated in vibrotactile frequency discrimination , the hmt+ in tactile motion perception [ 37 , 50 , 61 ] , and the ventral visual pathway in tactile object perception . the visual cortex is also involved in tactile perception of the tongue [ 42 , 62 , 63 ] . using the tongue display device ( tdu ) , a tactile - to - vision sensory substitution device that translates a visual image into electrotactile stimulation , several studies have shown that the specific visual areas were recruited to process different types of tongue tactile stimuli , such as the ventral stream for tactile - form recognition , the hmt+ complex for tactile motion discrimination , and the ventral lateral occipitotemporal cortex for virtual route recognition . moreover , rtms inhibition of the human hmt+ impaired the tactile speed discrimination , indicating that the recruitment of hmt+ is necessary for tactile motion processing . interestingly , tms stimulation of the visual cortex can induce subjective tongue - tactile sensations in the cb who is proficient at the use of the tdu , which indicates that the perceptual correlate of activity in the visual cortex reflects the characteristics of its novel sensory input source . similar with the tactile perception , the activation of the visual cortex by auditory perception was also frequently reported in cb subjects [ 54 , 65 , 66 ] . reported that the right dorsal visual cortex was activated by auditory perception task in the cb but not in the sc . using erp , several groups showed greater amplitude of the n1 component at the visual region in sound localization , suggesting the involvement of the visual cortex in early auditory processing in the eb [ 7577 ] . tms inhibition of the visual area can impair specific auditory performance in the eb [ 57 , 78 , 79 ] . in the cb , besides auditory and tactile perception , the visual cortex was also recruited in olfactory processing . in this study , a simple odor detection task , the authors found that cb subjects not only showed strong activation in the olfactory cortex , but also showed widespread activation in the visual cortex . combining earlier studies reported that superior olfactory perception in the cb [ 81 , 82 ] ; the recruitment of the visual cortex during odor detection suggests a preferential access of olfactory stimuli to this area in the cb . the visual cortex in eb subjects is not only involved in nonvisual perception , but also takes part in the process of higher - level cognitive tasks , such as language , attention , and working memory . converging evidence supports the involvement of the visual cortex in language processing in the cb or eb subjects . a recent study showed that the visual word form area ( vwfa ) , a component of the ventral stream that develops expertise for visual reading , can also process braille reading in the eb . further evidence for the involvement of the visual cortex in language processing in the eb has been provided by a combination of task activation and functional connectivity analyses . the authors found that ( 1 ) the responses of the visual regions and classic language regions across conditions were similar ; ( 2 ) language sensitivity was restricted to the left visual cortex ; ( 3 ) the left visual regions that responded to language had increased functional connectivity with classic language regions . besides the neuroimaging findings , in the cb , disruption of the visual cortex by tms or lesions impairs the performance of braille reading and verb generation [ 84 , 9295 ] . besides the language processing , the visual cortex that normally subserves vision is activated in the cb subjects when performing nonvisual attention - demanding tasks , such as spatial attention discrimination [ 32 , 35 , 36 ] . moreover , using three different kinds of working memory tasks ( verbal , tactile , and auditory ) , a recent study showed that the visual cortex of the eb subjects responded to all types of stimuli . two neural mechanisms have been proposed to explain the involvement of the visual cortex in the processing of nonvisual stimuli . one hypothesis holds that the visual cortex is supramodal in nature , which means that an occipital area relies on a common , abstract representation of the perceived stimuli irrespective of the sensory modality . another hypothesis is the cross - modal plasticity . in the cb or eb , the visual cortex that normally serves to process visual input shifts to cross - modal process nonvisual information via plastic reorganization of the inner structure and function . as discussed above , many pieces of evidence that showed the involvement of the visual areas in processing nonvisual inputs in both the sc and eb may support the supramodal hypothesis ( see sections 2.1 and 2.2 ) . the cross - modal involvement of the visual cortex in processing nonvisual stimuli means that the functional specialization of the visual areas is task - dependent rather than sensory modality - dependent . the supramodal hypothesis can explain that visual experience is not necessary to develop the normal functional organization of the visual areas , which has been confirmed in a variety of previous studies on the cb [ 74 , 90 , 109 , 110 ] , because the development of the functional organization may be driven by inputs from other sensory modalities . the superior performance has also been associated with the occipital activation in the cb [ 96 , 97 , 101 ] , suggesting the hypothesis of the cross - modal plasticity . this mechanism can also be applied to explain the involvement of the visual cortex in the higher - level cognitive tasks in the cb but not in the sc [ 84 , 9295 ] . in the eb , however , the entire hmt+ is involved in the representation of tactile motion , suggesting the coexistence of the supramodal ( anterior part ) and plastic ( posterior part ) mechanisms in this region . it should be noted that the improved nonvisual perception performance in the early blind subjects can also be the consequences of the experience - dependent plasticity of their auditory or somatosensory related cortices . for example , the mice that are binocularly enucleated from birth demonstrate remarkable expansion of their barrel cortex , which may be interpreted by increased usage of the whiskers after visual deprivation . cats that were deprived of vision from birth also show expanded primary somatosensory and auditory areas ; furthermore , the neurons in the anterior ectosylvian visual area ( aev ) that normally respond to visual stimuli are replaced by neurons of neighboring auditory ectosylvian area ( aea ) , which is accompanied by the improvement of auditory spatial tuning of this region than sighted controls [ 118 , 119 ] . as a result , the improved auditory / tactile perceptive performance might both be caused by the experience - dependent plasticity of the classic auditory / tactile regions with expanded cortical area , and by classic visual regions that turn to subserve the nonvisual information . as discussed above , much evidence supports the cross - modal processing of nonvisual signals in the visual cortex . it is then important to understand how the nonvisual sensory information reaches the occipital areas , especially in the cb subjects who have no visual experience about the external world . for example , the lateral geniculate nucleus ( lgn ) , the visual thalamic relay , mainly transfers visual signals to the primary visual cortex ( v1 ) ; the medial geniculate nucleus ( mgn ) is the auditory thalamic relay that connects the inferior colliculus ( ic ) and the primary auditory cortex ( a1 ) ; and the ventral posterior nuclei ( vp ) is the somatosensory thalamic relay that receives tactile signals and transmits them to the primary somatosensory cortex ( s1 ) [ 120 , 121 ] . recent findings have shown that the lgn and its output projections to the v1 are atrophied in early blind subjects [ 24 , 26 , 27 ] , which seems to contradict with this hypothesis . one putative explanation is that the atrophy of the lgn and the retinofugal pathway is the consequence of the interaction between disused neurodegeneration of the visual part of the pathway and cross - modal plasticity of the rewired nonvisual part of the pathway . in fact , pervious animal experiments have shown that the visual projections to the dorsal lateral geniculate nucleus are dramatically reduced in blind animals , while the auditory projections to the same region are strengthened [ 133 , 142 ] . another possibility is that the nonvisual signals bypass traditional retinofugal pathway ( from the lgn to the v1 ) and pass through the pulvinar - occipital pathway [ 143146 ] , or from the lgn to the higher visual areas ( such as the hmt+ and v4 ) [ 147150 ] . the dense anatomical connections between multisensory areas and both the visual and nonvisual sensory cortices have been identified in both animal and human studies [ 155 , 159 , 163 , 170 , 171 ] . in a recent study by our group , we found the rsfc between the early visual areas and s1 was dramatically decreased , while those between the higher - tier visual areas and s1 , and between the early and higher - tier visual areas , were relatively preserved or even strengthened in the cb . the existing thalamo - cortical and corticooccipital pathways found in the normal adult animals and humans provide anatomical evidence of the supramodal nature of the visual cortex . auditory and tactile information this anatomical connections pattern can explain the cross - modal involvement of visual cortex by nonvisual sensory tasks ; furthermore , it can explain the development of a portion of the visual areas does not depend on the visual experience because inputs from other sensory modalities are sufficient to support the development of these functional patterns . two competing hypotheses have been proposed to explain the neural mechanisms of cross - modal plasticity after early visual deprivation . for example , after experimental destruction of the superior colliculi and the visual cortex in neonatal hamsters , the authors observed a strong projection from the retina to the a1 , which can perceive the visual information [ 178 , 179 ] . studies in animals have also shown that when the brain is deprived of peripheral visual input at an early age , auditory inputs are re - routed to the visual cortex via the thalamooccipital pathway [ 133 , 139141 ] . in contrast , there are considerable studies showed that the whole segments of retinofugal pathway , including the optic tract , the lgn , and optic radiation , suffered atrophy and loss of integrity in humans after early visual deprivation [ 16 , 23 , 27 ] . as discussed in sections 3.1 and 3.2 , the tactile and auditory inputs can be conveyed to the visual cortex via the existing thalamooccipital pathway or corticooccipital pathway that have been confirmed in normal adult animals and humans . however , after early visual deprivation , nonvisual processing in the visual cortex is strengthened or unmasked because of the lack of visual input . the unmasking hypothesis is also supported by the cross - modal responses after short - term visual deprivation ( blindfolding ) . several hours to days blindfolding resulted in rapid , reversible improvement in task performance and recruitment of the visual cortex in nonvisual processing , such as tactile discrimination [ 181 , 182 ] , braille reading , and sound localization [ 184 , 185 ] . as shown in figure 1 , nonvisual signals can reach the visual cortex via thalamooccipital pathway , corticooccipital pathway , or combinations of these two pathways . an important but unsolved question is the nature of these nonvisual signals : stimulus - driven or top - down . the stimulus - driven signals refer to those from sensory organs or early sensory cortex , whereas the top - down signals are refer to those who came from the higher - level cortical regions . clarifying this question can help us to understand the neural mechanisms underlying cross - modal recruitment of the visual cortex and to design appropriate interventions to improve the adaptive capacity of blind subjects to the external environments . the following evidence supports stimulus - driven nature of the nonvisual signals that reach the visual cortex . these nonvisual signals can be conveyed from sensory organs to the visual cortex via rewired thalamic - cortical pathway [ 133139 ] and those from the nonvisual primary sensory cortices via the direct corticooccipital connections ( such as from a1 to v1 ) [ 153 , 156 , 157 ] , which bypass the higher - tier cognitive cortex , so the inputted nonvisual signals may not be modulated and reflect the pure stimulus - driven information . the involvement of the visual cortex in nonvisual perception in both sighted [ 45 , 46 , 72 ] and eb [ 37 , 42 , 50 , 6163 , 66 , 68 , 69 , 80 ] subjects also suggests the stimulus - driven nature of these nonvisual signals because the top - down effects such as visual imagery and attention were well controlled in these studies . the event - related potentials ( erps ) studies demonstrated that the n1 component of occipital response following nonvisual stimulation was as early as the typical component of the visual perception in eb subjects [ 76 , 77 , 187 ] . for example , a recent report showed that the shape - selective activity in the loc was present as early as 150 ms following the onset of tactile stimulation , which support the stimulus - driven somatosensory input to the loc . according to the top - down mechanism , the peripheral auditory / tactile signals are firstly modulated and refined by the higher - level cortical regions , such as the multisensory associate areas ( vlpfc , ppa , and sts ) , and then feed back to the visual cortex through the indirect corticooccipital pathway and cortico - thalamooccipital pathway ( figure 1 ) . furthermore , many task - evoked and lesion studies have demonstrated the modulation effects of the fronto - parietal network on the thalamus and visual cortex [ 165 , 188191 ] . it seems that in cb subjects , the top - down attention modulation of the occipital activity was strengthened [ 75 , 96 , 192195 ] . visual imagery , a complex mental process , can also activate the specific visual areas that are usually recruited by certain visual properties ( shape , space , and color ) , which supports the top - down controlling the visual activities [ 102108 ] . it should be noted that the earlier erp response can not exclude the effects of top - down modulation , because top - down attention can also module the early components of erp , for example , the n1 negativity or even earlier peak [ 196 , 197 ] . furthermore , a erp study demonstrated that the top - down attention modulation of the occipital activity was significantly strengthened in eb subjects . the following paragraphs state the two popular cognitive processes ( visual imagery and attention ) that might contribute to the top - down recruitment of the visual cortex . visual imagery mechanism supports the hypothesis of top - down recruitment of the visual cortex because the mental progress can recruit the visual cortex [ 102108 ] . top - down attention may be the most extensively studied cognitive process that can modulate the activation of the visual cortex [ 204208 ] . it is proposed that visual attention modulates visual processing even at an early stage ; it not only modulates the gain on incoming visual information , but also adds a pure top - down signal that increases baseline activity in the visual cortex ; moreover , attentional modulation can exert on different aspects of visual perception , such as locations , features , objects , or a combination . in the cb subjects , electrophysiological or neuroimaging studies have revealed that the top - down attention modulation was strengthened when they performing tactile / auditory attention - demanding tasks [ 75 , 111 , 112 , 212215 ] . in summary , the nature of the nonvisual signals to the visual cortex may be either stimulus - driven or top - down , or both . the cross - modal processing of nonvisual signals in the occipital areas in both sighted and eb subjects suggests that the functional organization of the visual cortex is supramodal in nature . the cross - modal plasticity can also account for parts of the findings in the eb subjects . the normally existing thalamo - cortical and corticooccipital pathways provide anatomical evidence for the supramodal nature of the visual cortex . the cross - modal plasticity in the eb might be driven by two neural mechanisms : rewiring and unmasking , although there is lack of in vivo evidence for the former . finally , the nature of the nonvisual signals to the visual cortex may be either stimulus - driven or top - down , or both .

life - threatening emergencies can happen at any school , at any time . in december of 2008 , the kuwaiti press reported the tragic death of a 16-year - old boy , following an epileptic seizure due to lack of first - aid seizure management and trained professionals within the school premises , teachers precisely . had those teachers simply known more about epilepsy and had there been better attitudes toward students with epilepsy , this boy could have been saved . this incident motivated us to undertake this study in order to raise awareness about epilepsy among school teachers and promote their attitudes toward students with epilepsy . epilepsy is one of the most common neurological problems of childhood , with a worldwide prevalence of 510 per 1000 people [ 1 , 2 ] . remarkably , about 3% of people will be diagnosed with epilepsy at some time in their life . studies have revealed that being labeled with epilepsy has a major effect on children , school children specifically , both academically and psychosocially . children suffering from epilepsy are often stigmatized because of fear of unexpected and public loss of self - control . moreover , children with epilepsy are at an increased risk for a number of education - related problems such as educational underachievement , learning disabilities , mental health problems , and social isolation . sometimes , social attitude and discrimination against children with epilepsy are more devastating and harmful than the disease itself . knowledge about epilepsy is an important issue in determining teachers ' attitudes toward children with epilepsy . in general , teachers do not receive any formal instructions on epilepsy during their education and training despite the fact that as much as 40% of the children 's developing life is spent at school . in kuwait , teachers are considered as social leaders and role models thus influencing the child 's critical period of social and psychological development . for that reason , studying teachers ' knowledge about epilepsy is beneficial for promoting our future generations . teachers ' attitude toward epilepsy is their predisposition or tendency to respond positively or negatively toward various issues related to students with epilepsy . these attitudes influence their choice of action and responses to challenges , incentives , and rewards . although attitude is a complex and abstract construct , recent studies have demonstrated the manner in which teachers ' attitudes may be translated into behaviors that can have problematic results for students with epilepsy . teachers ' perception of and approach to these students with epilepsy varies with the accuracy of their knowledge , which is often inadequate , limited , or even erroneous . kankirawatana conducted a survey of 360 schools in thailand in 1999 and reported that 15% of the respondents preferred to place all children with epilepsy in a special classroom . this preference may result from fear of handling the seizure of a student in the class . furthermore , half of the respondents who had experience with first - aid management of seizures used improper and potentially harmful measures . kankirawatana also concluded that , in addition to the proper management of epilepsy , there is a need for a general public education campaign about epilepsy to address and correct the existing biases . such biases have been reported by some studies , which concluded that some teachers believe that epilepsy could be enough reason to prevent marriage or have children or could even be a justification for divorce . also , it has been recognized that the mythical idea of epilepsy as a contagious disease seems to be one of the most relevant biases observed . furthermore , many teachers objected to the fact that persons with epilepsy can safely operate machinery and many thought that other children need to be protected from them . a few even thought that laws barring epileptic children from being adopted should be applied . the objectives of our study were as follows : evaluating middle and high school teachers ' knowledge about epilepsy.assessing teachers ' attitudes toward students with epilepsy.investigating the association of sociodemographic characteristics and teaching experience of teachers with their knowledge and attitudes . evaluating middle and high school teachers ' knowledge about epilepsy . assessing teachers ' attitudes toward students with epilepsy . investigating the association of sociodemographic characteristics and teaching experience of teachers with their knowledge and attitudes . the target population was middle and high school teachers in the 6 governorates of kuwait , namely , capital , hawalli , al - farwaniya , al - ahmadi , al - jahra , and mubarak al - kabeer . twenty - four schools ( 12 male and 12 female schools ) were randomly selected from the sampling frame obtained from the ministry of education . all available eligible teachers in selected schools during the data collection period were included in the study with schools as clusters . the total number of teachers who were approached was 850 , of whom 824 accepted to participate . an informed consent was obtained from each participant ; and it clearly stated that participation in this study is optional and that there is no risk as a result of participation in the study . in order to ensure the confidentiality , names of participants or other identifying information a human subject form was completed , and the research was approved by the department of community medicine ethics review board and the research ethics committee of health sciences center , kuwait university . permission for conducting the research was obtained from the ministry of education and the administration of each selected school . participants were asked to complete a self - administered questionnaire , comprising of 26 questions . the english version of the questionnaire was translated into arabic using simple and clear words that would convey the same meaning as the english version . the arabic version was back - translated into english by an independent bilingual person in order to ensure that the arabic version provides the same meaning as the english one . the questionnaire was pretested by administrating it to 10 teachers , in order to emphasize that its items were clear and to estimate the time required to complete the questionnaire , which was found to be approximately 10 minutes . sociodemographic characteristics ( questions 1 to 6 ) included information about the respondents ' age , gender , nationality , marital status , number of children , and highest level of education . teaching experience ( questions 7 to 10 ) included questions about the level of school the teacher is working at , the respondents ' position , how long he has been working as a teacher , and what subjects he teaches . section 3 . experience with epilepsy ( questions 11 to 24 ) included questions regarding persons with epilepsy and their relationship with the teacher : whether the teacher in question has ever dealt with a student with epilepsy , whether any member of his family has epilepsy , whether he has ever taught a student with epilepsy , and if he is currently teaching a student with epilepsy . additionally , this section included general - knowledge questions about epilepsy , such as whether they were aware of the life circumstances of persons with epilepsy and if they will be prepared to handle a seizure if it happens to one of their students in class . furthermore , this section targeted the teachers first - aid management of seizures , if they received any adequate training about seizure management and epilepsy in their educational training , if they are familiar with the different types of seizures and what they look like , and if they would prefer to have more information about how to handle seizures . this section also included questions on sources of their knowledge about epilepsy , its causes , and treatment . the aim of this section , knowledge about epilepsy ( question 25 ) , was to measure the level of knowledge about epilepsy . in order to compare our results with other populations and for the sake of standardization , we used a modified version of a valid and reliable research instrument to measure the level of knowledge about epilepsy . this section included the scale of attitudes toward persons with epilepsy ( atpe ) , a summated rating scale that measures both attitudes toward persons with epilepsy and knowledge about epilepsy [ 10 , 11 ] . teachers were asked to respond by any of 3 options : agree , disagree , or not sure , for each item . out of the 13 knowledge items , 6 items were considered correct if answered by agree , while the remaining 8 statements were correct if answered by disagree . if a respondent correctly answered an item , he was granted 1 , and he was granted 0 if his answer was incorrect . this section , attitudes toward students with epilepsy ( question 26 ) , targets and tests the teachers ' attitudes toward students with epilepsy . it included a modified version of the scale of attitudes toward persons with epilepsy ( atpe ) . teachers were asked to respond by any of 3 options : agree , disagree , or not sure , for each statement . out of the 15 attitude statements , 7 indicated a positive attitude if answered by disagree , while the remaining 8 indicated a positive attitude if answered by agree . hence , the range of positive attitude scale was 0 to 15 . additionally , the attitude score was divided into poor ( 1st tertile < 8 out of 15 ) , medium ( 2nd tertile 811 out of 15 ) , and positive ( 3rd tertile > 11 out of 15 ) . the statistical package for social sciences ( spss inc . , chicago , il , usa , 2010 ) version 19 was used for data entry and analysis . the p value 0.05 was used as the cut - off level for statistical significance . the nonparametric mann - whitney u test was used to compare two groups of nonnormally distributed variables , while kruskal - wallis one - way analysis of variance test was used to compare more than two groups . the nonparametric spearman rank correlation coefficient was used to assess the association between two quantitative nonnormally distributed variables . the unpaired t - test was used to compare the means of the normally distributed quantitative variables . the multivariable logistic regression for a binary outcome variable was applied to identify the independent determinants of poor knowledge about epilepsy , after adjustment for potential confounders . the dependent variable was binary ( 0 for > median knowledge score and 1 for median knowledge score ) . independent variables included sociodemographic variables , teaching experience , experience with epilepsy , and attitudes toward students with epilepsy . table 1 presents the sociodemographic characteristics and teaching experience as were self - reported by the respondents . most teachers ( 44.7% ) were at the age interval of 3039 , and 22.5% of them were at the age interval of 4049 years . the majority of respondents ( 58.7% ) were non - kuwaitis versus 41.3% kuwaiti nationals , making the kuwaiti : non - kuwaiti ratio 1 : 1.4 . the majority of respondents ( 83.9% ) were married , with children ranging in number from 0 to 11 ( median 3 ) . most participants ( 90.7% ) reported completing a university bachelor degree as their highest level of education . overall 86.5% of participants were teachers , 10.6% senior teachers , and 2.9% vice principals or principles . the median ( range ) of the number of years working as a teacher was 11 ( 140 ) . figure 1 illustrates the subjects taught by respondents . teaching arabic topped the list ( 18.1% ) followed by sciences ( 15.3% ) and social studies ( 13.2% ) . most respondents ( 86.7% ) positively answered the item would you like to have more information about how to respond when a student is having a seizure ? . similarly , 83.5% of them positively responded to the item would you like to have more general knowledge about epilepsy ? . in the meantime , 35.1% positively answered the question will you be prepared to handle a seizure if one of your students had a fit during class ? . besides , 29.3% positively responded to the question have you ever dealt with a person having epilepsy ? , and 24% of them replied by yes to the question have you been a teacher of a student with epilepsy ? . surprisingly , only 8.5% of participants thought that they had sufficient training in first - aid management of seizures . moreover , only 6.9% of them reported being aware of the different types of seizures and how they look like . in addition , only 4.5% reported that they had received adequate training about seizures management and epilepsy in their education curricula . figure 2 exhibits the proportion of persons with epilepsy with whom the respondents have dealt . almost one - fifth of participants reported that they have dealt with one student with epilepsy , 4.9% of them dealt with 2 students , and 1.2% dealt with 3 students . figure 3 charts the reported sources of teachers ' information for their knowledge about epilepsy . most of them ( 60.5% ) had their information from public media , followed by the internet ( 41.3% ) , education ( 25.4% ) , parents of students with epilepsy ( 19.2% ) , and health care professionals ( 19.3% ) . the majority of participants ( 73.0% ) reported genetic disorders , 47.4% head trauma , 47.3% brain disease , 24.0% possession of evil spirit , 14.7% insanity , 10.1% punishment from god , and 1.3% brain electricity . table 3 presents the 13 items of knowledge score about epilepsy together with the percentage of teachers who correctly answered each item . a high proportion ( 84.3% ) of participants correctly responded ( disagreed ) to the knowledge item individuals with epilepsy are also mentally retarded , 65.8% correctly answered the item when their seizures are controlled by medication , persons with epilepsy are just like anyone else , and 53.6% correctly answered the item epilepsy and epilepsy medications can have a significant effect on the affected students ' mood , memory , and learning . however , 31.3% of respondents correctly reported the item the offspring of parents with epilepsy will also have epilepsy , 12.4% correctly answered the item persons with epilepsy can safely operate machinery , 11.2% correctly answered the knowledge statement individuals with epilepsy are accident - prone , and 10.7% only correctly responded to the statement individuals with epilepsy can cope with a 40-hour work week . table 4 shows the association of knowledge score with sociodemographic characteristics and teaching experience of teachers . the knowledge score had a median of 5 and a range from 0 to 12 with a negatively skewed distribution ( figure 6 ) . the median knowledge score was significantly higher in senior teachers or vice - principles / principles than in teachers ( kruskal - wallis test , p = 0.012 ) . similarly , there was a significant association between knowledge about epilepsy and the number of years working as a teacher ( kruskal - wallis test , p = 0.042 ) . table 5 presents the association of knowledge score with teachers ' self - reported experience with epilepsy . the median knowledge score was significantly higher among respondents who had ever dealt with a person with epilepsy than those who never dealt with one ( mann - whitney u test , p < 0.001 ) . also , there was a significant difference in the median knowledge score with respect to does any member of your family have epilepsy ? ( mann - whitney u test , p < 0.001 ) , have you ever been a teacher of a student with epilepsy ? ( mann - whitney u test , p = 0.005 ) , will you be prepared to handle a seizure if one of your students had a fit during class ? ( mann - whitney u test , p < 0.001 ) , and do you think you have sufficient training in first - aid management of seizures ? table 6 demonstrates the 15 items of attitude score together with percentage of respondents with positive attitudes toward persons with epilepsy . a high proportion ( 91.3% ) of participants showed a positive attitude to the statement persons with epilepsy have the same rights as all people . similarly , 76.5% positively responded to the item the responsibility of educating children with epilepsy rests on the community , 75.5% showed a positive attitude to the item equal employment opportunities should be available to individuals with epilepsy , and 75.2% disagreed on the statement families of children with epilepsy should not be provided with supportive social services . however , only a low proportion ( 15.7% ) of respondents showed a positive attitude toward the statement persons with epilepsy should be prohibited from driving . table 7 shows the association of attitude score toward epilepsy with sociodemographic characteristics and teaching experience of respondents . the attitude score had a median of 10 and a range from 0 to 15 with a negatively skewed distribution ( figure 7 ) . the median attitude score was significantly higher in kuwaiti nationals than in non - kuwaitis ( mann - whitney u test , p < 0.001 ) , in married than single respondents ( kruskal - wallis test , p = 0.049 ) , and in senior teachers or principles than teachers ( kruskal - wallis test , p = 0.006 ) . also , as the number of years working as a teacher increases , the median attitude score significantly increases ( kruskal - wallis test , p = 0.023 ) . table 8 presents the association of attitude score toward epilepsy with teachers ' self - reported experience with epilepsy . the median attitude score was significantly higher among respondents who had ever dealt with a person having epilepsy than those who never dealt with an epileptic person ( mann - whitney u test , p < 0.001 ) . also , there was a significant difference in the median attitude score with respect to the attitude item does any member of your family have epilepsy ? ( mann - whitney u test , p = 0.002 ) , have you ever been a teacher of a student with epilepsy ? ( p = 0.002 ) , will you be prepared to handle a seizure if one of your students had a fit during class ? ( p < 0.001 ) , would you like to have more knowledge about epilepsy ? ( p < 0.001 ) , and would you like to have more information about how to respond when a student is having a seizure ? there was a significant association between the knowledge score and attitude score ( spearman correlation , rs = 0.0420 , p < 0.01 ) . table 9 displays the significant associated variables with good knowledge about epilepsy using the multivariable logistic regression analysis in order to adjust confounding between variables . the significant variables , which were independently associated with good knowledge about epilepsy after adjusting for sociodemographic characteristics , teaching experience , experience with epilepsy , and attitudes toward students with epilepsy , were having a member of the family with epilepsy ( adjusted odds ratio ( or ) 2.14 , 95% ci 1.213.77 , p = 0.009 ) , being aware of the life circumstances of persons with epilepsy ( adjusted or = 1.59 , 95% ci 1.302.51 , p = 0.048 ) , and having a positive attitude score ( adjusted or = 4.08 , 95% ci 2.476.75 , p < 0.001 ) . our results showed that most respondents ( 86.7% ) positively answered the item would you like to have more information about how to respond when a student is having a seizure ? . similarly , 83.5% of them positively responded to the item would you like to have more general knowledge about epilepsy ? these results are in concert with another study , which similarly concluded that most of the participants would like to have more information about epilepsy . this emphasizes the need for development and implementation of awareness programs about epilepsy and its management . in the meantime , 35.1% of our participants positively answered the question will you be prepared to handle a seizure if one of your students had a fit during class ? besides , 29.3% positively responded to the question have you ever dealt with a person with epilepsy ? . surprisingly , 8.5% of our participants answered that they had sufficient training in first - aid management of seizures . this result is in keeping with abbas and babikar , who concluded that recruited teachers had no previous training on epilepsy ; yet all of them had heard about epilepsy . only 5.7% of respondents reported being currently teachers of students with epilepsy , which is consistent with the previous study , which reported that about 3.5% of their respondents had a student in their schools with epilepsy . our study also showed that only 6.9% of respondents reported that they are aware of the different types of seizures and how they look like . a study carried out in nigeria interestingly found that more than half ( 52% ) of their respondents knew the different types of seizures and how they look like . in addition , only 4.5% of our participants reported that they had received adequate training about seizure management and epilepsy in their education curricula . this result is consistent with another study in which respondents reported using improper and potentially harmful measures and misconceptions for first - aid management of seizures . our results also showed that most of respondents ( 60.5% ) reported that their source of information about epilepsy was the public media , followed by the internet ( 41.3% ) , parents of students with epilepsy ( 19.2% ) , health care professionals ( 19.3% ) , and courses ( 0.6% ) . these data accord with another study , which concluded that the majority of respondents knew about epilepsy from public media or parents of the student with epilepsy ( 37.9% and 35.7% , resp . ) , and only a minority ( 0.6% ) acquired their knowledge about epilepsy from courses . in this study , the majority of our respondents ( 73% ) reported genetic disorders as causes for epilepsy , followed by head trauma ( 47.4% ) and brain disease ( 47.3% ) . unfortunately , 24% of our respondents reported that epilepsy can be caused by possession of evil spirits , consistently with ojinnaka , who concluded that 22.4% of their teachers believed that evil spirits can cause epilepsy . in addition , 14.7% of our respondents reported that epilepsy was due to insanity , and 10.1% attributed it to punishment from god , which is relatively high compared to a previous study . these results provide an evidence that a number of historically problematic and stigmatizing ideas about epilepsy still exist . concerning methods of treatment for epilepsy , a high proportion ( 73% ) of respondents reported holy qur'an and 9.1% of them reported meditation as possible treatment methods . this is an evidence that social norms , culture , and religion play important roles in the kuwaiti society . among our respondents , 16.8% of them reported the use of herbal medicine for treating epilepsy , consistently with another study . the present study showed that a high proportion of participants ( 84.3% ) disagreed with the knowledge statement individuals with epilepsy are also mentally retarded . this result does not accord with dantas et al . who found that many people still believe that epilepsy is a disease observed always in a mentally impaired person . in our study , 82.4% of respondents correctly answered the knowledge item epilepsy is not a contagious disease . however , abbas and babikar reported that a high number of people still think that epilepsy is a contagious disease . similar to a study conducted in istanbul , which revealed that epilepsy is a treatable disease , 65.8% of our respondents correctly answered the statement when their seizures are controlled by medication , persons with epilepsy are just like anyone else . in spite of the good education level of participating teachers as seen from the large proportion holding university bachelor degrees or higher , it seems that they did not receive adequate educational instructions about epilepsy and management of seizures . only 31.3% of our respondents correctly answered the knowledge item the offspring of parents with epilepsy will also have epilepsy . this result is consistent with other studies , which concluded that a high proportion of their participants thought that epilepsy usually passes to the offspring from an epileptic parent [ 15 , 16 ] . sociodemographic characteristics of teachers may affect their extent of knowledge and attitudes toward students with epilepsy . our data showed that the median knowledge score of participating teachers was 5 ( out of 13 ) with a range from 0 to 12 . it was significantly higher in senior teachers and in those with longer teaching years of experience . this finding is in keeping with another study , which concluded that as the level of education and income increase , knowledge about epilepsy improves in the society . in this study , the median knowledge score was significantly higher among respondents who had ever dealt with a person with epilepsy than those who never dealt with an epileptic person . reported that teachers with personal experience with epilepsy scored better results on most questions related to knowledge about epilepsy . concluded that teachers who had witnessed an epileptic seizure had a higher confidence for controlling a seizure . concluded that , witnessing a seizure , the presence of a greater number of epileptic students in class and participation in courses aimed at educating teachers about epilepsy led , in most cases , to being able to deal better with situations where a student had an epileptic seizure . our study indicated that a high proportion ( 91.3% ) of the participants showed positive attitudes to the statement persons with epilepsy have the same rights as all people . similarly , 66.9% positively responded to the item persons with epilepsy should not be prohibited from marrying , which accords with prior studies [ 5 , 13 ] but contrasts with the findings of daoud et al . . our participants equally showed positive attitudes for the item equal employment opportunities should be available to individuals with epilepsy which differs from a study conducted in korea , which found that a high proportion of their respondents objected to employing persons with epilepsy . interestingly , only 15.7% of our participants showed positive attitudes toward the item persons with epilepsy should be prohibited from driving , which is significantly lower than lee et al . who reported that about two - thirds of their participants stated that persons with epilepsy are accident - prone and should be prohibited from driving . previous research has shown that differences in sociocultural environments could account for differences in people 's experiences with epilepsy . in this study , the attitude score had a median of 10 ( out of 15 ) and a range from 0 to 15 . this result is in keeping with a previous study , in which most of their respondents who scored positive attitudes were married . this result may suggest that married participants may be more keen to acquire knowledge and hence developing better attitudes toward childhood related diseases , including epilepsy . moreover , the median attitude score was significantly higher in senior teachers or principles than teachers , which is also consistent with another study where teachers with higher educational levels and higher ranks showed more positive attitudes . additionally , as the number of years working as a teacher increased , the median attitude score significantly increased . this result is in concert with another study , which concluded a significant difference in attitude between teachers with a longer teaching experience than those with a shorter teaching experience . our data showed that the median attitude score was significantly higher among respondents who had ever dealt with an epileptic person than those who never dealt with a person having epilepsy . this result is in concert with another study , which concluded that teachers with more personal experience with epilepsy scored higher on most questions related to attitudes toward students with epilepsy . also , there was a significant difference in our median attitude score with respect to the attitude items would you like to have more knowledge about epilepsy ? and would you like to have more information about how to respond when a student is having a seizure ? . this result is consistent with another study , which similarly concluded that most of their participants were keen to have more information about epilepsy . it may be susceptible to information bias since we relied on self - reported data . besides , temporal relationships could not be established . because the respondents clearly knew that the purpose of the study was to measure attitude , their attitudes may be more positive than the actual attitudes because the teachers were aware of socially desirable responses . this may have led to inflation of the median attitude score and its imbalance in relation to the median knowledge score . in conclusion , this study assessed the level of knowledge about epilepsy and attitudes toward students with epilepsy among middle and high school teachers in kuwait . the median of the knowledge score about epilepsy was 5 ( out of 13 ) and ranges from 0 to 12 ( 38.4% ) , which is lower than that of other populations like north staffordshire ( 70% ) and kentucky , usa ( 70% ) [ 18 , 21 ] . on the other hand , the median of the attitude score toward epilepsy was 10 ( out of 15 ) and ranges from 0 to 15 . hence , the results suggest a somewhat positive picture of teachers ' attitudes toward epilepsy . although the level of education among the studied group of school teachers was relatively high , they did not receive formal training on epilepsy or first aid of seizures . this situation led to the imbalance between knowledge about epilepsy and attitudes toward persons with epilepsy . a number of historically problematic and stigmatizing ideas about epilepsy and persons with epilepsy remain prevalent . we recommend to increase the level of teachers ' knowledge about epilepsy and preparation to handle seizures throughout providing them with information about epilepsy and seizure first aid in the educational setting of teacher - in - training . in addition , further teacher attitude research and ongoing development and implementation of epilepsy education programs are needed . in order to dismiss myths surrounding epilepsy and to break the stigma associated with it , educators and other professionals must work together to convey accurate information and to enhance the development of positive attitudes in school teachers .

background and objectives . attitudes toward students with epilepsy and epilepsy - related knowledge of teachers are crucial for child 's safety in the school . the aim of this study was to evaluate teachers ' knowledge and attitudes toward epilepsy . methods . this cross - sectional study included 824 teachers from 24 randomly selected middle and high schools . scale of attitudes toward persons with epilepsy ( atpe ) was modified to assess teachers ' knowledge about epilepsy and attitudes toward students with epilepsy . results . median knowledge score about epilepsy was 5 ( out of 13 ) , while median attitude score was 10 ( out of 15 ) . both knowledge and attitude median scores were significantly higher in senior teachers with longer teaching experience and in respondents who dealt with a person with epilepsy . there was significant association between knowledge score and attitude score ( p < 0.01 ) . logistic regression showed that significant variables , independently associated with poor knowledge after adjusting for possible confounders , were not having a family member with epilepsy ( p = 0.009 ) , unawareness of life circumstances of persons with epilepsy ( p = 0.048 ) , and a poor attitude score ( p < 0.001 ) . conclusion . school teachers in kuwait have relatively poor knowledge about epilepsy but have positive attitudes toward students with epilepsy . a number of historical and stigmatizing ideas about epilepsy still exist . it is recommended to provide teachers with information about handling seizures in the educational setting through development and implementation of epilepsy education programs .

1. Introduction 2. Methods 3. Results 4. Discussion 5. Knowledge Score 6. Attitude Score 7. Limitations 8. Conclusions and Recommendations

had those teachers simply known more about epilepsy and had there been better attitudes toward students with epilepsy , this boy could have been saved . this incident motivated us to undertake this study in order to raise awareness about epilepsy among school teachers and promote their attitudes toward students with epilepsy . moreover , children with epilepsy are at an increased risk for a number of education - related problems such as educational underachievement , learning disabilities , mental health problems , and social isolation . knowledge about epilepsy is an important issue in determining teachers ' attitudes toward children with epilepsy . in kuwait , teachers are considered as social leaders and role models thus influencing the child 's critical period of social and psychological development . for that reason , studying teachers ' knowledge about epilepsy is beneficial for promoting our future generations . teachers ' attitude toward epilepsy is their predisposition or tendency to respond positively or negatively toward various issues related to students with epilepsy . although attitude is a complex and abstract construct , recent studies have demonstrated the manner in which teachers ' attitudes may be translated into behaviors that can have problematic results for students with epilepsy . teachers ' perception of and approach to these students with epilepsy varies with the accuracy of their knowledge , which is often inadequate , limited , or even erroneous . kankirawatana also concluded that , in addition to the proper management of epilepsy , there is a need for a general public education campaign about epilepsy to address and correct the existing biases . furthermore , many teachers objected to the fact that persons with epilepsy can safely operate machinery and many thought that other children need to be protected from them . the objectives of our study were as follows : evaluating middle and high school teachers ' knowledge about epilepsy.assessing teachers ' attitudes toward students with epilepsy.investigating the association of sociodemographic characteristics and teaching experience of teachers with their knowledge and attitudes . evaluating middle and high school teachers ' knowledge about epilepsy . assessing teachers ' attitudes toward students with epilepsy . investigating the association of sociodemographic characteristics and teaching experience of teachers with their knowledge and attitudes . the target population was middle and high school teachers in the 6 governorates of kuwait , namely , capital , hawalli , al - farwaniya , al - ahmadi , al - jahra , and mubarak al - kabeer . all available eligible teachers in selected schools during the data collection period were included in the study with schools as clusters . the total number of teachers who were approached was 850 , of whom 824 accepted to participate . an informed consent was obtained from each participant ; and it clearly stated that participation in this study is optional and that there is no risk as a result of participation in the study . sociodemographic characteristics ( questions 1 to 6 ) included information about the respondents ' age , gender , nationality , marital status , number of children , and highest level of education . teaching experience ( questions 7 to 10 ) included questions about the level of school the teacher is working at , the respondents ' position , how long he has been working as a teacher , and what subjects he teaches . experience with epilepsy ( questions 11 to 24 ) included questions regarding persons with epilepsy and their relationship with the teacher : whether the teacher in question has ever dealt with a student with epilepsy , whether any member of his family has epilepsy , whether he has ever taught a student with epilepsy , and if he is currently teaching a student with epilepsy . additionally , this section included general - knowledge questions about epilepsy , such as whether they were aware of the life circumstances of persons with epilepsy and if they will be prepared to handle a seizure if it happens to one of their students in class . furthermore , this section targeted the teachers first - aid management of seizures , if they received any adequate training about seizure management and epilepsy in their educational training , if they are familiar with the different types of seizures and what they look like , and if they would prefer to have more information about how to handle seizures . this section also included questions on sources of their knowledge about epilepsy , its causes , and treatment . the aim of this section , knowledge about epilepsy ( question 25 ) , was to measure the level of knowledge about epilepsy . in order to compare our results with other populations and for the sake of standardization , we used a modified version of a valid and reliable research instrument to measure the level of knowledge about epilepsy . this section included the scale of attitudes toward persons with epilepsy ( atpe ) , a summated rating scale that measures both attitudes toward persons with epilepsy and knowledge about epilepsy [ 10 , 11 ] . out of the 13 knowledge items , 6 items were considered correct if answered by agree , while the remaining 8 statements were correct if answered by disagree . this section , attitudes toward students with epilepsy ( question 26 ) , targets and tests the teachers ' attitudes toward students with epilepsy . it included a modified version of the scale of attitudes toward persons with epilepsy ( atpe ) . out of the 15 attitude statements , 7 indicated a positive attitude if answered by disagree , while the remaining 8 indicated a positive attitude if answered by agree . additionally , the attitude score was divided into poor ( 1st tertile < 8 out of 15 ) , medium ( 2nd tertile 811 out of 15 ) , and positive ( 3rd tertile > 11 out of 15 ) . the nonparametric mann - whitney u test was used to compare two groups of nonnormally distributed variables , while kruskal - wallis one - way analysis of variance test was used to compare more than two groups . the nonparametric spearman rank correlation coefficient was used to assess the association between two quantitative nonnormally distributed variables . the multivariable logistic regression for a binary outcome variable was applied to identify the independent determinants of poor knowledge about epilepsy , after adjustment for potential confounders . the dependent variable was binary ( 0 for > median knowledge score and 1 for median knowledge score ) . independent variables included sociodemographic variables , teaching experience , experience with epilepsy , and attitudes toward students with epilepsy . overall 86.5% of participants were teachers , 10.6% senior teachers , and 2.9% vice principals or principles . most respondents ( 86.7% ) positively answered the item would you like to have more information about how to respond when a student is having a seizure ? similarly , 83.5% of them positively responded to the item would you like to have more general knowledge about epilepsy ? besides , 29.3% positively responded to the question have you ever dealt with a person having epilepsy ? , and 24% of them replied by yes to the question have you been a teacher of a student with epilepsy ? figure 2 exhibits the proportion of persons with epilepsy with whom the respondents have dealt . almost one - fifth of participants reported that they have dealt with one student with epilepsy , 4.9% of them dealt with 2 students , and 1.2% dealt with 3 students . figure 3 charts the reported sources of teachers ' information for their knowledge about epilepsy . most of them ( 60.5% ) had their information from public media , followed by the internet ( 41.3% ) , education ( 25.4% ) , parents of students with epilepsy ( 19.2% ) , and health care professionals ( 19.3% ) . table 3 presents the 13 items of knowledge score about epilepsy together with the percentage of teachers who correctly answered each item . a high proportion ( 84.3% ) of participants correctly responded ( disagreed ) to the knowledge item individuals with epilepsy are also mentally retarded , 65.8% correctly answered the item when their seizures are controlled by medication , persons with epilepsy are just like anyone else , and 53.6% correctly answered the item epilepsy and epilepsy medications can have a significant effect on the affected students ' mood , memory , and learning . however , 31.3% of respondents correctly reported the item the offspring of parents with epilepsy will also have epilepsy , 12.4% correctly answered the item persons with epilepsy can safely operate machinery , 11.2% correctly answered the knowledge statement individuals with epilepsy are accident - prone , and 10.7% only correctly responded to the statement individuals with epilepsy can cope with a 40-hour work week . table 4 shows the association of knowledge score with sociodemographic characteristics and teaching experience of teachers . the knowledge score had a median of 5 and a range from 0 to 12 with a negatively skewed distribution ( figure 6 ) . the median knowledge score was significantly higher in senior teachers or vice - principles / principles than in teachers ( kruskal - wallis test , p = 0.012 ) . similarly , there was a significant association between knowledge about epilepsy and the number of years working as a teacher ( kruskal - wallis test , p = 0.042 ) . table 5 presents the association of knowledge score with teachers ' self - reported experience with epilepsy . the median knowledge score was significantly higher among respondents who had ever dealt with a person with epilepsy than those who never dealt with one ( mann - whitney u test , p < 0.001 ) . also , there was a significant difference in the median knowledge score with respect to does any member of your family have epilepsy ? ( mann - whitney u test , p < 0.001 ) , have you ever been a teacher of a student with epilepsy ? ( mann - whitney u test , p = 0.005 ) , will you be prepared to handle a seizure if one of your students had a fit during class ? ( mann - whitney u test , p < 0.001 ) , and do you think you have sufficient training in first - aid management of seizures ? table 6 demonstrates the 15 items of attitude score together with percentage of respondents with positive attitudes toward persons with epilepsy . similarly , 76.5% positively responded to the item the responsibility of educating children with epilepsy rests on the community , 75.5% showed a positive attitude to the item equal employment opportunities should be available to individuals with epilepsy , and 75.2% disagreed on the statement families of children with epilepsy should not be provided with supportive social services . however , only a low proportion ( 15.7% ) of respondents showed a positive attitude toward the statement persons with epilepsy should be prohibited from driving . table 7 shows the association of attitude score toward epilepsy with sociodemographic characteristics and teaching experience of respondents . the attitude score had a median of 10 and a range from 0 to 15 with a negatively skewed distribution ( figure 7 ) . the median attitude score was significantly higher in kuwaiti nationals than in non - kuwaitis ( mann - whitney u test , p < 0.001 ) , in married than single respondents ( kruskal - wallis test , p = 0.049 ) , and in senior teachers or principles than teachers ( kruskal - wallis test , p = 0.006 ) . also , as the number of years working as a teacher increases , the median attitude score significantly increases ( kruskal - wallis test , p = 0.023 ) . table 8 presents the association of attitude score toward epilepsy with teachers ' self - reported experience with epilepsy . the median attitude score was significantly higher among respondents who had ever dealt with a person having epilepsy than those who never dealt with an epileptic person ( mann - whitney u test , p < 0.001 ) . also , there was a significant difference in the median attitude score with respect to the attitude item does any member of your family have epilepsy ? ( mann - whitney u test , p = 0.002 ) , have you ever been a teacher of a student with epilepsy ? ( p = 0.002 ) , will you be prepared to handle a seizure if one of your students had a fit during class ? ( p < 0.001 ) , would you like to have more knowledge about epilepsy ? ( p < 0.001 ) , and would you like to have more information about how to respond when a student is having a seizure ? there was a significant association between the knowledge score and attitude score ( spearman correlation , rs = 0.0420 , p < 0.01 ) . table 9 displays the significant associated variables with good knowledge about epilepsy using the multivariable logistic regression analysis in order to adjust confounding between variables . the significant variables , which were independently associated with good knowledge about epilepsy after adjusting for sociodemographic characteristics , teaching experience , experience with epilepsy , and attitudes toward students with epilepsy , were having a member of the family with epilepsy ( adjusted odds ratio ( or ) 2.14 , 95% ci 1.213.77 , p = 0.009 ) , being aware of the life circumstances of persons with epilepsy ( adjusted or = 1.59 , 95% ci 1.302.51 , p = 0.048 ) , and having a positive attitude score ( adjusted or = 4.08 , 95% ci 2.476.75 , p < 0.001 ) . our results showed that most respondents ( 86.7% ) positively answered the item would you like to have more information about how to respond when a student is having a seizure ? similarly , 83.5% of them positively responded to the item would you like to have more general knowledge about epilepsy ? this emphasizes the need for development and implementation of awareness programs about epilepsy and its management . besides , 29.3% positively responded to the question have you ever dealt with a person with epilepsy ? our results also showed that most of respondents ( 60.5% ) reported that their source of information about epilepsy was the public media , followed by the internet ( 41.3% ) , parents of students with epilepsy ( 19.2% ) , health care professionals ( 19.3% ) , and courses ( 0.6% ) . these data accord with another study , which concluded that the majority of respondents knew about epilepsy from public media or parents of the student with epilepsy ( 37.9% and 35.7% , resp . ) , and only a minority ( 0.6% ) acquired their knowledge about epilepsy from courses . in addition , 14.7% of our respondents reported that epilepsy was due to insanity , and 10.1% attributed it to punishment from god , which is relatively high compared to a previous study . these results provide an evidence that a number of historically problematic and stigmatizing ideas about epilepsy still exist . this is an evidence that social norms , culture , and religion play important roles in the kuwaiti society . the present study showed that a high proportion of participants ( 84.3% ) disagreed with the knowledge statement individuals with epilepsy are also mentally retarded . similar to a study conducted in istanbul , which revealed that epilepsy is a treatable disease , 65.8% of our respondents correctly answered the statement when their seizures are controlled by medication , persons with epilepsy are just like anyone else . in spite of the good education level of participating teachers as seen from the large proportion holding university bachelor degrees or higher , it seems that they did not receive adequate educational instructions about epilepsy and management of seizures . sociodemographic characteristics of teachers may affect their extent of knowledge and attitudes toward students with epilepsy . our data showed that the median knowledge score of participating teachers was 5 ( out of 13 ) with a range from 0 to 12 . it was significantly higher in senior teachers and in those with longer teaching years of experience . this finding is in keeping with another study , which concluded that as the level of education and income increase , knowledge about epilepsy improves in the society . in this study , the median knowledge score was significantly higher among respondents who had ever dealt with a person with epilepsy than those who never dealt with an epileptic person . reported that teachers with personal experience with epilepsy scored better results on most questions related to knowledge about epilepsy . concluded that , witnessing a seizure , the presence of a greater number of epileptic students in class and participation in courses aimed at educating teachers about epilepsy led , in most cases , to being able to deal better with situations where a student had an epileptic seizure . our study indicated that a high proportion ( 91.3% ) of the participants showed positive attitudes to the statement persons with epilepsy have the same rights as all people . similarly , 66.9% positively responded to the item persons with epilepsy should not be prohibited from marrying , which accords with prior studies [ 5 , 13 ] but contrasts with the findings of daoud et al . our participants equally showed positive attitudes for the item equal employment opportunities should be available to individuals with epilepsy which differs from a study conducted in korea , which found that a high proportion of their respondents objected to employing persons with epilepsy . interestingly , only 15.7% of our participants showed positive attitudes toward the item persons with epilepsy should be prohibited from driving , which is significantly lower than lee et al . who reported that about two - thirds of their participants stated that persons with epilepsy are accident - prone and should be prohibited from driving . in this study , the attitude score had a median of 10 ( out of 15 ) and a range from 0 to 15 . this result is in keeping with a previous study , in which most of their respondents who scored positive attitudes were married . this result may suggest that married participants may be more keen to acquire knowledge and hence developing better attitudes toward childhood related diseases , including epilepsy . moreover , the median attitude score was significantly higher in senior teachers or principles than teachers , which is also consistent with another study where teachers with higher educational levels and higher ranks showed more positive attitudes . additionally , as the number of years working as a teacher increased , the median attitude score significantly increased . this result is in concert with another study , which concluded a significant difference in attitude between teachers with a longer teaching experience than those with a shorter teaching experience . our data showed that the median attitude score was significantly higher among respondents who had ever dealt with an epileptic person than those who never dealt with a person having epilepsy . this result is in concert with another study , which concluded that teachers with more personal experience with epilepsy scored higher on most questions related to attitudes toward students with epilepsy . also , there was a significant difference in our median attitude score with respect to the attitude items would you like to have more knowledge about epilepsy ? this result is consistent with another study , which similarly concluded that most of their participants were keen to have more information about epilepsy . this may have led to inflation of the median attitude score and its imbalance in relation to the median knowledge score . in conclusion , this study assessed the level of knowledge about epilepsy and attitudes toward students with epilepsy among middle and high school teachers in kuwait . the median of the knowledge score about epilepsy was 5 ( out of 13 ) and ranges from 0 to 12 ( 38.4% ) , which is lower than that of other populations like north staffordshire ( 70% ) and kentucky , usa ( 70% ) [ 18 , 21 ] . on the other hand , the median of the attitude score toward epilepsy was 10 ( out of 15 ) and ranges from 0 to 15 . hence , the results suggest a somewhat positive picture of teachers ' attitudes toward epilepsy . this situation led to the imbalance between knowledge about epilepsy and attitudes toward persons with epilepsy . a number of historically problematic and stigmatizing ideas about epilepsy and persons with epilepsy remain prevalent . we recommend to increase the level of teachers ' knowledge about epilepsy and preparation to handle seizures throughout providing them with information about epilepsy and seizure first aid in the educational setting of teacher - in - training . in addition , further teacher attitude research and ongoing development and implementation of epilepsy education programs are needed . in order to dismiss myths surrounding epilepsy and to break the stigma associated with it , educators and other professionals must work together to convey accurate information and to enhance the development of positive attitudes in school teachers .

complete resection is often curative for benign meningiomas and the progression - free survival of approximately 95% at 5 years and 90% at 10 years has been reported22,23 ) . however , meningiomas that envelop critical neural or vascular structures can not be completely resected due to post - resective neurological deterioration . over the past two decades , radiosurgery has been conducted as an adjuvant or alternative procedure to resective surgery1 ) . gamma knife radiosurgery ( gkrs ) can be considered for a tumor that is not suitable for resection because gkrs has shown effective local tumor control rates and acceptable complication rates comparable to those achieved with surgical resections13,14,21 ) . however , radiation - induced complications , such as aggravation of pre - existing peritumoral edema ( pte ) have also been reported to cause neurological deterioration2 ) . radiation - induced intratumoral necrosis ( rin ) is one of the signs of treatment response after radiosurgery , however , it can exacerbate the mass effect especially when the tumor is located adjacent to critical structures or the cerebrospinal fluid pathway until the tumor starts to shrink by rin . until now , relatively little has been published regarding rin or pte after gkrs in patients with intracranial meningiomas . therefore , we aimed to study the incidence , clinical significance , and relevant factors of rin and pte during the early period after gkrs for intracranial meningiomas . sixty - four patients with a single intracranial meningioma underwent gkrs in our hospital during the period between august 2008 and january 2011 . the mean age at the time of gkrs was 59.7 years ( range , 29 - 90 ) . among the 64 patients , gkrs was performed as a primary treatment modality in 50 patients , and as an adjunctive therapy after resective surgery for who grade i meningioma in 14 patients ( 7 for the residual , 7 for the recurrent tumor ) . among 64 meningiomas , 34 lesions were located in the non - skull base and 30 in the skull base . the locations were the convexity ( n=22 ) , petrous apex ( n=12 ) , falx ( n=7 ) , olfactory groove ( n=6 ) , sphenoid wing ( n=6 ) , parasagittal ( n=5 ) , cavernous sinus ( n=5 ) , and tentorium ( n=1 ) . gkrs was performed using a model c leksell gamma knife ( elekta instruments ab , stockholm , sweden ) . the planning system was a leksell gamma plan version 8.3.1 ( elekta instruments ab ) . for magnetic resonance ( mr ) imaging of radiosurgery planning , t1-weighted axial images with contrast and t2-weighted axial images were obtained with 2 mm slice thickness without gaps . the mean volume of 64 meningiomas was 4.9 cc ( range , 0.3 - 20 ) . the mean prescription dose of 13.4 gy ( range , 11 - 18 ) was delivered to the mean 49.9% ( range , 45 - 50 ) isodose line . tumors with a volume of < 1 cc , 1 - 5 cc , 5 - 10 cc and > 10 cc were treated with 18 - 16 gy , 16 - 14 gy , 14 - 13 gy and 13 - 11 gy , respectively , until september 2009 . however , the prescription dose was lowered starting october 2009 because of severe pte in our cases . tumors with a volume of < 1 cc , 1 - 5 cc , 5 - 10 cc and > 10 cc were treated with 14 gy , 13 gy , 12 gy and 11 gy , respectively , according to the literature6 ) . the radiosurgical prescription parameters ( rpp ) evaluated were paddick 's conformity index ( ci ) , shaw 's ci , and gradient index ( gi)18,25 ) . mr imaging was performed every 6 months , including continuous thin cut t1 enhanced images , which was the same technique as mr imaging for gkrs . tumor volume was calculated from enhancing lesions in t1 enhanced images , and pte volume was calculated from t2 abnormal signal volume minus the tumor volume . volume measurements of tumors and pte were performed using the co - registration program ( leksell gamma plan , version 8.3.1 ) . complete response ( cr ) was defined as a complete disappearance of all enhancing tumors , partial response ( pr ) as 50% decrease in enhancing tumor volume , progressive disease ( pd ) as 25% increase in enhancing tumor volume , and stable disease ( sd ) as < 50% decrease or < 25% increase in enhancing tumor volume . intratumoral necrosis was defined as low signal intensity area on contrast - enhanced t1-weighted images7,8 ) . radiation - induced intratumoral necrosis ( rin ) was defined as newly developed intratumoral necrosis or aggravation of pre - existing necrosis of more than 25% on the follow - up imaging after gkrs . aggravation of pre - existing pte was defined as at least 25% increase of the volume , compared to the baseline data measured at the time of gkrs . statistical analysis was performed using spss version 12.0 ( spss , chicago , il , usa ) . local control rate was calculated from the time of gkrs . to investigate relevant factors , kaplan - meier analysis was used for categorical variables , and cox regression model was used for continuous variables and multivariate analysis . sixty - four patients with a single intracranial meningioma underwent gkrs in our hospital during the period between august 2008 and january 2011 . the mean age at the time of gkrs was 59.7 years ( range , 29 - 90 ) . among the 64 patients , gkrs was performed as a primary treatment modality in 50 patients , and as an adjunctive therapy after resective surgery for who grade i meningioma in 14 patients ( 7 for the residual , 7 for the recurrent tumor ) . among 64 meningiomas , 34 lesions were located in the non - skull base and 30 in the skull base . the locations were the convexity ( n=22 ) , petrous apex ( n=12 ) , falx ( n=7 ) , olfactory groove ( n=6 ) , sphenoid wing ( n=6 ) , parasagittal ( n=5 ) , cavernous sinus ( n=5 ) , and tentorium ( n=1 ) . gkrs was performed using a model c leksell gamma knife ( elekta instruments ab , stockholm , sweden ) . the planning system was a leksell gamma plan version 8.3.1 ( elekta instruments ab ) . for magnetic resonance ( mr ) imaging of radiosurgery planning , t1-weighted axial images with contrast and t2-weighted axial images were obtained with 2 mm slice thickness without gaps . the mean volume of 64 meningiomas was 4.9 cc ( range , 0.3 - 20 ) . the mean prescription dose of 13.4 gy ( range , 11 - 18 ) was delivered to the mean 49.9% ( range , 45 - 50 ) isodose line . tumors with a volume of < 1 cc , 1 - 5 cc , 5 - 10 cc and > 10 cc were treated with 18 - 16 gy , 16 - 14 gy , 14 - 13 gy and 13 - 11 gy , respectively , until september 2009 . however , the prescription dose was lowered starting october 2009 because of severe pte in our cases . tumors with a volume of < 1 cc , 1 - 5 cc , 5 - 10 cc and > 10 cc were treated with 14 gy , 13 gy , 12 gy and 11 gy , respectively , according to the literature6 ) . the radiosurgical prescription parameters ( rpp ) evaluated were paddick 's conformity index ( ci ) , shaw 's ci , and gradient index ( gi)18,25 ) . mr imaging was performed every 6 months , including continuous thin cut t1 enhanced images , which was the same technique as mr imaging for gkrs . tumor volume was calculated from enhancing lesions in t1 enhanced images , and pte volume was calculated from t2 abnormal signal volume minus the tumor volume . volume measurements of tumors and pte were performed using the co - registration program ( leksell gamma plan , version 8.3.1 ) . complete response ( cr ) was defined as a complete disappearance of all enhancing tumors , partial response ( pr ) as 50% decrease in enhancing tumor volume , progressive disease ( pd ) as 25% increase in enhancing tumor volume , and stable disease ( sd ) as < 50% decrease or < 25% increase in enhancing tumor volume . intratumoral necrosis was defined as low signal intensity area on contrast - enhanced t1-weighted images7,8 ) . radiation - induced intratumoral necrosis ( rin ) was defined as newly developed intratumoral necrosis or aggravation of pre - existing necrosis of more than 25% on the follow - up imaging after gkrs . aggravation of pre - existing pte was defined as at least 25% increase of the volume , compared to the baseline data measured at the time of gkrs . statistical analysis was performed using spss version 12.0 ( spss , chicago , il , usa ) . local control rate was calculated from the time of gkrs . to investigate relevant factors , kaplan - meier analysis was used for categorical variables , and cox regression model was used for continuous variables and multivariate analysis . the median clinical follow - up duration was 19.9 months ( range , 3.7 - 35.1 ) . sixty - four meningiomas were assessed by at least one follow - up imaging with a mean imaging follow - up duration of 12.9 months ( range , 5.4 - 33.4 ) . results of local tumor control at the time of the last follow - up were cr in 0 ( 0% ) , pr in 3 ( 4.7% ) , sd in 60 ( 93.7% ) , and pd in 1 ( 1.6% ) . the one progressed case was not a true failure because rin was observed at the last follow - up ( 17.8 months after gkrs ) and tumor shrinkage is expected with further follow - up . the actuarial local tumor control rate at 5 years was 97.9%13 ) . at the time of gkrs , intratumoral necrosis was observed in 5 ( 7.8% ) out of 64 meningiomas but rin after gkrs was observed in 21 ( 32.8% ) lesions . among 21 lesions , new radiation - induced intratumoral necrosis developed in 16 ( 76.2% ) , and 5 ( 23.8% ) showed aggravation of pre - existing intratumoral necrosis of more than 25% . however , there was no significant change of tumor volume itself between the time of gkrs and rin ( mean , 7.0 vs. 7.8 cc ) . the median interval to the development of rin was 6.50.4 months ( range , 3.7 - 17.0 ) . age , paddick 's ci , marginal dose , maximum dose , target volume , locations , pre - existing intratumoral necrosis and treatment modality were assessed for factors related to rin . target volume > 4.5 cc ( p=0.0099 ) , margin dose > 13gy ( p=0.0079 ) , maximum dose > 25 gy ( p=0.0002 ) , paddick 's ci 0.85 ( p=0.0343 ) and pre - existing intratumoral necrosis ( yes ) ( p=0.0095 ) were significant factors related to rin in univariate analysis . both the maximum dose > 25 gy ( p=0.001 , odds ratio=6.313 , 95% confidence interval : 2.089 - 19.080 using the forward stepwise method ) and target volume > 4.5 cc remained significant in multivariate analysis ( p=0.016 , odds ratio=0.287 , 95% confidence interval : 0.104 - 0.794 using the forward stepwise method ) ( table 1 ) . rin rates of target volume > 4.5 cc were 3.3% , 10.0% and 46.7% at 6 , 12 and 24 months , respectively . however , rin rates of target volume 4.5 cc were 2.3% , 2.3% and 13.6% at 6 , 12 and 24 months , respectively . pte was observed in 11 ( 17.2% ) out of 64 meningiomas at the time of gkrs , but in 18 lesions ( 28.1% ) after gkrs during the follow - up period . among 18 lesions , pte was newly developed in seven lesions and six showed aggravation of pre - existing pte , meaning that new or aggravated pte was observed in 20.3% ( 13/64 ) of meningiomas . the median interval to new development or aggravation of pte was 7.00.7 months ( range , 1.1 - 13.1 ) , which was a little later than the median interval to rin . among the factors , rin ( yes ) ( p=0.0008 ) , paddick 's ci 0.85 ( p=0.0449 ) , pre - existing rin ( yes ) ( p=0.0129 ) and maximum dose > 24 gy ( p=0.0113 ) were the significant factors related to new or aggravated pte in univariate analysis . both rin ( yes ) ( p=0.001 , odds ratio=0.086 , 95% confidence interval : 0.019 - 0.387 using the forward stepwise method ) and maximum dose > 24 gy ( p=0.016 , odds ratio=10.000 , 95% confidence interval : 1.535 - 65.137 using the forward stepwise method ) also remained significant in multivariate analysis . however , pre - existing pte , target volume and marginal dose were not significantly related to new or aggravated pte in univariate and multivariate analyses ( table 2 ) . among 21 lesions with developed rin after gkrs , pte was observed in 7 lesions at the time of gkrs and in 14 after gkrs . the mean volume of 7 pre - existing pte was 8.7 cc ( range , 0.1 - 17.5 ) , however , the mean volume of 14 pte after gkrs was significantly increased to 15.5 cc ( range , 0.8 - 38.6 ) ( p=0.013 , wilcoxon signed ranks test ) . among 21 lesions that showed rin with or without pte and seven lesions that showed pte without rin , 9 lesions were symptomatic during the median follow - up period of 18.22.4 months ( range , 6.8 - 29.4 ) after gkrs . therefore , symptomatic complications developed during the early period after gkrs in 14.1% ( 9/64 ) of the patients in our series . rin or pte - related symptoms were headache in 4 , dizziness in 3 and seizure in 2 . the median onset time was 6.00.3 months ( range , 3.7 - 13.5 ) after gkrs . among 9 patients , symptoms resolved in six patients after the median time of 5.4 months ( range , 0.6 - 12.2 ) but a 51-year old male patient underwent gkrs for a meningioma of 15.4 cc in volume with a prescription dose of 13 gy at 50% isodose line . the patient complained of dizziness and the follow up mr imaging taken at 5.6 months after gkrs showed rin and aggravation of pte . even after administration of oral steroid , a follow - up mr taken at 11.8 months after gkrs showed a further increase of tumor volume and pte to 18.7 cc and 38.6 cc , respectively ( fig . results of local tumor control at the time of the last follow - up were cr in 0 ( 0% ) , pr in 3 ( 4.7% ) , sd in 60 ( 93.7% ) , and pd in 1 ( 1.6% ) . the one progressed case was not a true failure because rin was observed at the last follow - up ( 17.8 months after gkrs ) and tumor shrinkage is expected with further follow - up . at the time of gkrs , intratumoral necrosis was observed in 5 ( 7.8% ) out of 64 meningiomas but rin after gkrs was observed in 21 ( 32.8% ) lesions . among 21 lesions , new radiation - induced intratumoral necrosis developed in 16 ( 76.2% ) , and 5 ( 23.8% ) showed aggravation of pre - existing intratumoral necrosis of more than 25% . however , there was no significant change of tumor volume itself between the time of gkrs and rin ( mean , 7.0 vs. 7.8 cc ) . the median interval to the development of rin was 6.50.4 months ( range , 3.7 - 17.0 ) . age , paddick 's ci , marginal dose , maximum dose , target volume , locations , pre - existing intratumoral necrosis and treatment modality were assessed for factors related to rin . target volume > 4.5 cc ( p=0.0099 ) , margin dose > 13gy ( p=0.0079 ) , maximum dose > 25 gy ( p=0.0002 ) , paddick 's ci 0.85 ( p=0.0343 ) and pre - existing intratumoral necrosis ( yes ) ( p=0.0095 ) were significant factors related to rin in univariate analysis . both the maximum dose > 25 gy ( p=0.001 , odds ratio=6.313 , 95% confidence interval : 2.089 - 19.080 using the forward stepwise method ) and target volume > 4.5 cc remained significant in multivariate analysis ( p=0.016 , odds ratio=0.287 , 95% confidence interval : 0.104 - 0.794 using the forward stepwise method ) ( table 1 ) . rin rates of target volume > 4.5 cc were 3.3% , 10.0% and 46.7% at 6 , 12 and 24 months , respectively . however , rin rates of target volume 4.5 cc were 2.3% , 2.3% and 13.6% at 6 , 12 and 24 months , respectively . pte was observed in 11 ( 17.2% ) out of 64 meningiomas at the time of gkrs , but in 18 lesions ( 28.1% ) after gkrs during the follow - up period . among 18 lesions , pte was newly developed in seven lesions and six showed aggravation of pre - existing pte , meaning that new or aggravated pte was observed in 20.3% ( 13/64 ) of meningiomas . the median interval to new development or aggravation of pte was 7.00.7 months ( range , 1.1 - 13.1 ) , which was a little later than the median interval to rin . among the factors , rin ( yes ) ( p=0.0008 ) , paddick 's ci 0.85 ( p=0.0449 ) , pre - existing rin ( yes ) ( p=0.0129 ) and maximum dose > 24 gy ( p=0.0113 ) were the significant factors related to new or aggravated pte in univariate analysis . both rin ( yes ) ( p=0.001 , odds ratio=0.086 , 95% confidence interval : 0.019 - 0.387 using the forward stepwise method ) and maximum dose > 24 gy ( p=0.016 , odds ratio=10.000 , 95% confidence interval : 1.535 - 65.137 using the forward stepwise method ) also remained significant in multivariate analysis . however , pre - existing pte , target volume and marginal dose were not significantly related to new or aggravated pte in univariate and multivariate analyses ( table 2 ) . among 21 lesions with developed rin after gkrs , pte was observed in 7 lesions at the time of gkrs and in 14 after gkrs . the mean volume of 7 pre - existing pte was 8.7 cc ( range , 0.1 - 17.5 ) , however , the mean volume of 14 pte after gkrs was significantly increased to 15.5 cc ( range , 0.8 - 38.6 ) ( p=0.013 , wilcoxon signed ranks test ) . among 21 lesions that showed rin with or without pte and seven lesions that showed pte without rin , 9 lesions were symptomatic during the median follow - up period of 18.22.4 months ( range , 6.8 - 29.4 ) after gkrs . therefore , symptomatic complications developed during the early period after gkrs in 14.1% ( 9/64 ) of the patients in our series . rin or pte - related symptoms were headache in 4 , dizziness in 3 and seizure in 2 . the median onset time was 6.00.3 months ( range , 3.7 - 13.5 ) after gkrs . among 9 patients , symptoms resolved in six patients after the median time of 5.4 months ( range , 0.6 - 12.2 ) but a 51-year old male patient underwent gkrs for a meningioma of 15.4 cc in volume with a prescription dose of 13 gy at 50% isodose line . the patient complained of dizziness and the follow up mr imaging taken at 5.6 months after gkrs showed rin and aggravation of pte . even after administration of oral steroid , a follow - up mr taken at 11.8 months after gkrs showed a further increase of tumor volume and pte to 18.7 cc and 38.6 cc , respectively ( fig . stereotactic radiosurgery offers a relatively non - invasive and effective method in the management for intracranial tumors , including meningiomas14 ) . they concluded that both conventional radiotherapy and gkrs were safe and efficient , but gkrs provided a better radiological response , and was more compatible with most patients than conventional radiotherapy . hsieh et al.10 ) compared the treatment results of meningioma between gkrs and linear accelerator - based radiosurgery . they reported that the mean complication rate was 15.2% ( range , 8.3 - 23.6 ) in the gkrs treated group treated , and 25.9% ( range , 17.0 - 34.9 ) in those treated using a linear accelerator . therefore , gkrs can be said to provide more beneficial effects in terms of radiation - induced complications . radiation - induced imaging changes can be the signs showing treatment response after gkrs , but these changes can also exacerbate patients ' symptoms or neurological signs by increasing the mass effect . these unwanted effects can prompt additional treatments , such as steroid administration or decompressive surgery , especially in benign tumors . therefore , some researchers have studied radiation - induced imaging changes after stereotactic radiosurgery for benign intracranial tumors and relevant factors . chang et al.3 ) reported that patients treated with gkrs for meningiomas experienced peritumorous imaging changes at a rate of 23.6% . in their series , tumor location , maximal dose , and margin dose were related to the occurrence of imaging changes after gkrs in univariate analysis . however , only tumor location ( convexity , parasagittal and falx cerebri ) remained significant in multivariate analysis . other studies have reported regarding the significance of tumor location and the location of the non - skull base meningiomas seemed to encounter more adverse radiation effects compared to the skull base lesions4,5,12,16,19 ) . however , there was no correlation between tumor location and adverse radiation effects in our study . among the imaging changes that can lead to clinical deterioration after gkrs cai et al.2 ) reported that worsened pre - existing pte , or new pte occurred in about 25% of patients who underwent gkrs for intracranial meningiomas . tumor - brain contact interface area was a strong predictor for the occurrence of new pte in their series . kollov et al.13 ) analyzed 400 meningiomas and found that tumors with edema before gkrs , tumor volume > 10.0 cc , tumors treated with a maximum dose > 30 gy and margin dose > 16 gy were significantly related to post - gkrs edema ( aggravation or new development ) . we thought that rin may lead to neurological deterioration because rin can result in transient tumor volume increase , especially when meningiomas are located adjacent to the critical structures , such as the cerebrospinal fluid pathway . in our series , a target volume > 4.5 cc and maximum dose > 25 gy were significantly related to the development of rin , but there was no significant increase of tumor volume itself in the 21 meningiomas that developed rin after gkrs . however , rin was significantly related to new development or aggravation of pte in both univariate and multivariate analyses . the median interval to the development of rin ( 6.50.4 months ) was a little shorter compared to that of pte ( 7.00.7 months ) . these results may suggest that rin can be a warning sign of the development or aggravation of pte . besides rin , maximum dose > 24 gy was also a significant factor related to new development or aggravation of pte in our study . flickinger et al.6 ) reported on the possible relations between marginal dose and complications occurring after gkrs of meningiomas . they reported that the actuarial rates of any symptomatic post - radiosurgical sequelae ( at 10 years ) were 5.32.3% in the median marginal dose 14 gy ( range , 8.9 - 20 ) and 22.99.3% in the median marginal dose 17 gy ( range , 10 - 20 ) . in addition , they insisted that complications correlated with the volume of tissue receiving 12 gy . however , marginal dose was not significantly related to new development or aggravation of pte in our study . this result may be caused by the lowered prescription dose from october 2009 after having patients experience severe pte after gkrs . hur et al.11 ) reported on the difference of the maximum dose ranges from 0.7 - 7% by matrix size and target volume . their results are coherent with our results which showed that maximum dose was significantly related to new development or aggravation of pte , instead of marginal dose . among 64 lesions , 11 lesions were treated with marginal dose of 12 gy showed the range of maximum dose from 23.6 - 24.3 gy ( mean , 24.1 ) in our study . in our series , target volume > 4.5 cc and maximum dose > 25 gy were significant factors for the development of rin after gkrs for intracranial meningiomas . rin and maximum dose > 24 gy were significantly related to new development or aggravation of pte , and rin preceded new development or aggravation of pte . therefore , we suggest that close observation is required for meningiomas treated with a maximum dose > 24 gy and showing rin after gkrs , because accompanying pte may deteriorate neurological conditions when they are located adjacent to critical structures or the cerebrospinal fluid pathway .

objectiveto study the clinical significance and relevant factors of radiation - induced intratumoral necrosis ( rin ) and peritumoral edema ( pte ) after gamma knife radiosurgery ( gkrs ) for intracranial meningiomas.methodswe retrospectively analyzed the data of 64 patients who underwent gkrs for intracranial meningioma . the mean lesion volume was 4.9 cc ( range , 0.3 - 20 ) , and the mean prescription dose of 13.4 gy ( range , 11 - 18 ) was delivered to the mean 49.9% ( range , 45 - 50 ) isodose line . rin was defined as newly developed or enlarged intratumoral necrosis after gkrs.resultsrin and new development or aggravation of pte were observed in 21 ( 32.8% ) and 18 ( 28.1% ) cases of meningioma , respectively during the median follow - up duration of 19.91.0 months . among various factors , maximum dose ( > 25 gy ) and target volume ( > 4.5 cc ) were significantly related to rin , and rin and maximum dose ( > 24 gy ) were significantly related to the development or aggravation of pte . in 21 meningiomas with development of rin after gkrs , there was no significant change of the tumor volume itself between the times of gkrs and rin . however , the pte volume increased significantly compared to that at the time of gkrs ( p=0.013 ) . the median interval to rin after gkrs was 6.50.4 months and the median interval to new or aggravated pte was 7.00.7 months.conclusiona close observation is required for meningiomas treated with a maximum dose > 24 gy and showing rin after gkrs , since following or accompanying pte may deteriorate neurological conditions especially when the location involves adjacent critical structures .

INTRODUCTION MATERIALS AND METHODS Patient characteristics Radiosurgical treatment Local tumor control, radiation-induced intratumoral necrosis and peritumoral edema Statistical analysis RESULTS Local tumor control Radiation-induced intratumoral necrosis Peritumoral edema Radiation-induced intratumoral necrosis or peritumoral edema-related symptoms DISCUSSION CONCLUSION

gamma knife radiosurgery ( gkrs ) can be considered for a tumor that is not suitable for resection because gkrs has shown effective local tumor control rates and acceptable complication rates comparable to those achieved with surgical resections13,14,21 ) . however , radiation - induced complications , such as aggravation of pre - existing peritumoral edema ( pte ) have also been reported to cause neurological deterioration2 ) . radiation - induced intratumoral necrosis ( rin ) is one of the signs of treatment response after radiosurgery , however , it can exacerbate the mass effect especially when the tumor is located adjacent to critical structures or the cerebrospinal fluid pathway until the tumor starts to shrink by rin . therefore , we aimed to study the incidence , clinical significance , and relevant factors of rin and pte during the early period after gkrs for intracranial meningiomas . the mean age at the time of gkrs was 59.7 years ( range , 29 - 90 ) . the mean volume of 64 meningiomas was 4.9 cc ( range , 0.3 - 20 ) . the mean prescription dose of 13.4 gy ( range , 11 - 18 ) was delivered to the mean 49.9% ( range , 45 - 50 ) isodose line . tumors with a volume of < 1 cc , 1 - 5 cc , 5 - 10 cc and > 10 cc were treated with 18 - 16 gy , 16 - 14 gy , 14 - 13 gy and 13 - 11 gy , respectively , until september 2009 . tumors with a volume of < 1 cc , 1 - 5 cc , 5 - 10 cc and > 10 cc were treated with 14 gy , 13 gy , 12 gy and 11 gy , respectively , according to the literature6 ) . tumor volume was calculated from enhancing lesions in t1 enhanced images , and pte volume was calculated from t2 abnormal signal volume minus the tumor volume . complete response ( cr ) was defined as a complete disappearance of all enhancing tumors , partial response ( pr ) as 50% decrease in enhancing tumor volume , progressive disease ( pd ) as 25% increase in enhancing tumor volume , and stable disease ( sd ) as < 50% decrease or < 25% increase in enhancing tumor volume . radiation - induced intratumoral necrosis ( rin ) was defined as newly developed intratumoral necrosis or aggravation of pre - existing necrosis of more than 25% on the follow - up imaging after gkrs . aggravation of pre - existing pte was defined as at least 25% increase of the volume , compared to the baseline data measured at the time of gkrs . the mean age at the time of gkrs was 59.7 years ( range , 29 - 90 ) . the mean volume of 64 meningiomas was 4.9 cc ( range , 0.3 - 20 ) . the mean prescription dose of 13.4 gy ( range , 11 - 18 ) was delivered to the mean 49.9% ( range , 45 - 50 ) isodose line . tumors with a volume of < 1 cc , 1 - 5 cc , 5 - 10 cc and > 10 cc were treated with 18 - 16 gy , 16 - 14 gy , 14 - 13 gy and 13 - 11 gy , respectively , until september 2009 . tumors with a volume of < 1 cc , 1 - 5 cc , 5 - 10 cc and > 10 cc were treated with 14 gy , 13 gy , 12 gy and 11 gy , respectively , according to the literature6 ) . tumor volume was calculated from enhancing lesions in t1 enhanced images , and pte volume was calculated from t2 abnormal signal volume minus the tumor volume . complete response ( cr ) was defined as a complete disappearance of all enhancing tumors , partial response ( pr ) as 50% decrease in enhancing tumor volume , progressive disease ( pd ) as 25% increase in enhancing tumor volume , and stable disease ( sd ) as < 50% decrease or < 25% increase in enhancing tumor volume . radiation - induced intratumoral necrosis ( rin ) was defined as newly developed intratumoral necrosis or aggravation of pre - existing necrosis of more than 25% on the follow - up imaging after gkrs . aggravation of pre - existing pte was defined as at least 25% increase of the volume , compared to the baseline data measured at the time of gkrs . the median clinical follow - up duration was 19.9 months ( range , 3.7 - 35.1 ) . sixty - four meningiomas were assessed by at least one follow - up imaging with a mean imaging follow - up duration of 12.9 months ( range , 5.4 - 33.4 ) . results of local tumor control at the time of the last follow - up were cr in 0 ( 0% ) , pr in 3 ( 4.7% ) , sd in 60 ( 93.7% ) , and pd in 1 ( 1.6% ) . the one progressed case was not a true failure because rin was observed at the last follow - up ( 17.8 months after gkrs ) and tumor shrinkage is expected with further follow - up . at the time of gkrs , intratumoral necrosis was observed in 5 ( 7.8% ) out of 64 meningiomas but rin after gkrs was observed in 21 ( 32.8% ) lesions . among 21 lesions , new radiation - induced intratumoral necrosis developed in 16 ( 76.2% ) , and 5 ( 23.8% ) showed aggravation of pre - existing intratumoral necrosis of more than 25% . however , there was no significant change of tumor volume itself between the time of gkrs and rin ( mean , 7.0 vs. 7.8 cc ) . the median interval to the development of rin was 6.50.4 months ( range , 3.7 - 17.0 ) . age , paddick 's ci , marginal dose , maximum dose , target volume , locations , pre - existing intratumoral necrosis and treatment modality were assessed for factors related to rin . target volume > 4.5 cc ( p=0.0099 ) , margin dose > 13gy ( p=0.0079 ) , maximum dose > 25 gy ( p=0.0002 ) , paddick 's ci 0.85 ( p=0.0343 ) and pre - existing intratumoral necrosis ( yes ) ( p=0.0095 ) were significant factors related to rin in univariate analysis . both the maximum dose > 25 gy ( p=0.001 , odds ratio=6.313 , 95% confidence interval : 2.089 - 19.080 using the forward stepwise method ) and target volume > 4.5 cc remained significant in multivariate analysis ( p=0.016 , odds ratio=0.287 , 95% confidence interval : 0.104 - 0.794 using the forward stepwise method ) ( table 1 ) . rin rates of target volume > 4.5 cc were 3.3% , 10.0% and 46.7% at 6 , 12 and 24 months , respectively . however , rin rates of target volume 4.5 cc were 2.3% , 2.3% and 13.6% at 6 , 12 and 24 months , respectively . pte was observed in 11 ( 17.2% ) out of 64 meningiomas at the time of gkrs , but in 18 lesions ( 28.1% ) after gkrs during the follow - up period . among 18 lesions , pte was newly developed in seven lesions and six showed aggravation of pre - existing pte , meaning that new or aggravated pte was observed in 20.3% ( 13/64 ) of meningiomas . the median interval to new development or aggravation of pte was 7.00.7 months ( range , 1.1 - 13.1 ) , which was a little later than the median interval to rin . among the factors , rin ( yes ) ( p=0.0008 ) , paddick 's ci 0.85 ( p=0.0449 ) , pre - existing rin ( yes ) ( p=0.0129 ) and maximum dose > 24 gy ( p=0.0113 ) were the significant factors related to new or aggravated pte in univariate analysis . both rin ( yes ) ( p=0.001 , odds ratio=0.086 , 95% confidence interval : 0.019 - 0.387 using the forward stepwise method ) and maximum dose > 24 gy ( p=0.016 , odds ratio=10.000 , 95% confidence interval : 1.535 - 65.137 using the forward stepwise method ) also remained significant in multivariate analysis . however , pre - existing pte , target volume and marginal dose were not significantly related to new or aggravated pte in univariate and multivariate analyses ( table 2 ) . among 21 lesions with developed rin after gkrs , pte was observed in 7 lesions at the time of gkrs and in 14 after gkrs . the mean volume of 7 pre - existing pte was 8.7 cc ( range , 0.1 - 17.5 ) , however , the mean volume of 14 pte after gkrs was significantly increased to 15.5 cc ( range , 0.8 - 38.6 ) ( p=0.013 , wilcoxon signed ranks test ) . among 21 lesions that showed rin with or without pte and seven lesions that showed pte without rin , 9 lesions were symptomatic during the median follow - up period of 18.22.4 months ( range , 6.8 - 29.4 ) after gkrs . the median onset time was 6.00.3 months ( range , 3.7 - 13.5 ) after gkrs . among 9 patients , symptoms resolved in six patients after the median time of 5.4 months ( range , 0.6 - 12.2 ) but a 51-year old male patient underwent gkrs for a meningioma of 15.4 cc in volume with a prescription dose of 13 gy at 50% isodose line . even after administration of oral steroid , a follow - up mr taken at 11.8 months after gkrs showed a further increase of tumor volume and pte to 18.7 cc and 38.6 cc , respectively ( fig . results of local tumor control at the time of the last follow - up were cr in 0 ( 0% ) , pr in 3 ( 4.7% ) , sd in 60 ( 93.7% ) , and pd in 1 ( 1.6% ) . the one progressed case was not a true failure because rin was observed at the last follow - up ( 17.8 months after gkrs ) and tumor shrinkage is expected with further follow - up . at the time of gkrs , intratumoral necrosis was observed in 5 ( 7.8% ) out of 64 meningiomas but rin after gkrs was observed in 21 ( 32.8% ) lesions . among 21 lesions , new radiation - induced intratumoral necrosis developed in 16 ( 76.2% ) , and 5 ( 23.8% ) showed aggravation of pre - existing intratumoral necrosis of more than 25% . however , there was no significant change of tumor volume itself between the time of gkrs and rin ( mean , 7.0 vs. 7.8 cc ) . the median interval to the development of rin was 6.50.4 months ( range , 3.7 - 17.0 ) . age , paddick 's ci , marginal dose , maximum dose , target volume , locations , pre - existing intratumoral necrosis and treatment modality were assessed for factors related to rin . target volume > 4.5 cc ( p=0.0099 ) , margin dose > 13gy ( p=0.0079 ) , maximum dose > 25 gy ( p=0.0002 ) , paddick 's ci 0.85 ( p=0.0343 ) and pre - existing intratumoral necrosis ( yes ) ( p=0.0095 ) were significant factors related to rin in univariate analysis . both the maximum dose > 25 gy ( p=0.001 , odds ratio=6.313 , 95% confidence interval : 2.089 - 19.080 using the forward stepwise method ) and target volume > 4.5 cc remained significant in multivariate analysis ( p=0.016 , odds ratio=0.287 , 95% confidence interval : 0.104 - 0.794 using the forward stepwise method ) ( table 1 ) . rin rates of target volume > 4.5 cc were 3.3% , 10.0% and 46.7% at 6 , 12 and 24 months , respectively . however , rin rates of target volume 4.5 cc were 2.3% , 2.3% and 13.6% at 6 , 12 and 24 months , respectively . pte was observed in 11 ( 17.2% ) out of 64 meningiomas at the time of gkrs , but in 18 lesions ( 28.1% ) after gkrs during the follow - up period . among 18 lesions , pte was newly developed in seven lesions and six showed aggravation of pre - existing pte , meaning that new or aggravated pte was observed in 20.3% ( 13/64 ) of meningiomas . the median interval to new development or aggravation of pte was 7.00.7 months ( range , 1.1 - 13.1 ) , which was a little later than the median interval to rin . among the factors , rin ( yes ) ( p=0.0008 ) , paddick 's ci 0.85 ( p=0.0449 ) , pre - existing rin ( yes ) ( p=0.0129 ) and maximum dose > 24 gy ( p=0.0113 ) were the significant factors related to new or aggravated pte in univariate analysis . both rin ( yes ) ( p=0.001 , odds ratio=0.086 , 95% confidence interval : 0.019 - 0.387 using the forward stepwise method ) and maximum dose > 24 gy ( p=0.016 , odds ratio=10.000 , 95% confidence interval : 1.535 - 65.137 using the forward stepwise method ) also remained significant in multivariate analysis . however , pre - existing pte , target volume and marginal dose were not significantly related to new or aggravated pte in univariate and multivariate analyses ( table 2 ) . among 21 lesions with developed rin after gkrs , pte was observed in 7 lesions at the time of gkrs and in 14 after gkrs . the mean volume of 7 pre - existing pte was 8.7 cc ( range , 0.1 - 17.5 ) , however , the mean volume of 14 pte after gkrs was significantly increased to 15.5 cc ( range , 0.8 - 38.6 ) ( p=0.013 , wilcoxon signed ranks test ) . among 21 lesions that showed rin with or without pte and seven lesions that showed pte without rin , 9 lesions were symptomatic during the median follow - up period of 18.22.4 months ( range , 6.8 - 29.4 ) after gkrs . the median onset time was 6.00.3 months ( range , 3.7 - 13.5 ) after gkrs . among 9 patients , symptoms resolved in six patients after the median time of 5.4 months ( range , 0.6 - 12.2 ) but a 51-year old male patient underwent gkrs for a meningioma of 15.4 cc in volume with a prescription dose of 13 gy at 50% isodose line . the patient complained of dizziness and the follow up mr imaging taken at 5.6 months after gkrs showed rin and aggravation of pte . even after administration of oral steroid , a follow - up mr taken at 11.8 months after gkrs showed a further increase of tumor volume and pte to 18.7 cc and 38.6 cc , respectively ( fig . they reported that the mean complication rate was 15.2% ( range , 8.3 - 23.6 ) in the gkrs treated group treated , and 25.9% ( range , 17.0 - 34.9 ) in those treated using a linear accelerator . among the imaging changes that can lead to clinical deterioration after gkrs cai et al.2 ) reported that worsened pre - existing pte , or new pte occurred in about 25% of patients who underwent gkrs for intracranial meningiomas . kollov et al.13 ) analyzed 400 meningiomas and found that tumors with edema before gkrs , tumor volume > 10.0 cc , tumors treated with a maximum dose > 30 gy and margin dose > 16 gy were significantly related to post - gkrs edema ( aggravation or new development ) . in our series , a target volume > 4.5 cc and maximum dose > 25 gy were significantly related to the development of rin , but there was no significant increase of tumor volume itself in the 21 meningiomas that developed rin after gkrs . however , rin was significantly related to new development or aggravation of pte in both univariate and multivariate analyses . the median interval to the development of rin ( 6.50.4 months ) was a little shorter compared to that of pte ( 7.00.7 months ) . these results may suggest that rin can be a warning sign of the development or aggravation of pte . besides rin , maximum dose > 24 gy was also a significant factor related to new development or aggravation of pte in our study . they reported that the actuarial rates of any symptomatic post - radiosurgical sequelae ( at 10 years ) were 5.32.3% in the median marginal dose 14 gy ( range , 8.9 - 20 ) and 22.99.3% in the median marginal dose 17 gy ( range , 10 - 20 ) . however , marginal dose was not significantly related to new development or aggravation of pte in our study . their results are coherent with our results which showed that maximum dose was significantly related to new development or aggravation of pte , instead of marginal dose . among 64 lesions , 11 lesions were treated with marginal dose of 12 gy showed the range of maximum dose from 23.6 - 24.3 gy ( mean , 24.1 ) in our study . in our series , target volume > 4.5 cc and maximum dose > 25 gy were significant factors for the development of rin after gkrs for intracranial meningiomas . rin and maximum dose > 24 gy were significantly related to new development or aggravation of pte , and rin preceded new development or aggravation of pte . therefore , we suggest that close observation is required for meningiomas treated with a maximum dose > 24 gy and showing rin after gkrs , because accompanying pte may deteriorate neurological conditions when they are located adjacent to critical structures or the cerebrospinal fluid pathway .

ginger ( zingiber officinale roscoe ) is a perennial herb belonging to the family zingiberaceae , primarily grown in asia and tropical regions , and is one of the most important and widely consumed herbs worldwide . cultivated for its edible under - ground stem ( rhizome ) , ginger has been used since antiquity both as a spice and as a herbal medicine to treat a variety of primarily gastrointestinal ailments , such as nausea , vomiting ( emesis ) , diarrhea , and dyspepsia , and also diverse ailments , including arthritis , muscular aches , and fever.1 this long and established history of medicinal use in humans has stimulated ongoing clinical trials to scientifically assess the effectiveness of ginger as an adjuvant therapy or as a complementary and alternative medicine ( cam ) in a number of indications related to nausea and vomiting ; the most studied of these include nausea and vomiting in pregnancy ( nvp ) , chemotherapy - induced nausea and vomiting ( cinv ) , postoperative nausea and vomiting , and , to a lesser extent , motion sickness.2 ginger is considered as a safe herb for human consumption.3 ginger appears on the us food and drug administration generally recognized as safe list and is included in the pharmacopeias of many western countries . the british herbal compendium lists ginger as a remedy for vomiting with pregnancy along with other indications.4 indeed , ginger capsules have been available in uk for more than 40 years as a remedy for motion sickness and as a carminative . in 2012 , the european medicines agency published an assessment report from the committee of herbal medicinal products describing the use of ginger in the prevention of nausea and vomiting , concluding that plausible clinical evidence exists for the beneficial effects of dry powdered rhizome on a number of conditions related to nausea and vomiting.5 this review summarizes the development of ginger as an antiemetic for npv and cinv and will also focus more on a critical appraisal of the different preparations and presentations of ginger available for patients and the posology used . the rhizome comprises 1%4% of volatile oils and an oleoresin.6 the distinctive odor and flavor of ginger are due to these volatile oils and also nonvolatile phenolic compounds , which have pungent properties.7 the volatile ( steam extracted ) oils consist mainly of sequiterpene hydrocarbons , predominantly zingiberol , which gives rise to the characteristic aroma of ginger . the nonvolatile phenolic phytochemicals of ginger consist of gingerols , shogaols , paradols , and zingerone , and more than 30 gingerol - related compounds can be fractionated from crude ginger.8 gingerols correspond to a series of chemical homologs differentiated by the length of their unbranched alkyl chains ( n6n12 ) . of all the gingerols , 6-gingerol is the most abundant and well - investigated ginger phytochemical.2 the major pharmacological activity of ginger appears to be attributed to gingerols and shogaols , which are the dehydrated products of gingerols . consequently , gingerols are the major components in the fresh ginger rhizome , whereas shogaols , especially 6-shogaol , are the most abundant polyphenolic constituents of dried ginger.7 in relation to its antiemetic properties , ginger ( and its constituents ) acts peripherally , within the gastrointestinal tract , by increasing the gastric tone and motility due to anticholinenergic and antiserotonergic actions.9,10 it is also reported to increase gastric emptying.11 this combination of functions explains the widely accepted ability of ginger to relieve symptoms of functional gastrointestinal disorders , such as dyspepsia , abdominal pain , and nausea , which is often associated with decreased gastric motility . although the exact mode of action of ginger in relation to its antiemetic properties is still being unraveled , three recent studies have investigated the action of ginger on serotonin ( 5-hydroxytryptamine , 5-ht3 , and 5-ht4 ) and cholinergic ( m3 ) receptor activities.1214 working on the evidence that emetogenic chemotherapeutic drugs increase 5-ht concentration and activate visceral vagal afferent nerve activity , jin et al used patch - clamp methods to study the effects of ginger and its pungent constituents on 5-ht - evoked inward currents in rat nodose ganglia neurons . results showed that 6-shogaol , 6-gingerol , and zingerone could inhibit the 5-ht response in a concentration - dependent manner , with 6-shogaol exhibiting the greatest potency.12 furthermore , the inhibition of 5-ht activity occurred in a noncompetitive manner , validating the earlier work.10 using a different methodological approach ( calcium influx and radioligand - binding assays ) , walstab et al.13 used heterologous expression to demonstrate , for the first time , the inhibitory effect of 6-shogaol and 6-gingeral on recombinant human 5-ht3 receptors and also native receptors from human gut enteric neurons . this inhibition was found to be noncompetitive since a 5-ht3 receptor antagonist , gr65630 , was not displaced by the ginger extract . interestingly , both studies posited that since binding of ginger to 5-ht receptors occurs at a site other than the orthosteric - binding site of competitive 5-ht antagonists , combination therapy with known pharmaceutical 5-ht antagonists might increase the antiemetic efficacy . additionally , using a bioassay for contractile ( m ) 3 receptors ( guinea pig ileum ) , pertz et al.14 demonstrated that 6- , 8- , and 10-gingerol and 6-shogaol could slightly but significantly depress carbachol - induced contractions . collectively , these studies provide molecular evidence that ginger antagonizes activation of ( m ) 3 and 5-ht3 receptors , thereby inhibiting afferent inputs to the central nervous system that are stimulated by specific neurotransmitters , such as serotonin , released from the gastrointestinal tract . ginger has also been studied extensively in vitro and in animal models of hypertension , oxidative stress , fungal infection , and cancer ; consequently , ginger has been investigated in the treatment of many different disease states , including cancer , osteoarthritis , and diabetes . these are beyond the scope of this review but have been the subject of several recent reviews.1517 ginger is used in numerous forms , including fresh , dried , pickled , preserved , crystallized , candied , and powdered or ground . evidently , the concentrations of active ingredients ( gingerols and shogaols ) will differ between the different preparations and the processing steps involved . indeed , gingerols are thermally labile , and the extent of gingerol - to - shogaol conversion will likely impact significantly on the medicinal benefits since the two compounds vary in their bioavailability and pharmacological properties.18 a recent methodological analysis using high - performance liquid chromatography ( hplc ) coupled to time - of - flight mass spectrometry demonstrated that dried ginger powder products contained the highest quantity of gingerol - related compounds ( 714 mg / g ) , followed by fresh ginger ( 22.8 mg / g ) and powdered ginger tea products ( 0.8 mg / g).19 attempts to assess the efficacy of ginger in many clinical trials might have been conceivably weakened by the inconsistency in the form of ginger used ( fresh or dried ) and also the dosing regimen . of the 12 studies reviewed in a recent meta - analysis on the use of ginger in nvp , various preparations were described , including ginger biscuits , ginger powder capsules , ginger essence capsules , ginger extract capsules , and ginger syrup in water.20 also , the daily dosage varied from 600 to 2500 mg . similarly , in a recent systematic review on the use of ginger in cinv , typical dosing regimens were 12 g of ginger.21 to obtain patient compliance , it would be necessary to formulate ginger into the dosage forms that are practical to use , while retaining the active components . in this respect , capsules are the common dosage form considered for many oral drugs and different methodologies exist for the preparation of active gingerols ( and shogaols ) in capsule form.22 in the above meta - analysis of nvp , 10 out of 12 studies used ginger in a capsule form and 7 out of 7 the studies reviewed for cinv used encapsulated ginger.20 considering the dosage , there is a remarkable lack of information on the concentration of active ingredients in the various preparations used in clinical trials ; none of the nvp studies reviewed above described any form of chemical analysis to quantify the concentration of active ingredients , and only 2 out of 7 cinv studies did so . this is obviously essential information as commercial preparations of ginger may have widely different concentrations of gingerols . in a study of dietary ginger root supplements , schwertner et al used hplc to measure the concentrations of active ingredients in locally purchased ginger capsules . results ranged from 0.0 to 9.43 mg / g for 6-gingerol , 0.16 to 2.18 mg / g for 6-shogaol , 0.00 to 1.1 mg / g for 8-gingerol , and 0.00 to 1.40 mg / g for 10-gingerol , and somewhat worryingly , the suggested daily dose varied from 250 mg to nearly 5 g.23 the absence of standardized ginger constituents has also been highlighted in a recent study protocol to assess ginger in the setting of chemotherapy - induced nausea.24 in this study design , the authors have chosen to use a commercially prepared ginger extract capsule that has been standardized to contain 5% gingerols ( referring to the total gingerol strength ) , which is equivalent to 15 mg of active ingredient per 300 mg ginger extract and is an amount utilized in some previous clinical trials.24 in addition to differences in the quantity of ginger used between studies , the dosing intervals also vary between clinical trials . in this regard , two recent clinical studies have investigated the pharmacokinetics of different ginger preparations in humans.25,26 in the first study , healthy volunteers were given a single oral dose of ginger , ranging from 100 mg to 2 g , and the blood samples were periodically taken up to 72 hours . results showed that no free gingerols or shogaol could be detected in the plasma ; however , these analytes were readily detected as predominantly glucuronide and sulfate conjugates in serum , indicating that gingerols undergo oxidation of their phenolic side chain.25 extending this analysis , the same authors developed a more sensitive technique and established that free forms of 10-gingerol and 6-shogaol , as well as glucuronide metabolites of 6- , 8- , and 10-gingerol and 6-shogaol could be identified one hour after oral dosing with 2 g of the ginger extract.26 interestingly , the half - life of these compounds and their metabolites was determined to be between one and three hours in human plasma , and multiple dosing experiments established that no accumulation of metabolites occurred . given these results , it may be prudent to extend the frequency of dosing , within the expectations of patient compliance . although there is no consensus agreement on the correct dosage of ginger , most clinical studies recommend a safe daily dose of 1000 mg , at least in the setting on nvp.2729 accordingly , 7 out of 12 studies described in the viljoen et al.20 meta - analysis used this final amount , and a subgroup analysis in this report favored the lower daily dosage of < 1500 mg for nausea relief . as a demonstration , ding et al.30 calculated that 1000 mg is equivalent to one teaspoon ( 5 g ) of freshly grated ginger extract , 2 ml of liquid ginger extract , four cups ( 237 ml each ) of prepackaged ginger tea , two teaspoons of ginger syrup ( 10 ml ) , or two pieces of crystallized ginger ( 1 in2 ) . the european medicines agency monograph summarized the following most often used dosages ( through june 2010 ) : nvp 500 mg three times daily for three to five days , postoperative nausea and vomiting 1000 mg for one hour before induction of anesthesia , and motion sickness 1000 mg for one hour before start of travel.5 the us food and drug administration generally recognized as safe list states that up to 4 g of ginger can be consumed daily , although these amounts are generally not reached in studies . indeed , the illustration by ding et al.30 serves to apprise patients of the ease of exceeding the maximum daily dose subject to the type of ginger consumed . interestingly , these regimens often consider the prophylactic use of ginger to delay acute emesis , presumably working in a manner associated with priming of receptors . indeed , successful treatments for cinv have been proven to be those given before chemotherapy treatment starts.31 as stated earlier , the most common and well - established use of ginger is undoubtedly its utilization in alleviating symptoms of nausea and vomiting . in this sense , it is perhaps appropriate to mention the differences between nausea and vomiting . nausea is characterized by an uncomfortable sensation experienced in the throat and epigastrum that may or may not result in the expulsion of contents from the stomach , while vomiting is the involuntary , forceful expulsion of contents from the stomach . nausea and vomiting can occur separately , although since vomiting is nearly always preceeded by nausea , they are often considered components of a single entity.32 nausea is a nonobservable phenomenon , while vomiting is objective , and the occurrence and the frequency of vomiting may be measured . in the clinical setting , various instruments are used to assess nausea and vomiting based on self - reporting.33 the inv-2 or rhodes index of nausea , vomiting , and retching is an eight - item self - report questionnaire that measures nausea and vomiting as separate entities and is used frequently as an outcome measure in controlled studies.34 another common tool to measure nausea is a visual analog scale ( vas ) of 010 cm to score severity . it is thought that up to 80% of women have nvp in some degree during the first trimester of pregnancy , and for the majority of women , symptoms typically resolve by 1214 weeks gestation.35,36 colloquially known as morning sickness , this term is clearly a misnomer because symptoms can occur at any time of the day.37 in a small percentage of pregnancies ( 0.2%5% ) , persistent and excessive nausea and vomiting resulting in dehydration , electrolyte imbalance , and weight loss ( termed hyperemesis gravidarum ) can occur and is a leading cause of hospital admissions during the first half of pregnancy.38 evidently , this often debilitating condition can have a significant impact on the quality of a woman s life , both personally and professionally , and can be emotionally traumatic . the exact cause of nvp remains unclear and probably depends on several factors.39 among these , a commonly accepted etiology is ascribed to hormonal changes that occur during pregnancy , such as elevation of serum human chorionic gonadotrophin,40 estrogens,41 and also helicobacter pylori infection.42 several medications are currently available for the treatment of nvp.43,44 emesis can be treated with drugs known as antiemetics , most notably serotonin ( 5-ht3 ) receptor antagonists . however , many women are cautious of medicines for fear of harming the fetus , especially given that nvp usually occurs during the vulnerable period of embryonic organogenesis . accordingly , the popularity of cam , including nonpharmacological medicines and herbal extracts , has grown considerably in recent years , and a high frequency of cam use during pregnancy has been noted.45 a recent multi - national study on the prevalence of herbal medicine use in pregnancy found that over 28% of participating women used herbals ( 2735/9459).46 of the 134 different herbs used , ginger and cranberry accounted for the majority of herbals ( 23.5% and 22.7% , respectively ) , with valerian and raspberry being also popular choices . the impact of the use of ginger as an antiemetic in nvp has been extensively investigated in clinical studies for at least 30 years.47 because of the heterogeneity inherent in the study design and occasional problems with quality , not all randomized clinical trials can be incorporated into a meta - analysis . nonetheless , two meta - analyses of randomized clinical trials ( level i evidence ) have been published very recently.20,48 in the smaller of the two meta - analyses , six studies conducted from 1991 to 2009 fulfilled the inclusion criteria , and 508 subjects were randomly assigned to receive ginger ( 1000 mg daily ) or placebo.48 predictably , these studies varied in the formulation and dosage of ginger : three studies administered 250 mg ginger capsules four times daily28,29,49 ; one study used 350 mg ginger capsules four times daily,50 one study administered 250 mg ginger syrup four times daily,51 and one study administered 500 mg ginger powder in biscuit , five biscuits daily.52 although the duration of study also varied ( between four days and three weeks ) , using an end point of improvement of early nvp , the meta - analysis demonstrated that ginger was better than placebo in improving nvp when given at a dose of 1000 mg / d for at least four days . the authors of the meta - analysis concluded that ginger was an effective nonpharmacological option for nvp and was better than placebo . in the second systematic review and meta - analysis , viljoen et al.20 studied the efficacy of orally administered ginger as treatment for nvp in pregnant women at any stage of pregnancy and reviewed randomized studies from 1991 to 2011 . from 302 records identified through database searching , 12 studies met the criteria established by the authors , involving 1278 pregnant women and included the six studies reviewed by thomson et al.3 the six additional studies used ginger capsules of different dosages : 125 mg four times daily,53 200 mg three times daily,54 325 mg ( 2 ) three times daily,55 and 500 mg two times56,57 or three times58 daily . ginger versus placebo was assessed in 7 out of 12 studies.28,29,49,5154 individual results from all seven studies concluded that ginger was more effective than placebo in relieving the intensity of nvp in general ; however , only three from the seven studies concluded that ginger was more effective in reducing the number of vomiting episodes ( although there was a trend for improvement).28,29,48 in four studies assessing ginger versus vitamin b6 supplementation , a common first - line treatment for nausea , three studies reported no difference between the two groups,50,57,58 and one study showed that ginger significantly improved nausea and vomiting symptoms.55 besides this meta - analysis , a recently published study also found no significant differences between the ginger group ( 47 patients treated with 250 mg ginger four times daily ) and the vitamin b6 group ( 40 mg twice daily).59 one study assessed the efficacy of ginger against the anti - histaminic drug dimenhydrinate and found ginger to be just as effective , with fewer side effects.56 the effects of ginger were not significantly different from those obtained to metoclopramide54 in conclusion , viljoen et al acknowledged the limited number of studies and low quality of evidence , but based on the evidence , ginger could be a possibly effective option for women with nvp , although large standardized trials are necessary . cinv is a major adverse effect of chemotherapy , and cancer patients rate of nausea is the most distressing side effect of chemotherapy.60 the risk of suffering cinv is dependent on the emetic potential of the chemotherapy.61 some chemotherapeutic agents , including cyclophosphamide and cisplatin , can lead to an extremely high incidence of cinv , upward of 90%.31 the standard of care for chemotherapy - induced vomiting is antiemetics , most notably sertononin ( 5-ht3 ) receptor anatagonists and glucocorticoids , such as dexamethasone ; however , efforts to control nausea have been less successful.62 with respect to chemotherapy - induced nausea , the following three separate stages can be categorized : anticipatory nausea ( before initiation of chemotherapy ) , acute nausea ( occurring within 24 hours of chemotherapy ) , and delayed nausea ( 24 hours to five days postchemotherapy).63 in a recent double - blind multicenter trial , 576 adult cancer patients were assigned to receive : ( 1 ) placebo , ( 2 ) 0.5 g ginger , ( 3 ) 1.0 g ginger , or ( 4 ) 1.5 g ginger , on top of antiemetic treatment ( 5-ht3 receptor antagonists).64 patients received the regimen for two six - day periods ; ginger administration started three days prior to chemotherapy . significantly , this study , the largest to date , standardized the ginger constituents used in the trial and the capsules contained a purified liquid extract of ginger root with concentrated 8.5 mg of combined gingerols , zingerone , and shogaol content ( equivalent to 250 mg of ginger root ) . results from mixed - model analyses showed that all concentrations of ginger significantly reduced the incidence of acute , but not delayed , nausea , with 0.5 and 1.0 g being the most effective.64 a prior study of cinv also standardized the ginger concentration to a combination of active ingredients.65 in this case , hplc analysis was used to verify the concentrations , which were found to be 2.15% 6-gingerol , 0.72% 8-gingerol , 1.78% 10-gingerol , and 0.37% 6-shogaol . participants ( 129 ) were randomized to receive 1 g ( four capsules of 250 mg ) of ginger daily , 2 g ( eight capsules ) of ginger daily , or matching placebo for three days together with an antiemetic ( 5-ht3 receptor antagonist and/or aprepitant , a neurokinin 1 receptor blocker ) , and the duration of intervention was three days postchemotherapy . although well tolerated , ginger provided no additional benefit for reduction of the prevalence or severity of acute or delayed cinv when used as an adjuvant therapy . these two studies were included in a recent review of controlled clinical trials by marx et al.21 who performed a systematic search of the literature until april 2012 . from 27 records analyzed , seven studies fitted the inclusion criteria . the five additional studies used the following encapsulated ginger of different dosages : 250 mg four times daily,66,67 500 mg three times68 or four times69 daily , and 167 mg six times daily or 400 mg five times daily depending on patient weight.70 of the five additional studies , one reported no additional benefit to standard emetic control,66 two reported some benefit,68,70 and two reported that ginger performed equally well as metoclopramide.67,69 thus , from the seven trials analyzed , five reported favorable results , while results from the other two clinical trials were unfavorable . similar to the conclusions in the study by viljoen et al.20 , marx et al posited that the mixed results from the trials could perhaps be explained by the nonstandardized preparations of ginger used and inconsistencies in study methods and outcomes . it is thought that up to 80% of women have nvp in some degree during the first trimester of pregnancy , and for the majority of women , symptoms typically resolve by 1214 weeks gestation.35,36 colloquially known as morning sickness , this term is clearly a misnomer because symptoms can occur at any time of the day.37 in a small percentage of pregnancies ( 0.2%5% ) , persistent and excessive nausea and vomiting resulting in dehydration , electrolyte imbalance , and weight loss ( termed hyperemesis gravidarum ) can occur and is a leading cause of hospital admissions during the first half of pregnancy.38 evidently , this often debilitating condition can have a significant impact on the quality of a woman s life , both personally and professionally , and can be emotionally traumatic . the exact cause of nvp remains unclear and probably depends on several factors.39 among these , a commonly accepted etiology is ascribed to hormonal changes that occur during pregnancy , such as elevation of serum human chorionic gonadotrophin,40 estrogens,41 and also helicobacter pylori infection.42 several medications are currently available for the treatment of nvp.43,44 emesis can be treated with drugs known as antiemetics , most notably serotonin ( 5-ht3 ) receptor antagonists . however , many women are cautious of medicines for fear of harming the fetus , especially given that nvp usually occurs during the vulnerable period of embryonic organogenesis . accordingly , the popularity of cam , including nonpharmacological medicines and herbal extracts , has grown considerably in recent years , and a high frequency of cam use during pregnancy has been noted.45 a recent multi - national study on the prevalence of herbal medicine use in pregnancy found that over 28% of participating women used herbals ( 2735/9459).46 of the 134 different herbs used , ginger and cranberry accounted for the majority of herbals ( 23.5% and 22.7% , respectively ) , with valerian and raspberry being also popular choices . the impact of the use of ginger as an antiemetic in nvp has been extensively investigated in clinical studies for at least 30 years.47 because of the heterogeneity inherent in the study design and occasional problems with quality , not all randomized clinical trials can be incorporated into a meta - analysis . nonetheless , two meta - analyses of randomized clinical trials ( level i evidence ) have been published very recently.20,48 in the smaller of the two meta - analyses , six studies conducted from 1991 to 2009 fulfilled the inclusion criteria , and 508 subjects were randomly assigned to receive ginger ( 1000 mg daily ) or placebo.48 predictably , these studies varied in the formulation and dosage of ginger : three studies administered 250 mg ginger capsules four times daily28,29,49 ; one study used 350 mg ginger capsules four times daily,50 one study administered 250 mg ginger syrup four times daily,51 and one study administered 500 mg ginger powder in biscuit , five biscuits daily.52 although the duration of study also varied ( between four days and three weeks ) , using an end point of improvement of early nvp , the meta - analysis demonstrated that ginger was better than placebo in improving nvp when given at a dose of 1000 mg / d for at least four days . the authors of the meta - analysis concluded that ginger was an effective nonpharmacological option for nvp and was better than placebo . in the second systematic review and meta - analysis , viljoen et al.20 studied the efficacy of orally administered ginger as treatment for nvp in pregnant women at any stage of pregnancy and reviewed randomized studies from 1991 to 2011 . from 302 records identified through database searching , 12 studies met the criteria established by the authors , involving 1278 pregnant women and included the six studies reviewed by thomson et al.3 the six additional studies used ginger capsules of different dosages : 125 mg four times daily,53 200 mg three times daily,54 325 mg ( 2 ) three times daily,55 and 500 mg two times56,57 or three times58 daily . ginger versus placebo was assessed in 7 out of 12 studies.28,29,49,5154 individual results from all seven studies concluded that ginger was more effective than placebo in relieving the intensity of nvp in general ; however , only three from the seven studies concluded that ginger was more effective in reducing the number of vomiting episodes ( although there was a trend for improvement).28,29,48 in four studies assessing ginger versus vitamin b6 supplementation , a common first - line treatment for nausea , three studies reported no difference between the two groups,50,57,58 and one study showed that ginger significantly improved nausea and vomiting symptoms.55 besides this meta - analysis , a recently published study also found no significant differences between the ginger group ( 47 patients treated with 250 mg ginger four times daily ) and the vitamin b6 group ( 40 mg twice daily).59 one study assessed the efficacy of ginger against the anti - histaminic drug dimenhydrinate and found ginger to be just as effective , with fewer side effects.56 the effects of ginger were not significantly different from those obtained to metoclopramide54 in conclusion , viljoen et al acknowledged the limited number of studies and low quality of evidence , but based on the evidence , ginger could be a possibly effective option for women with nvp , although large standardized trials are necessary . cinv is a major adverse effect of chemotherapy , and cancer patients rate of nausea is the most distressing side effect of chemotherapy.60 the risk of suffering cinv is dependent on the emetic potential of the chemotherapy.61 some chemotherapeutic agents , including cyclophosphamide and cisplatin , can lead to an extremely high incidence of cinv , upward of 90%.31 the standard of care for chemotherapy - induced vomiting is antiemetics , most notably sertononin ( 5-ht3 ) receptor anatagonists and glucocorticoids , such as dexamethasone ; however , efforts to control nausea have been less successful.62 with respect to chemotherapy - induced nausea , the following three separate stages can be categorized : anticipatory nausea ( before initiation of chemotherapy ) , acute nausea ( occurring within 24 hours of chemotherapy ) , and delayed nausea ( 24 hours to five days postchemotherapy).63 in a recent double - blind multicenter trial , 576 adult cancer patients were assigned to receive : ( 1 ) placebo , ( 2 ) 0.5 g ginger , ( 3 ) 1.0 g ginger , or ( 4 ) 1.5 g ginger , on top of antiemetic treatment ( 5-ht3 receptor antagonists).64 patients received the regimen for two six - day periods ; ginger administration started three days prior to chemotherapy . significantly , this study , the largest to date , standardized the ginger constituents used in the trial and the capsules contained a purified liquid extract of ginger root with concentrated 8.5 mg of combined gingerols , zingerone , and shogaol content ( equivalent to 250 mg of ginger root ) . results from mixed - model analyses showed that all concentrations of ginger significantly reduced the incidence of acute , but not delayed , nausea , with 0.5 and 1.0 g being the most effective.64 a prior study of cinv also standardized the ginger concentration to a combination of active ingredients.65 in this case , hplc analysis was used to verify the concentrations , which were found to be 2.15% 6-gingerol , 0.72% 8-gingerol , 1.78% 10-gingerol , and 0.37% 6-shogaol . participants ( 129 ) were randomized to receive 1 g ( four capsules of 250 mg ) of ginger daily , 2 g ( eight capsules ) of ginger daily , or matching placebo for three days together with an antiemetic ( 5-ht3 receptor antagonist and/or aprepitant , a neurokinin 1 receptor blocker ) , and the duration of intervention was three days postchemotherapy . although well tolerated , ginger provided no additional benefit for reduction of the prevalence or severity of acute or delayed cinv when used as an adjuvant therapy . these two studies were included in a recent review of controlled clinical trials by marx et al.21 who performed a systematic search of the literature until april 2012 . from 27 records analyzed , the five additional studies used the following encapsulated ginger of different dosages : 250 mg four times daily,66,67 500 mg three times68 or four times69 daily , and 167 mg six times daily or 400 mg five times daily depending on patient weight.70 of the five additional studies , one reported no additional benefit to standard emetic control,66 two reported some benefit,68,70 and two reported that ginger performed equally well as metoclopramide.67,69 thus , from the seven trials analyzed , five reported favorable results , while results from the other two clinical trials were unfavorable . similar to the conclusions in the study by viljoen et al.20 , marx et al posited that the mixed results from the trials could perhaps be explained by the nonstandardized preparations of ginger used and inconsistencies in study methods and outcomes . adverse effects after ingestion of ginger are uncommon but can include mild gastrointestinal complications , such as pyrosis ( heartburn or reflux)71 and eructation ( belching).50 in a study of 27 healthy volunteers who were given a single oral dose of ginger ( ranging from 100 mg to 2 g ) , minor gastrointestinal upsets were the major treatment associated toxicities.25 despite previous studies indicating that ginger could interfere with platelet aggregation and cause excessive bleeding,72 in a randomized crossover study of 12 healthy volunteers taking 1.2 g of dried rhizome three times daily for two weeks , ginger did not affect platelet aggregation and had no effect on the pharmacokinetics or pharmacodynamics of a single 25 mg dose of warfarin taken on day 7.73 of note , the meta - analysis of nvp by viljoen et al also reviewed the safety of ginger as a secondary objective . the authors found that ginger did [ not ] pose a risk for side - effects or adverse events during pregnancy.20 aside from level i evidence , safety of ginger in nvp has been studied in at least two level ii ( nonrandomized or cohort ) studies . in the first prospective study , the pregnancy outcome of 187 women from toronto who were exposed to ginger during the first trimester of pregnancy was compared with women who had been exposed to nonteratogenic drugs that were not antiemetics.74 there were no statistically significant differences between the two groups in terms of live births , spontaneous abortions , therapeutic abortions , birth weight , or gestational age . a more recent and larger population - based cohort study in norway ( 68,522 women ) found that the use of ginger during pregnancy ( 1020 women , 1.5% ) was not associated with an increased risk of congenital malformations , still birth / perinatal birth , low birth weight , or preterm birth.75 ginger is an ancient herb used widely in history for its many natural medicinal properties and particularly as an antiemetic . the best available evidence demonstrates that ginger is an effective and inexpensive treatment for nausea and vomiting and is safe . given the attainability of ginger preparations with known active ingredients , it would be interesting to perform preclinical studies to understand the efficacy of principal ginger constituents , including gingerols and shogaols . dose - finding studies using varied standardized extracts should also be undertaken to accurately determine the effective dose and preparation of ginger . the results from these studies could be used to optimize the design of clinical trials to enhance the efficacy of ginger in nausea and vomiting .

the rhizomes of zingiber officinale ( ginger ) have been used since ancient times as a traditional remedy for gastrointestinal complaints . the most active ingredients in ginger are the pungent principles , particularly gingerols and shogaols . various preclinical and clinical studies have evaluated ginger as an effective and safe treatment for nausea and vomiting in the context of pregnancy and as an adjuvant treatment for chemotherapy - induced nausea and vomiting . here , we provide an update and analysis of ginger use for the prevention of nausea and vomiting , with a focus on the types and presentations of ginger available . we also examine the pharmacokinetic properties of ginger and highlight the type and posology of ginger and its metabolites .

Introduction Ginger Chemistry and Pharmacological Effects Presentations of Ginger Clinical Effectiveness of Ginger Nausea and vomiting in pregnancy Chemotherapy-induced nausea and vomiting Safety Issues of Ginger Conclusion

cultivated for its edible under - ground stem ( rhizome ) , ginger has been used since antiquity both as a spice and as a herbal medicine to treat a variety of primarily gastrointestinal ailments , such as nausea , vomiting ( emesis ) , diarrhea , and dyspepsia , and also diverse ailments , including arthritis , muscular aches , and fever.1 this long and established history of medicinal use in humans has stimulated ongoing clinical trials to scientifically assess the effectiveness of ginger as an adjuvant therapy or as a complementary and alternative medicine ( cam ) in a number of indications related to nausea and vomiting ; the most studied of these include nausea and vomiting in pregnancy ( nvp ) , chemotherapy - induced nausea and vomiting ( cinv ) , postoperative nausea and vomiting , and , to a lesser extent , motion sickness.2 ginger is considered as a safe herb for human consumption.3 ginger appears on the us food and drug administration generally recognized as safe list and is included in the pharmacopeias of many western countries . the british herbal compendium lists ginger as a remedy for vomiting with pregnancy along with other indications.4 indeed , ginger capsules have been available in uk for more than 40 years as a remedy for motion sickness and as a carminative . in 2012 , the european medicines agency published an assessment report from the committee of herbal medicinal products describing the use of ginger in the prevention of nausea and vomiting , concluding that plausible clinical evidence exists for the beneficial effects of dry powdered rhizome on a number of conditions related to nausea and vomiting.5 this review summarizes the development of ginger as an antiemetic for npv and cinv and will also focus more on a critical appraisal of the different preparations and presentations of ginger available for patients and the posology used . of all the gingerols , 6-gingerol is the most abundant and well - investigated ginger phytochemical.2 the major pharmacological activity of ginger appears to be attributed to gingerols and shogaols , which are the dehydrated products of gingerols . consequently , gingerols are the major components in the fresh ginger rhizome , whereas shogaols , especially 6-shogaol , are the most abundant polyphenolic constituents of dried ginger.7 in relation to its antiemetic properties , ginger ( and its constituents ) acts peripherally , within the gastrointestinal tract , by increasing the gastric tone and motility due to anticholinenergic and antiserotonergic actions.9,10 it is also reported to increase gastric emptying.11 this combination of functions explains the widely accepted ability of ginger to relieve symptoms of functional gastrointestinal disorders , such as dyspepsia , abdominal pain , and nausea , which is often associated with decreased gastric motility . although the exact mode of action of ginger in relation to its antiemetic properties is still being unraveled , three recent studies have investigated the action of ginger on serotonin ( 5-hydroxytryptamine , 5-ht3 , and 5-ht4 ) and cholinergic ( m3 ) receptor activities.1214 working on the evidence that emetogenic chemotherapeutic drugs increase 5-ht concentration and activate visceral vagal afferent nerve activity , jin et al used patch - clamp methods to study the effects of ginger and its pungent constituents on 5-ht - evoked inward currents in rat nodose ganglia neurons . indeed , gingerols are thermally labile , and the extent of gingerol - to - shogaol conversion will likely impact significantly on the medicinal benefits since the two compounds vary in their bioavailability and pharmacological properties.18 a recent methodological analysis using high - performance liquid chromatography ( hplc ) coupled to time - of - flight mass spectrometry demonstrated that dried ginger powder products contained the highest quantity of gingerol - related compounds ( 714 mg / g ) , followed by fresh ginger ( 22.8 mg / g ) and powdered ginger tea products ( 0.8 mg / g).19 attempts to assess the efficacy of ginger in many clinical trials might have been conceivably weakened by the inconsistency in the form of ginger used ( fresh or dried ) and also the dosing regimen . in this respect , capsules are the common dosage form considered for many oral drugs and different methodologies exist for the preparation of active gingerols ( and shogaols ) in capsule form.22 in the above meta - analysis of nvp , 10 out of 12 studies used ginger in a capsule form and 7 out of 7 the studies reviewed for cinv used encapsulated ginger.20 considering the dosage , there is a remarkable lack of information on the concentration of active ingredients in the various preparations used in clinical trials ; none of the nvp studies reviewed above described any form of chemical analysis to quantify the concentration of active ingredients , and only 2 out of 7 cinv studies did so . results ranged from 0.0 to 9.43 mg / g for 6-gingerol , 0.16 to 2.18 mg / g for 6-shogaol , 0.00 to 1.1 mg / g for 8-gingerol , and 0.00 to 1.40 mg / g for 10-gingerol , and somewhat worryingly , the suggested daily dose varied from 250 mg to nearly 5 g.23 the absence of standardized ginger constituents has also been highlighted in a recent study protocol to assess ginger in the setting of chemotherapy - induced nausea.24 in this study design , the authors have chosen to use a commercially prepared ginger extract capsule that has been standardized to contain 5% gingerols ( referring to the total gingerol strength ) , which is equivalent to 15 mg of active ingredient per 300 mg ginger extract and is an amount utilized in some previous clinical trials.24 in addition to differences in the quantity of ginger used between studies , the dosing intervals also vary between clinical trials . in this regard , two recent clinical studies have investigated the pharmacokinetics of different ginger preparations in humans.25,26 in the first study , healthy volunteers were given a single oral dose of ginger , ranging from 100 mg to 2 g , and the blood samples were periodically taken up to 72 hours . although there is no consensus agreement on the correct dosage of ginger , most clinical studies recommend a safe daily dose of 1000 mg , at least in the setting on nvp.2729 accordingly , 7 out of 12 studies described in the viljoen et al.20 meta - analysis used this final amount , and a subgroup analysis in this report favored the lower daily dosage of < 1500 mg for nausea relief . indeed , successful treatments for cinv have been proven to be those given before chemotherapy treatment starts.31 as stated earlier , the most common and well - established use of ginger is undoubtedly its utilization in alleviating symptoms of nausea and vomiting . in the clinical setting , various instruments are used to assess nausea and vomiting based on self - reporting.33 the inv-2 or rhodes index of nausea , vomiting , and retching is an eight - item self - report questionnaire that measures nausea and vomiting as separate entities and is used frequently as an outcome measure in controlled studies.34 another common tool to measure nausea is a visual analog scale ( vas ) of 010 cm to score severity . it is thought that up to 80% of women have nvp in some degree during the first trimester of pregnancy , and for the majority of women , symptoms typically resolve by 1214 weeks gestation.35,36 colloquially known as morning sickness , this term is clearly a misnomer because symptoms can occur at any time of the day.37 in a small percentage of pregnancies ( 0.2%5% ) , persistent and excessive nausea and vomiting resulting in dehydration , electrolyte imbalance , and weight loss ( termed hyperemesis gravidarum ) can occur and is a leading cause of hospital admissions during the first half of pregnancy.38 evidently , this often debilitating condition can have a significant impact on the quality of a woman s life , both personally and professionally , and can be emotionally traumatic . accordingly , the popularity of cam , including nonpharmacological medicines and herbal extracts , has grown considerably in recent years , and a high frequency of cam use during pregnancy has been noted.45 a recent multi - national study on the prevalence of herbal medicine use in pregnancy found that over 28% of participating women used herbals ( 2735/9459).46 of the 134 different herbs used , ginger and cranberry accounted for the majority of herbals ( 23.5% and 22.7% , respectively ) , with valerian and raspberry being also popular choices . the impact of the use of ginger as an antiemetic in nvp has been extensively investigated in clinical studies for at least 30 years.47 because of the heterogeneity inherent in the study design and occasional problems with quality , not all randomized clinical trials can be incorporated into a meta - analysis . nonetheless , two meta - analyses of randomized clinical trials ( level i evidence ) have been published very recently.20,48 in the smaller of the two meta - analyses , six studies conducted from 1991 to 2009 fulfilled the inclusion criteria , and 508 subjects were randomly assigned to receive ginger ( 1000 mg daily ) or placebo.48 predictably , these studies varied in the formulation and dosage of ginger : three studies administered 250 mg ginger capsules four times daily28,29,49 ; one study used 350 mg ginger capsules four times daily,50 one study administered 250 mg ginger syrup four times daily,51 and one study administered 500 mg ginger powder in biscuit , five biscuits daily.52 although the duration of study also varied ( between four days and three weeks ) , using an end point of improvement of early nvp , the meta - analysis demonstrated that ginger was better than placebo in improving nvp when given at a dose of 1000 mg / d for at least four days . in the second systematic review and meta - analysis , viljoen et al.20 studied the efficacy of orally administered ginger as treatment for nvp in pregnant women at any stage of pregnancy and reviewed randomized studies from 1991 to 2011 . ginger versus placebo was assessed in 7 out of 12 studies.28,29,49,5154 individual results from all seven studies concluded that ginger was more effective than placebo in relieving the intensity of nvp in general ; however , only three from the seven studies concluded that ginger was more effective in reducing the number of vomiting episodes ( although there was a trend for improvement).28,29,48 in four studies assessing ginger versus vitamin b6 supplementation , a common first - line treatment for nausea , three studies reported no difference between the two groups,50,57,58 and one study showed that ginger significantly improved nausea and vomiting symptoms.55 besides this meta - analysis , a recently published study also found no significant differences between the ginger group ( 47 patients treated with 250 mg ginger four times daily ) and the vitamin b6 group ( 40 mg twice daily).59 one study assessed the efficacy of ginger against the anti - histaminic drug dimenhydrinate and found ginger to be just as effective , with fewer side effects.56 the effects of ginger were not significantly different from those obtained to metoclopramide54 in conclusion , viljoen et al acknowledged the limited number of studies and low quality of evidence , but based on the evidence , ginger could be a possibly effective option for women with nvp , although large standardized trials are necessary . cinv is a major adverse effect of chemotherapy , and cancer patients rate of nausea is the most distressing side effect of chemotherapy.60 the risk of suffering cinv is dependent on the emetic potential of the chemotherapy.61 some chemotherapeutic agents , including cyclophosphamide and cisplatin , can lead to an extremely high incidence of cinv , upward of 90%.31 the standard of care for chemotherapy - induced vomiting is antiemetics , most notably sertononin ( 5-ht3 ) receptor anatagonists and glucocorticoids , such as dexamethasone ; however , efforts to control nausea have been less successful.62 with respect to chemotherapy - induced nausea , the following three separate stages can be categorized : anticipatory nausea ( before initiation of chemotherapy ) , acute nausea ( occurring within 24 hours of chemotherapy ) , and delayed nausea ( 24 hours to five days postchemotherapy).63 in a recent double - blind multicenter trial , 576 adult cancer patients were assigned to receive : ( 1 ) placebo , ( 2 ) 0.5 g ginger , ( 3 ) 1.0 g ginger , or ( 4 ) 1.5 g ginger , on top of antiemetic treatment ( 5-ht3 receptor antagonists).64 patients received the regimen for two six - day periods ; ginger administration started three days prior to chemotherapy . results from mixed - model analyses showed that all concentrations of ginger significantly reduced the incidence of acute , but not delayed , nausea , with 0.5 and 1.0 g being the most effective.64 a prior study of cinv also standardized the ginger concentration to a combination of active ingredients.65 in this case , hplc analysis was used to verify the concentrations , which were found to be 2.15% 6-gingerol , 0.72% 8-gingerol , 1.78% 10-gingerol , and 0.37% 6-shogaol . it is thought that up to 80% of women have nvp in some degree during the first trimester of pregnancy , and for the majority of women , symptoms typically resolve by 1214 weeks gestation.35,36 colloquially known as morning sickness , this term is clearly a misnomer because symptoms can occur at any time of the day.37 in a small percentage of pregnancies ( 0.2%5% ) , persistent and excessive nausea and vomiting resulting in dehydration , electrolyte imbalance , and weight loss ( termed hyperemesis gravidarum ) can occur and is a leading cause of hospital admissions during the first half of pregnancy.38 evidently , this often debilitating condition can have a significant impact on the quality of a woman s life , both personally and professionally , and can be emotionally traumatic . accordingly , the popularity of cam , including nonpharmacological medicines and herbal extracts , has grown considerably in recent years , and a high frequency of cam use during pregnancy has been noted.45 a recent multi - national study on the prevalence of herbal medicine use in pregnancy found that over 28% of participating women used herbals ( 2735/9459).46 of the 134 different herbs used , ginger and cranberry accounted for the majority of herbals ( 23.5% and 22.7% , respectively ) , with valerian and raspberry being also popular choices . the impact of the use of ginger as an antiemetic in nvp has been extensively investigated in clinical studies for at least 30 years.47 because of the heterogeneity inherent in the study design and occasional problems with quality , not all randomized clinical trials can be incorporated into a meta - analysis . nonetheless , two meta - analyses of randomized clinical trials ( level i evidence ) have been published very recently.20,48 in the smaller of the two meta - analyses , six studies conducted from 1991 to 2009 fulfilled the inclusion criteria , and 508 subjects were randomly assigned to receive ginger ( 1000 mg daily ) or placebo.48 predictably , these studies varied in the formulation and dosage of ginger : three studies administered 250 mg ginger capsules four times daily28,29,49 ; one study used 350 mg ginger capsules four times daily,50 one study administered 250 mg ginger syrup four times daily,51 and one study administered 500 mg ginger powder in biscuit , five biscuits daily.52 although the duration of study also varied ( between four days and three weeks ) , using an end point of improvement of early nvp , the meta - analysis demonstrated that ginger was better than placebo in improving nvp when given at a dose of 1000 mg / d for at least four days . in the second systematic review and meta - analysis , viljoen et al.20 studied the efficacy of orally administered ginger as treatment for nvp in pregnant women at any stage of pregnancy and reviewed randomized studies from 1991 to 2011 . ginger versus placebo was assessed in 7 out of 12 studies.28,29,49,5154 individual results from all seven studies concluded that ginger was more effective than placebo in relieving the intensity of nvp in general ; however , only three from the seven studies concluded that ginger was more effective in reducing the number of vomiting episodes ( although there was a trend for improvement).28,29,48 in four studies assessing ginger versus vitamin b6 supplementation , a common first - line treatment for nausea , three studies reported no difference between the two groups,50,57,58 and one study showed that ginger significantly improved nausea and vomiting symptoms.55 besides this meta - analysis , a recently published study also found no significant differences between the ginger group ( 47 patients treated with 250 mg ginger four times daily ) and the vitamin b6 group ( 40 mg twice daily).59 one study assessed the efficacy of ginger against the anti - histaminic drug dimenhydrinate and found ginger to be just as effective , with fewer side effects.56 the effects of ginger were not significantly different from those obtained to metoclopramide54 in conclusion , viljoen et al acknowledged the limited number of studies and low quality of evidence , but based on the evidence , ginger could be a possibly effective option for women with nvp , although large standardized trials are necessary . cinv is a major adverse effect of chemotherapy , and cancer patients rate of nausea is the most distressing side effect of chemotherapy.60 the risk of suffering cinv is dependent on the emetic potential of the chemotherapy.61 some chemotherapeutic agents , including cyclophosphamide and cisplatin , can lead to an extremely high incidence of cinv , upward of 90%.31 the standard of care for chemotherapy - induced vomiting is antiemetics , most notably sertononin ( 5-ht3 ) receptor anatagonists and glucocorticoids , such as dexamethasone ; however , efforts to control nausea have been less successful.62 with respect to chemotherapy - induced nausea , the following three separate stages can be categorized : anticipatory nausea ( before initiation of chemotherapy ) , acute nausea ( occurring within 24 hours of chemotherapy ) , and delayed nausea ( 24 hours to five days postchemotherapy).63 in a recent double - blind multicenter trial , 576 adult cancer patients were assigned to receive : ( 1 ) placebo , ( 2 ) 0.5 g ginger , ( 3 ) 1.0 g ginger , or ( 4 ) 1.5 g ginger , on top of antiemetic treatment ( 5-ht3 receptor antagonists).64 patients received the regimen for two six - day periods ; ginger administration started three days prior to chemotherapy . results from mixed - model analyses showed that all concentrations of ginger significantly reduced the incidence of acute , but not delayed , nausea , with 0.5 and 1.0 g being the most effective.64 a prior study of cinv also standardized the ginger concentration to a combination of active ingredients.65 in this case , hplc analysis was used to verify the concentrations , which were found to be 2.15% 6-gingerol , 0.72% 8-gingerol , 1.78% 10-gingerol , and 0.37% 6-shogaol . adverse effects after ingestion of ginger are uncommon but can include mild gastrointestinal complications , such as pyrosis ( heartburn or reflux)71 and eructation ( belching).50 in a study of 27 healthy volunteers who were given a single oral dose of ginger ( ranging from 100 mg to 2 g ) , minor gastrointestinal upsets were the major treatment associated toxicities.25 despite previous studies indicating that ginger could interfere with platelet aggregation and cause excessive bleeding,72 in a randomized crossover study of 12 healthy volunteers taking 1.2 g of dried rhizome three times daily for two weeks , ginger did not affect platelet aggregation and had no effect on the pharmacokinetics or pharmacodynamics of a single 25 mg dose of warfarin taken on day 7.73 of note , the meta - analysis of nvp by viljoen et al also reviewed the safety of ginger as a secondary objective . the best available evidence demonstrates that ginger is an effective and inexpensive treatment for nausea and vomiting and is safe . given the attainability of ginger preparations with known active ingredients , it would be interesting to perform preclinical studies to understand the efficacy of principal ginger constituents , including gingerols and shogaols .

the use of flat detectors allows display of vessel anatomy with submillimeter resolution and a high contrast - to - noise ratio ( cnr ) . three - dimensional ( 3d ) cone beam imaging using a flat detector in a c - arc system was adopted and eventually displayed an image quality approaching that of ct with respect to contrast resolution [ 1 , 2 ] . however , and despite the above improvement , 3d imaging of objects with high x - ray transparency and small detail may still be difficult . materials such as nitinol with its excellent biocompatibility and self deployment by shape memory are widely used for intracranial stents , and generally yield good clinical results . the usage of nitinol stents has become a common practice in the endovascular treatment as a coiling scaffold [ 68 ] to prevent wire herniation . the visualization of nitinol stents in treatment of atherosclerotic stenoses [ 10 , 11 ] is challenging and necessitates a highly developed x - ray imaging technique . generally , in 2d clinical imaging protocols , only the stent end - markers made of tantalum or platinum can be recognized , but the stent body itself and struts are barely visible due to the low absorption of the constituents . to improve visualization , we need a high contrast resolution combined with high spatial resolution imaging . with 3d cone beam imaging based on flat detectors , both cnr and spatial resolution can be tailored such that fine detail rendition is sufficient to visualize the stent 's struts . we describe a vascular imaging technique validated by an image quality assessment , using phantom objects with a variety of commercially available nitinol stents . the purpose of this study is to visualize details of a stent , to support an improved analysis of its placement , by exploiting a joint optimization of all components of the imaging chain and the associated 3d vascular imaging platform . a proprietary sw package calculates the image quality in terms of signal flow through the entire imaging chain , using image quality descriptors like modulation transfer function ( mtf ) , impulse response , noise power spectrum , low - contrast and high - contrast ( spatial ) resolution . associated with these descriptors , the acquired dose can be calculated at any point in the physical chain , including a computed tomography dose index ( ctdi ) type of dose assessment for cone beam imaging . the analytical model also considers all the intra- and inter - component relations within the imaging system ( also before and after detection ) . the quality description also incorporates an image quality degradation resulting from , e.g. , system remnant blurring upon geometrical calibration and arc movement . the analytical model , comprehensively described by kroon et al . [ 12 , 13 ] , allows us to vary the system parameters in x - ray generation , absorption , dose , and detection as well as in image processing quality , yielding accurate verified results . a number of self - expandable nitinol stents ( table 1 ) , as well as one steel - based stent for reference , were deployed in plastic ( infuse ) tubes with an inner diameter of 3.5 mm which were inserted in a channel of an anthropomorphic head phantom ( cirs , norfolk , virginia ; model 603 ) , placed in the system isocenter . the tubes were filled with a diluted contrast agent : 500 ml h2o and 50 ml visip 270 ( ge ) , in order to produce a 550 hu density mimicking a contrast - filled vessel . the remainder of the phantom channel was filled with diluted contrast agent : 5,000 ml h2o and 50 ml visip 270 , producing 31-hu density . another set of experiments was carried out by filling the tubes with 31-hu diluted contrast agent and the remaining volume again with 31 hu , representing blood . yet , another experiment was performed by inserting platinum coiling wires close to the stents , in an air - filled cavity in the phantom , enabling an assessment of streaking effects . table 1an overview of the stent propertiestypemanufacturerlength ( mm)diameter ( mm)strut cross - section ( m)materialneuroformboston scientific153w68*t66nitinolnatick , maenterprisecordis374.5nitinolbridgewater , njsolitaireev3204 80nitinolplymouth , mnwingspanboston scientific203.5w68*t73nitinolnatick , masilkbalt extrusion253.5nitinolmontmorency , frfour pt wiresleo+balt extrusion252.5nitinolmontmorency , frtwo pt wirespharosmicrus / biotroniks252.75 60steelsan jose , cacr co alloythe manufacturers did not supply the dimensions an overview of the stent properties the manufacturers did not supply the dimensions we used a philips xper vascular system equipped with a large ( 30 40 cm ) flat detector and a 3d workstation for producing the 3d rendered images and hosting the typical 3d image post - processing modules . images were acquired with a standard 3d protocol ( with 4549 mgy ctdi dose ) using a large detector zoom format in combination with a large tube focus , or in a zoomed detector format ( 22-cm imaging diagonal ) and a small tube focus . a set of 620 images was acquired with a 30 fr / s , resulting in a 20-s scan - time over a 200-degree arc - travel . ctdi was measured using a standard measurement protocol with a 16-cm diameter polymethyl methacrylate ( pmma ) cylinder , and dose was measured with the unfors ( billdal , sweden ) mult - o - meter 601-pms . we determined the values using the largest irradiation field while keeping the technique factors identical to those for the high - resolution case with its smaller field . the ctdi protocol would be meaningless for a smaller beam format , as it is not irradiating the complete cylinder with a diameter of 16 cm , and thus not the peripheral probe positions . spatial resolution was measured with a catphan 500 phantom ( the phantom factory , salem ny ) . the resolution reading was limited by the phantom maximum of 2.1 lp / mm . this led us to the following protocols : standard 3d neuro protocol ( 120 kv ) , intracranial stent protocol ( ics , 80 kv ) and ics high resolution ( hires , 80 kv , zoomed detector format ) . the reconstructions were carried out in a zoomed mode , i.e. , a region of interest was chosen as a fraction of the maximum volume , determined by the detector size and the projection geometry . the corresponding technical parameters are shown in table 2 , where linear reconstruction zoom factors of 50% and 33% for the hires protocol and 33% and 17% for the standard protocol are listed . the final images were rendered by maximum intensity projection with a slice thickness of 5.0 mm , accommodating the stent 's radial dimensions . the zoomed secondary reconstructions were carried out by panning the volume such that the relevant stent phantom portions were centered therein . table 2volume and voxel dimensions for the standard protocol and the high - resolution / high - contrast imaging protocolreconstruction zooming ( % ) volume ( 256 voxel matrix ) ( mm)voxel size ( mm)hires5052.8 52.8 52.80.213334.4 34.4 34.40.13standard3382.7 82.7 63.90.321742.6 42.6 32.90.17 volume and voxel dimensions for the standard protocol and the high - resolution / high - contrast imaging protocol the high contrast limiting resolution has been optimized by balancing the relevant mtf of the imaging components , taking noise transfer into account . focus size , detector pixel size , and the reconstruction voxel size are matched such that the transfer contributions of each parameter are balanced , resulting in an optimal voxel size and voxel number . the nearest predefined volume is chosen such that it matches the size of the stent under test . a further reduction of the voxel size would increase the image noise , whereas an increase of the voxel size would result in a spatial resolution loss . the true resolution of the high - resolution protocol is better than the maximum reading of the catphan 500 phantom and could thus not be read by this phantom . deviations of 1020% between measured and simulated values were found , which can be accounted for by the choice of the threshold modulation of 4% . we optimized for the mtf and the inherent system impulse response , including the stent strut diameter of up to 0.08 mm , as well as for contrast and noise . since the basic materials of the stents are identical for all types ( see table 1 ) , we decided to only use one stent type , namely a wingspan nitinol stent . only in case of the high - resolution / high - contrast case , the imaging results of all stents are shown , including the pharos reference stent . table 3limiting high contrast spatial resolution as measured with a catphan 500 phantom ( the phantom factory ) and simulated with the iq analysis programspatial resolutionvolume definitionmeasured ( lp / mm)simulated ( lp / mm)standardnon - zoomed reconstruction volume0.50.533% reconstruction volume1.31.617% reconstruction volume1.61.8hiresnon - zoomed reconstruction volume1.11.250% reconstruction volume2.02.533% reconstruction volume2.13.2catphan 500 phantom range limit limiting high contrast spatial resolution as measured with a catphan 500 phantom ( the phantom factory ) and simulated with the iq analysis program catphan 500 phantom range limit figures 1 , 2 , 3 , 4 , and 5 show snapshots of reconstructed images in planes through the stent 's axes and centered within the volume , where window settings on the work station were such that contrast and noise were comparable . for absolute stent dimensions , see table 1 . figure 1 shows the results of the three protocols for 31 hu , where for reference , an optical image of the stent is shown also . the reconstruction scaling for the high- and standard resolution cases is found in table 2 . figure 2 shows the images for the high contrast tube filling of 550 hu . apart from the object contrast , the conditions are identical to those described for fig . 1 . the effect of varying the tube voltage is shown in fig . 3 , referring to a sequence of reconstructed images , displaying the image contrast for the infuse tubes filled with 31-hu contrast liquid . figure 4 depicts the effect of streaking due to highly opaque platinum coils for 31-hu filling . finally , in fig . 5 , a number of stent types illustrate the performance in the high - resolution / high - contrast mode , for again 31-hu contrast liquid . figure 5 also shows example results of the more radiopaque material platinum in the silk and leo stents and the co cr steel alloy - based pharos stent . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . in all cases , an intra - tube contrast agent of 31 hu was used . the relevant reconstruction ( sub ) volumes are denoted as 50% and 33% for the hires case . for the standard resolution , 17% and for comparison , a contrast inverted optical image is shown on the far leftfig . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . in all cases , an intra - tube high - density contrast agent of 550 volumes are denoted as 50% and 33% for the hires case . for the standard resolution , 17% and 33% images a comparison between the high - resolution cases for high - contrast ( 80 kv ) ( a ) , intermediate contrast ( 100 kv ) ( b ) , and standard contrast ( 120 kv ) ( c ) . 4streak artifacts for pt coils neighboring the neuroform stent . a high - resolution / high - contrast , b standard resolution / high - contrast case , and c the standard resolution / standard contrast casefig . 5high - resolution / high - contrast cases for 50% and 33% reconstruction volumes and various stent types . the enterprise stent at 33% reconstruction volume was intentionally left out due to its large length not fitting this reduced volume . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . in all cases , an intra - tube contrast agent of 31 hu was used . the relevant reconstruction ( sub ) volumes are denoted as 50% and 33% for the hires case . for the standard resolution , 17% and for comparison , a contrast inverted optical image is shown on the far left a wingspan nitinol stent . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . in all cases , an intra - tube high - density contrast agent of 550 volumes are denoted as 50% and 33% for the hires case . for the standard resolution , 17% and 33% images are shown a wingspan nitinol stent . a comparison between the high - resolution cases for high - contrast ( 80 kv ) ( a ) , intermediate contrast ( 100 kv ) ( b ) , and standard contrast ( 120 kv ) ( c ) . the applied intra - tube density was 31 hu streak artifacts for pt coils neighboring the neuroform stent . a high - resolution / high - contrast , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case high - resolution / high - contrast cases for 50% and 33% reconstruction volumes and various stent types . the enterprise stent at 33% reconstruction volume was intentionally left out due to its large length not fitting this reduced volume . the steel - based pharos stent was included for reference measured and modeled limiting resolutions are summarized in table 3 . the results on measured resolution were found by reading the reconstructed images from a catphan 500 phantom in a transaxial plane . by adjusting the technique factors , a ctdi - type weighted dose is kept at the desired low value of 50 mgy for all protocols . the actual dose measurement and the analysis table 4measured and simulated ctdi dose for the standard protocol and the high - resolution / high - contrast protocolctdimeasured dose ( mgy)simulated dose ( mgy)standard4543hires4955 measured and simulated ctdi dose for the standard protocol and the high - resolution / high - contrast protocol figures 1 , 2 , 3 , 4 , and 5 show snapshots of reconstructed images in planes through the stent 's axes and centered within the volume , where window settings on the work station were such that contrast and noise were comparable . for absolute stent dimensions , see table 1 . figure 1 shows the results of the three protocols for 31 hu , where for reference , an optical image of the stent is shown also . the reconstruction scaling for the high- and standard resolution cases is found in table 2 . figure 2 shows the images for the high contrast tube filling of 550 hu . apart from the object contrast , the conditions are identical to those described for fig . 1 . the effect of varying the tube voltage is shown in fig . 3 , referring to a sequence of reconstructed images , displaying the image contrast for the infuse tubes filled with 31-hu contrast liquid . figure 4 depicts the effect of streaking due to highly opaque platinum coils for 31-hu filling . finally , in fig . 5 , a number of stent types illustrate the performance in the high - resolution / high - contrast mode , for again 31-hu contrast liquid . figure 5 also shows example results of the more radiopaque material platinum in the silk and leo stents and the co cr steel alloy - based pharos stent . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . in all cases , an intra - tube contrast agent of 31 hu was used . the relevant reconstruction ( sub ) volumes are denoted as 50% and 33% for the hires case . for the standard resolution , 17% and for comparison , a contrast inverted optical image is shown on the far leftfig . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . in all cases , an intra - tube high - density contrast agent of 550 volumes are denoted as 50% and 33% for the hires case . for the standard resolution , 17% and 33% images a comparison between the high - resolution cases for high - contrast ( 80 kv ) ( a ) , intermediate contrast ( 100 kv ) ( b ) , and standard contrast ( 120 kv ) ( c ) . 4streak artifacts for pt coils neighboring the neuroform stent . a high - resolution / high - contrast , b standard resolution / high - contrast case , and c the standard resolution / standard contrast casefig . 5high - resolution / high - contrast cases for 50% and 33% reconstruction volumes and various stent types . the enterprise stent at 33% reconstruction volume was intentionally left out due to its large length not fitting this reduced volume . the steel - based pharos stent was included for reference a wingspan nitinol stent . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . in all cases , an intra - tube contrast agent of 31 hu was used . the relevant reconstruction ( sub ) volumes are denoted as 50% and 33% for the hires case . for the standard resolution , 17% and a contrast inverted optical image is shown on the far left a wingspan nitinol stent . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . in all cases , an intra - tube high - density contrast agent of 550 volumes are denoted as 50% and 33% for the hires case . for the standard resolution , 17% and 33% images are shown a wingspan nitinol stent . a comparison between the high - resolution cases for high - contrast ( 80 kv ) ( a ) , intermediate contrast ( 100 kv ) ( b ) , and standard contrast ( 120 kv ) ( c ) . the applied intra - tube density was 31 hu streak artifacts for pt coils neighboring the neuroform stent . a high - resolution / high - contrast , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case high - resolution / high - contrast cases for 50% and 33% reconstruction volumes and various stent types . the enterprise stent at 33% reconstruction volume was intentionally left out due to its large length not fitting this reduced volume . the results on measured resolution were found by reading the reconstructed images from a catphan 500 phantom in a transaxial plane . by adjusting the technique factors , a ctdi - type weighted dose is kept at the desired low value of 50 mgy for all protocols . the actual dose measurement and the analysis table 4measured and simulated ctdi dose for the standard protocol and the high - resolution / high - contrast protocolctdimeasured dose ( mgy)simulated dose ( mgy)standard4543hires4955 measured and simulated ctdi dose for the standard protocol and the high - resolution / high - contrast protocol intracranial stenting became feasible as a routine treatment by the introduction of very flexible stents and have become a single treatment for a number of neurovascular conditions ( biondi et al . the stents are manufactured from a very thin material and most are self expanding , i.e. , made from an alloy such as nitinol . however , most nitinol stents are difficult to visualize with fluoroscopy or c - arm cone beam ct . visualization can be improved by increasing the density of the stent , more radiation , or enhanced image acquisition settings and processing . increasing the density will add material and therefore unfavorably alters the stent characteristics and is therefore undesirable . consequently , the visibility is preferably improved by further optimizing the imaging chain . to support and judge correct positioning , the nitinol stent should be visualized in its wall apposition , while conforming to varying diameters throughout the stent 's length . to this end , it is advantageous to view details with virtually optical quality . during the past 6 years , the image quality of ct - like reconstructions , using a c - arm interventional x - ray system equipped with a flat detector , became increasingly notable . high - resolution imaging , with submillimeter isotropic spatial resolution , outperformed digital radiography , fluoroscopy , and even conventional ct ( kamran et al . ) . a continuous effort to enhance image quality enabled imaging of details with low absorption . evidence on high - quality in vitro imaging of the proper deployment of nitinol stents , related to area coverage , kinking , prolapse , and flattening , is given by aurboonyawat , ebrahimi and alvarado . the stent conformity in curved vascular models and simulated aneurysm necks could be studied in detail . in a clinical setting , imagery of balloon mounted stents , based on a cr co steel alloy , with intravenous administration of contrast medium , was carried out by buhk et al . . recently , patel et al . demonstrated high - resolution and contrast - enhanced simultaneous imaging of intraarterial cerebrovascular stents and their host arteries . in our study , a wingspan stent was chosen as an arbitrary example object for an evaluation through reconstructed images . clearly the spatial impulse response improved by introducing the hires protocol , as is shown in fig . 1 for the 31-hu contrast - filled tube , where the cnr was improved as well by the increased object contrast . these two measures lead to an improved visibility of the stent 's struts , when comparing the standard protocol ( 17% zooming ) with the results of the high - resolution / high - contrast protocol ( 33% zooming ) . the open cell structure of the stent is clearly discerned by virtually continuous strokes of the struts , so that its x - ray quality is approaching an optical quality . in fig . 2 , a comparison can be made for the 550-hu contrast filling . in this case , the contrast between struts and background is lower due to the denser filling of the tube , giving an accordingly reduced cnr . the sensitivity to object contrast can be readily viewed in fig . 3 , where the comparison is made for a varying tube voltage : the obvious increase in the cnr makes the stent more pronounced with respect to the ( noisy ) background and accounts for the choice of the lowest voltage , 80 kv , for the ics and hires protocols . in fig . 4 , although the window settings are optimal for stent perception , details are still rendered with a sufficient contrast to artifact distance . finally , fig . 5 displays reconstructions of all stent types for the high - resolution / high - contrast case , showing the detail rendering of the protocol . as the cross - sections of the struts of the investigated types vary between 60 and 80 m , the rendered contrasts vary accordingly , since the optimized system impulse response dominates for all cases . the silk and leo stents stand out more by high object contrast of the platinum auxiliary wires . in spite of the minimal strut diameter , the co cr steel alloy accounts for an increased visibility due to its inherent higher absorption . the remaining stent types are comparable in their visualization quality , which is obvious , considering the similarity in dimensions and composition . for all protocols under test , the maximum weighted ctdi dose of 50 mgy is lower than the european guidelines , as according to eur 16262 . a dose of 60 mgy is recommended for routine head examinations . due to the limited anatomical coverage , by using a smaller irradiated field in the high - resolution case , moreover , we found that the difference between modeled and measured doses is sufficiently small . we have optimized nitinol stent imaging , in the framework of a full - scale 3d imaging model for x - ray imaging . by balancing the sharpness transfer and noise transfer of the imaging components and tuning the contrast - rendering capabilities of a 3d x - ray imaging system , we showed that thin nitinol stent struts can be viewed with a high cnr and good detail rendering . while optimizing , the ctdi radiation doses were kept below recommended values . the quality of 3d images , produced with optimum system settings , proved that independent of the type or manufacturer of the stents , the detail rendering is adequate to assess the post - deployment shape .

introductionto assess an optimized 3d imaging protocol for intracranial nitinol stents in 3d c - arm flat detector imaging . for this purpose , an image quality simulation and an in vitro study was carried out.methodsnitinol stents of various brands were placed inside an anthropomorphic head phantom , using iodine contrast . experiments with objects were preceded by image quality and dose simulations . we varied x - ray imaging parameters in a commercially interventional x - ray system to set 3d image quality in the contrast noise sharpness space . beam quality was varied to evaluate contrast of the stents while keeping absorbed dose below recommended values . two detector formats were used , paired with an appropriate pixel size and x - ray focus size . zoomed reconstructions were carried out and snapshot images acquired . high contrast spatial resolution was assessed with a ct phantom.resultswe found an optimal protocol for imaging intracranial nitinol stents . contrast resolution was optimized for nickel titanium - containing stents . a high spatial resolution larger than 2.1 lp / mm allows struts to be visualized . we obtained images of stents of various brands and a representative set of images is shown . independent of the make , struts can be imaged with virtually continuous strokes . measured absorbed doses are shown to be lower than 50 mgy computed tomography dose index ( ctdi).conclusionby balancing the modulation transfer of the imaging components and tuning the high - contrast imaging capabilities , we have shown that thin nitinol stent wires can be reconstructed with high contrast - to - noise ratio and good detail , while keeping radiation doses within recommended values . experimental results compare well with imaging simulations .

Introduction Materials and methods Results Reconstructed stent images inside a head phantom High contrast limiting spatial resolution Dose CTDI Discussion Conclusions

the use of flat detectors allows display of vessel anatomy with submillimeter resolution and a high contrast - to - noise ratio ( cnr ) . three - dimensional ( 3d ) cone beam imaging using a flat detector in a c - arc system was adopted and eventually displayed an image quality approaching that of ct with respect to contrast resolution [ 1 , 2 ] . however , and despite the above improvement , 3d imaging of objects with high x - ray transparency and small detail may still be difficult . materials such as nitinol with its excellent biocompatibility and self deployment by shape memory are widely used for intracranial stents , and generally yield good clinical results . the usage of nitinol stents has become a common practice in the endovascular treatment as a coiling scaffold [ 68 ] to prevent wire herniation . the visualization of nitinol stents in treatment of atherosclerotic stenoses [ 10 , 11 ] is challenging and necessitates a highly developed x - ray imaging technique . generally , in 2d clinical imaging protocols , only the stent end - markers made of tantalum or platinum can be recognized , but the stent body itself and struts are barely visible due to the low absorption of the constituents . to improve visualization , we need a high contrast resolution combined with high spatial resolution imaging . with 3d cone beam imaging based on flat detectors , both cnr and spatial resolution can be tailored such that fine detail rendition is sufficient to visualize the stent 's struts . we describe a vascular imaging technique validated by an image quality assessment , using phantom objects with a variety of commercially available nitinol stents . the purpose of this study is to visualize details of a stent , to support an improved analysis of its placement , by exploiting a joint optimization of all components of the imaging chain and the associated 3d vascular imaging platform . a proprietary sw package calculates the image quality in terms of signal flow through the entire imaging chain , using image quality descriptors like modulation transfer function ( mtf ) , impulse response , noise power spectrum , low - contrast and high - contrast ( spatial ) resolution . associated with these descriptors , the acquired dose can be calculated at any point in the physical chain , including a computed tomography dose index ( ctdi ) type of dose assessment for cone beam imaging . the quality description also incorporates an image quality degradation resulting from , e.g. [ 12 , 13 ] , allows us to vary the system parameters in x - ray generation , absorption , dose , and detection as well as in image processing quality , yielding accurate verified results . a number of self - expandable nitinol stents ( table 1 ) , as well as one steel - based stent for reference , were deployed in plastic ( infuse ) tubes with an inner diameter of 3.5 mm which were inserted in a channel of an anthropomorphic head phantom ( cirs , norfolk , virginia ; model 603 ) , placed in the system isocenter . the tubes were filled with a diluted contrast agent : 500 ml h2o and 50 ml visip 270 ( ge ) , in order to produce a 550 hu density mimicking a contrast - filled vessel . the remainder of the phantom channel was filled with diluted contrast agent : 5,000 ml h2o and 50 ml visip 270 , producing 31-hu density . another set of experiments was carried out by filling the tubes with 31-hu diluted contrast agent and the remaining volume again with 31 hu , representing blood . yet , another experiment was performed by inserting platinum coiling wires close to the stents , in an air - filled cavity in the phantom , enabling an assessment of streaking effects . table 1an overview of the stent propertiestypemanufacturerlength ( mm)diameter ( mm)strut cross - section ( m)materialneuroformboston scientific153w68*t66nitinolnatick , maenterprisecordis374.5nitinolbridgewater , njsolitaireev3204 80nitinolplymouth , mnwingspanboston scientific203.5w68*t73nitinolnatick , masilkbalt extrusion253.5nitinolmontmorency , frfour pt wiresleo+balt extrusion252.5nitinolmontmorency , frtwo pt wirespharosmicrus / biotroniks252.75 60steelsan jose , cacr co alloythe manufacturers did not supply the dimensions an overview of the stent properties the manufacturers did not supply the dimensions we used a philips xper vascular system equipped with a large ( 30 40 cm ) flat detector and a 3d workstation for producing the 3d rendered images and hosting the typical 3d image post - processing modules . images were acquired with a standard 3d protocol ( with 4549 mgy ctdi dose ) using a large detector zoom format in combination with a large tube focus , or in a zoomed detector format ( 22-cm imaging diagonal ) and a small tube focus . a set of 620 images was acquired with a 30 fr / s , resulting in a 20-s scan - time over a 200-degree arc - travel . ctdi was measured using a standard measurement protocol with a 16-cm diameter polymethyl methacrylate ( pmma ) cylinder , and dose was measured with the unfors ( billdal , sweden ) mult - o - meter 601-pms . we determined the values using the largest irradiation field while keeping the technique factors identical to those for the high - resolution case with its smaller field . spatial resolution was measured with a catphan 500 phantom ( the phantom factory , salem ny ) . the resolution reading was limited by the phantom maximum of 2.1 lp / mm . the reconstructions were carried out in a zoomed mode , i.e. , a region of interest was chosen as a fraction of the maximum volume , determined by the detector size and the projection geometry . the zoomed secondary reconstructions were carried out by panning the volume such that the relevant stent phantom portions were centered therein . table 2volume and voxel dimensions for the standard protocol and the high - resolution / high - contrast imaging protocolreconstruction zooming ( % ) volume ( 256 voxel matrix ) ( mm)voxel size ( mm)hires5052.8 52.8 52.80.213334.4 34.4 34.40.13standard3382.7 82.7 63.90.321742.6 42.6 32.90.17 volume and voxel dimensions for the standard protocol and the high - resolution / high - contrast imaging protocol the high contrast limiting resolution has been optimized by balancing the relevant mtf of the imaging components , taking noise transfer into account . focus size , detector pixel size , and the reconstruction voxel size are matched such that the transfer contributions of each parameter are balanced , resulting in an optimal voxel size and voxel number . a further reduction of the voxel size would increase the image noise , whereas an increase of the voxel size would result in a spatial resolution loss . the true resolution of the high - resolution protocol is better than the maximum reading of the catphan 500 phantom and could thus not be read by this phantom . deviations of 1020% between measured and simulated values were found , which can be accounted for by the choice of the threshold modulation of 4% . since the basic materials of the stents are identical for all types ( see table 1 ) , we decided to only use one stent type , namely a wingspan nitinol stent . only in case of the high - resolution / high - contrast case , the imaging results of all stents are shown , including the pharos reference stent . table 3limiting high contrast spatial resolution as measured with a catphan 500 phantom ( the phantom factory ) and simulated with the iq analysis programspatial resolutionvolume definitionmeasured ( lp / mm)simulated ( lp / mm)standardnon - zoomed reconstruction volume0.50.533% reconstruction volume1.31.617% reconstruction volume1.61.8hiresnon - zoomed reconstruction volume1.11.250% reconstruction volume2.02.533% reconstruction volume2.13.2catphan 500 phantom range limit limiting high contrast spatial resolution as measured with a catphan 500 phantom ( the phantom factory ) and simulated with the iq analysis program catphan 500 phantom range limit figures 1 , 2 , 3 , 4 , and 5 show snapshots of reconstructed images in planes through the stent 's axes and centered within the volume , where window settings on the work station were such that contrast and noise were comparable . figure 1 shows the results of the three protocols for 31 hu , where for reference , an optical image of the stent is shown also . figure 2 shows the images for the high contrast tube filling of 550 hu . 5 , a number of stent types illustrate the performance in the high - resolution / high - contrast mode , for again 31-hu contrast liquid . figure 5 also shows example results of the more radiopaque material platinum in the silk and leo stents and the co cr steel alloy - based pharos stent . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . for the standard resolution , 17% and for comparison , a contrast inverted optical image is shown on the far leftfig . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . in all cases , an intra - tube high - density contrast agent of 550 volumes are denoted as 50% and 33% for the hires case . for the standard resolution , 17% and 33% images a comparison between the high - resolution cases for high - contrast ( 80 kv ) ( a ) , intermediate contrast ( 100 kv ) ( b ) , and standard contrast ( 120 kv ) ( c ) . a high - resolution / high - contrast , b standard resolution / high - contrast case , and c the standard resolution / standard contrast casefig . 5high - resolution / high - contrast cases for 50% and 33% reconstruction volumes and various stent types . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . for the standard resolution , 17% and for comparison , a contrast inverted optical image is shown on the far left a wingspan nitinol stent . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . in all cases , an intra - tube high - density contrast agent of 550 volumes are denoted as 50% and 33% for the hires case . for the standard resolution , 17% and 33% images are shown a wingspan nitinol stent . a comparison between the high - resolution cases for high - contrast ( 80 kv ) ( a ) , intermediate contrast ( 100 kv ) ( b ) , and standard contrast ( 120 kv ) ( c ) . a high - resolution / high - contrast , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case high - resolution / high - contrast cases for 50% and 33% reconstruction volumes and various stent types . the actual dose measurement and the analysis table 4measured and simulated ctdi dose for the standard protocol and the high - resolution / high - contrast protocolctdimeasured dose ( mgy)simulated dose ( mgy)standard4543hires4955 measured and simulated ctdi dose for the standard protocol and the high - resolution / high - contrast protocol figures 1 , 2 , 3 , 4 , and 5 show snapshots of reconstructed images in planes through the stent 's axes and centered within the volume , where window settings on the work station were such that contrast and noise were comparable . figure 1 shows the results of the three protocols for 31 hu , where for reference , an optical image of the stent is shown also . figure 2 shows the images for the high contrast tube filling of 550 hu . the effect of varying the tube voltage is shown in fig . 5 , a number of stent types illustrate the performance in the high - resolution / high - contrast mode , for again 31-hu contrast liquid . figure 5 also shows example results of the more radiopaque material platinum in the silk and leo stents and the co cr steel alloy - based pharos stent . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . for the standard resolution , 17% and for comparison , a contrast inverted optical image is shown on the far leftfig . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . in all cases , an intra - tube high - density contrast agent of 550 volumes are denoted as 50% and 33% for the hires case . for the standard resolution , 17% and 33% images a comparison between the high - resolution cases for high - contrast ( 80 kv ) ( a ) , intermediate contrast ( 100 kv ) ( b ) , and standard contrast ( 120 kv ) ( c ) . a high - resolution / high - contrast , b standard resolution / high - contrast case , and c the standard resolution / standard contrast casefig . 5high - resolution / high - contrast cases for 50% and 33% reconstruction volumes and various stent types . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . for the standard resolution , 17% and a contrast inverted optical image is shown on the far left a wingspan nitinol stent . a comparison between a the high - resolution / high - contrast case , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case . in all cases , an intra - tube high - density contrast agent of 550 volumes are denoted as 50% and 33% for the hires case . for the standard resolution , 17% and 33% images are shown a wingspan nitinol stent . a comparison between the high - resolution cases for high - contrast ( 80 kv ) ( a ) , intermediate contrast ( 100 kv ) ( b ) , and standard contrast ( 120 kv ) ( c ) . a high - resolution / high - contrast , b standard resolution / high - contrast case , and c the standard resolution / standard contrast case high - resolution / high - contrast cases for 50% and 33% reconstruction volumes and various stent types . the actual dose measurement and the analysis table 4measured and simulated ctdi dose for the standard protocol and the high - resolution / high - contrast protocolctdimeasured dose ( mgy)simulated dose ( mgy)standard4543hires4955 measured and simulated ctdi dose for the standard protocol and the high - resolution / high - contrast protocol intracranial stenting became feasible as a routine treatment by the introduction of very flexible stents and have become a single treatment for a number of neurovascular conditions ( biondi et al . the stents are manufactured from a very thin material and most are self expanding , i.e. however , most nitinol stents are difficult to visualize with fluoroscopy or c - arm cone beam ct . visualization can be improved by increasing the density of the stent , more radiation , or enhanced image acquisition settings and processing . consequently , the visibility is preferably improved by further optimizing the imaging chain . to support and judge correct positioning , the nitinol stent should be visualized in its wall apposition , while conforming to varying diameters throughout the stent 's length . to this end , it is advantageous to view details with virtually optical quality . during the past 6 years , the image quality of ct - like reconstructions , using a c - arm interventional x - ray system equipped with a flat detector , became increasingly notable . high - resolution imaging , with submillimeter isotropic spatial resolution , outperformed digital radiography , fluoroscopy , and even conventional ct ( kamran et al . ) a continuous effort to enhance image quality enabled imaging of details with low absorption . evidence on high - quality in vitro imaging of the proper deployment of nitinol stents , related to area coverage , kinking , prolapse , and flattening , is given by aurboonyawat , ebrahimi and alvarado . in a clinical setting , imagery of balloon mounted stents , based on a cr co steel alloy , with intravenous administration of contrast medium , was carried out by buhk et al . demonstrated high - resolution and contrast - enhanced simultaneous imaging of intraarterial cerebrovascular stents and their host arteries . these two measures lead to an improved visibility of the stent 's struts , when comparing the standard protocol ( 17% zooming ) with the results of the high - resolution / high - contrast protocol ( 33% zooming ) . the open cell structure of the stent is clearly discerned by virtually continuous strokes of the struts , so that its x - ray quality is approaching an optical quality . in this case , the contrast between struts and background is lower due to the denser filling of the tube , giving an accordingly reduced cnr . 3 , where the comparison is made for a varying tube voltage : the obvious increase in the cnr makes the stent more pronounced with respect to the ( noisy ) background and accounts for the choice of the lowest voltage , 80 kv , for the ics and hires protocols . 4 , although the window settings are optimal for stent perception , details are still rendered with a sufficient contrast to artifact distance . 5 displays reconstructions of all stent types for the high - resolution / high - contrast case , showing the detail rendering of the protocol . the silk and leo stents stand out more by high object contrast of the platinum auxiliary wires . for all protocols under test , the maximum weighted ctdi dose of 50 mgy is lower than the european guidelines , as according to eur 16262 . due to the limited anatomical coverage , by using a smaller irradiated field in the high - resolution case , moreover , we found that the difference between modeled and measured doses is sufficiently small . we have optimized nitinol stent imaging , in the framework of a full - scale 3d imaging model for x - ray imaging . by balancing the sharpness transfer and noise transfer of the imaging components and tuning the contrast - rendering capabilities of a 3d x - ray imaging system , we showed that thin nitinol stent struts can be viewed with a high cnr and good detail rendering . while optimizing , the ctdi radiation doses were kept below recommended values . the quality of 3d images , produced with optimum system settings , proved that independent of the type or manufacturer of the stents , the detail rendering is adequate to assess the post - deployment shape .

the simultaneous measurement of expression levels of tens of thousands of genes in a biological sample enabled by dna microarray technology has provided a new and powerful way to characterize the molecular basis of diseases such as cancer [ 1 , 2 ] . in the past decade , mrna expression profiles of tumor tissues have been successfully used to distinguish tumor types or subtypes [ 35 ] . they also appear to hold great promise as a method for predicting clinical outcomes [ 68 ] . for example , gene expression profiles have been used to classify lung adenocarcinoma into subgroups that correlated with the degree of tumor differentiation as well as patient survival . gene expression profile analysis initially emphasized the identification of groups of genes that are differentially regulated in different experimental conditions or patient samples . coexpression across a variety of samples implied coregulation or similar function [ 10 , 11 ] . an approach complementary to this gene - centered view is to take a sample - centered perspective in which one treats the genome - wide profiles of each sample as the entities to be classified with respect to their gene expression patterns . the goal here is to assign samples ( rather than genes ) to groups based on the high - dimensional molecular signature determined by the thousands of individual gene expression values . while the gene - centered perspective is useful for understanding the molecular pathways in which individual genes are involved , the sample - centered view is more relevant for biological and clinical questions , such as in the study of the developmental and pathogenetic relationship between tissues as a whole [ 12 , 13 ] or the identification of prognostic or diagnostic signatures of tumors based on entire gene expression profile portraits [ 4 , 1419 ] . the notion of molecular portraits has gained importance as gene expression profiles for increasingly large numbers of samples or conditions ( eg , experimental variables , patients , treatment groups , etc ) have become available [ 18 , 20 , 21 ] . however , the analysis of large numbers of gene expression profiles as integrated entities poses a challenge in terms of how to best organize and graphically present the high - dimensional data without loss of the notion of an individual profile as an independent entity . it would be desirable to capture the global picture of sample clusters within one visual representation while simultaneously presenting the specific expression pattern within each individual sample , and hence , simultaneously allowing gene - specific analysis . current representations , such as the widely used heat maps in two - way hierarchical clustering [ 22 , 23 ] or coordinate systems in principal component analysis ( pca ) , multidimensional scaling ( mds ) and their variants [ 2426 ] , compress the expression profile information of a sample into a single quantity , such as a scalar value for the distance ( dissimilarity ) between the sample , a branch in a dendrogram , a narrow column in a heat - map , or a point in reduced - dimensional space . such aggregate displays discard possibly relevant information immanent in the complex , higher - order ( system - level ) genome - wide expression pattern . this intrinsic but hidden information reflects the collective behavior of genes orchestrated by genome - scale gene regulatory networks that govern cell behavior . as pathology and radiology teach us , the implicit visual cues present within a complex image ( eg , histological section , radiograph ) can not be reduced to a set of numerical variables without loss of system - level information content . thus , it is possible that some irreducible information contained within high - dimensional gene profiles of patient or experimental samples may be lost in current clustering and representation methods . in the absence of specific questions or hypotheses , it would therefore be desirable to be able to directly compare microarray results of individual tumor samples with their complete feature - richness in the same holistic way as pathologists compare histological tumor samples , namely , based on human gestalt perception . in contrast to histological patterns , the thousands of expression values in a microarray measurement are too dense and irregular to be directly interpreted in a holistic manner . hence , they must be presented in a form appropriate for human pattern recognition without discarding the global , higher - order information . self - organizing maps ( soms ) have the capacity to display information - rich diagrams . in the case of microarray data they can present individual samples as an entity and , at the same time , display high - resolution patterns within the transcriptome . a self - organizing map is a neural network algorithm for unsupervised machine learning with a strong visualization capability . in brief , it assigns a set of n input objects ( eg , genes ) to a number k ( k < n ) rectangular or hexagonal tiles ( som nodes ) , each of which represents a cluster of objects ( genes ) , arranged so as to form a coherent pattern within a two - dimensional mosaic ( som grid ) . the patterns arise because the distances between the tiles on the mosaic are a function of the similarity between the gene clusters that the tiles represent , with most similar clusters being adjacent to each other in the mosaic . early applications of soms for visualization of gene expression profiles emphasized the gene - centered perspective ( clustering of genes ) and used each tile to represent one cluster of genes in order to identify gene clusters with interesting expression patterns or to link them to gene functions . as in k - means clustering , the number of clusters k is chosen in this approach to approximate the number of expected number of gene clusters , for example , k = 12 on a 3 4 = 12 node grid . other studies used som in the sample - centered mode to map individual tumor samples onto the som grid and thereby classify tumor samples into a small number of diagnostic or prognostic groups [ 32 , 33 ] . in both cases , an entire experiment consisting of multiple samples ( expression profiles ) was represented by one single som grid , and the sample - specific visualization capabilities of som were not explored . in another study , soms were used in the gene - centered mode to analyze lymphoma samples , but the number of clusters ( k = 22 14 ) was much larger than the expected number of biological clusters . the characteristic som mosaics contained coherent patterns generated by the colored tiles ordered so as to reflect the clustered gene expression profile of the individual samples . but while this approach used the visual representation of som , it still focused on finding subset of genes for classifying tumors . in these cases the som maps were used as graphical representation mostly to illustrate a particular algorithm of analysis , much as dendrograms serve to give evidence of hierarchical clustering , but are not actually read by the human eye to obtain specific information . instead , we propose that som displays can be specifically treated as new , complex objects for a next level analysis , namely , visual gestalt recognition . thus , we do not use computer algorithm in the sense of artificial intelligence , but more as intelligence enhancement for the human brain in the holistic comparison of the transcriptomes . to enable such an integrative analysis based on visualization of each tumor sample as a unique and complex molecular portrait , we adapted gedi a som - based tool developed for visualizing the dynamics of genome - wide gene expression profiles to represent static microarray samples as two - dimensional high - resolution som mosaics . using published gene expression profiles from a large set of lung tumor samples , we offer a first assessment of the usefulness of this type of holistic visual analysis of tumor gene expression profiles . these studies reveal that human gestalt perception can lead to discovery of novel biological features without a preconceived hypothesis , and uncover new relationships between lung tumor subtypes that had previously escaped the analysis using conventional algorithmic classification techniques [ 5 , 36 ] . starting from an n m matrix of data from the analysis of n genes across m samples ( mrna expression profiles ) , gedi transforms each sample 's expression profile into a map that contains a visually recognizable color pattern , referred to as a gedi map . these maps are mosaics generated by self - organizing maps ( soms ) ( see materials and methods ) . in brief , this was achieved by ( i ) a moderate reduction of dimensionality with respect to the genes , from n genes into k gene clusters , which are represented by metagenes , and ( ii ) by a spatial reordering of these metagenes onto a two - dimensional space represented by an a - by - b grid ( with a b = k ) using som . the expression values of the metagenes are the centroids of the corresponding clusters and are displayed as one of the k colored since the soms assign the same metagene to the same tile for each mosaic , they can be compared to each other . moreover , since metagenes that exhibit a similar behavior with respect to the m samples are placed next or close to each other on the mosaic , the tiles collectively create a coherent pattern on each mosaic that is characteristic for each sample [ 29 , 38 ] . importantly , in contrast to conventional cluster analysis using k - means or som , where typically k < 30 clusters , here k is many folds higher than the expected or desired number of biologically significant clusters , and hence , each of the k metagenes can be viewed as representing a minicluster of just a few genes ( with a typical median of around 10 genes ) . a minicluster is thus not meant to represent some biologically relevant gene cluster . instead , the som algorithm is used to pixelate the expression profile into k pixels and rearrange them , which is why k is required to be high : typically , k = 100 s to 1000 s [ 12 , 13 ] . these miniclusters consequently contain an order of magnitude fewer genes than in conventional gene clustering and are hence more homogeneous , warranting the representation as a metagene . accordingly , the patterns formed by the metagenes on a gedi map will be referred to as metapattern . based on the characteristic visual metapatterns , gedi maps allow the direct comparison of the biological samples , as well as immediate identification of biologically interesting groups of genes . the genome - wide gene expression profiles of 25 samples of normal lung tissue ( lung ) and different pulmonary tumors , carcinoid ( car ) , squamous cell carcinoma ( sq ) , and small - cell cancer ( smc ) , were visualized as 25 gedi maps , each consisting of a 31-by-30 mosaic , representing 930 miniclusters . discrete differences in patterns of gene expression between normal lung and tumor samples are immediately detected upon visual inspection of the gedi maps ( figure 1 ) . each sample exhibits characteristic spatial and color patterns , reflecting genome - wide transcriptional behavior of the respective tissue sample . the visual patterns of the gedi maps of these different tissue samples remained distinct when the analysis was performed with a wide range of som parameters and the soms were run to convergence ( not shown ) . inspection of gedi maps allows a straightforward classification of the samples into subgroups without the aid of a clustering algorithm , but simply based on the visual differences in the metapatterns . samples grouped together with members of the same category , with the exception of one outlier , a small - cell lung cancer sample , smc6 whose gedi map looked different ( figure 1 ) . as previously demonstrated , hierarchical cluster analysis reliably arranged these lung tumor samples into distinct clusters which corresponded well to the different clusters identified using gedi ( figure 1 ) . a known drawback of hierarchical clustering is that the linear arrangement of the clustered objects ( samples ) at the terminal branches of the dendrogram can be presented in multiple ways ( orderings ) . this can make the unbiased global assessment of intersample similarity across all the samples difficult . although this arbitrariness can be eliminated by using a one - dimensional som , k - means clustering , or other optimization algorithm to achieve some objective branch ordering [ 39 , 40 ] , this method is not often used . by contrast , because there is no a priori clustering structure in gedi , sample clustering is directly obvious and robust and avoids bias suggestion of relatedness a known problem with hierarchical clustering . another shortcoming of hierarchical clustering is that the hierarchical relationship displayed in the dendrograms does not necessarily have a biological meaning . for example , hierarchical clustering forces the randomly permuted data into a tree structure with similar overall structure ( albeit with a higher distance score between the branches ) even though the samples have now random attributes and have no meaningful relation ( figure 2 ) . in contrast , in this case the gedi maps immediately reveal the poor quality of clustering : the samples that were clustered together by hierarchical clustering do not exhibit any consistent global pattern ( figure 2 ) . therefore , gedi also provides a first - line sample - centered quality control for traditional clustering methods . because gedi maps provide a global view of the gene expression profiles of each sample , they immediately present an explanation for why a particular sample behaves as an outlier ( when sample diagnosis is known ) and which genes account for that behavior . for example , the dramatic difference between the gedi map of an outlier , smc6 ( figure 1 ) , relative to samples within the cluster of nominal small - cell lung cancers immediately reveals that smc6 deviates from the other small - cell carcinomas and the different pattern of tiles explains why . in addition to visually comparing gedi maps as individual entities , one can extract the numerical centroid values yk in sample j of each metagene k to analyze gedi maps quantitatively . by utilizing the metagenes instead of the real genes to characterize a transcriptome , the complexity is reduced , in our case from the original data matrix n m = 12562 25 to 930 25 . to evaluate the fidelity of gedi mosaic patterns in representing the expression profiles established by all the genes , we calculated the correlation coefficients rjk for every pair of samples ( j , k ) using either ( 1 ) the expression data for all of the individual genes or ( 2 ) the metagenes . if the gedi mosaic patterns of metagenes faithfully represent the genome - wide gene expression profiles , the correlation coefficients for all sample pairs calculated in these two ways will be similar . in fact , the gedi patterns preserved the correlation between samples obtained from the real gene expression data ( figures 3(a ) , 3(b ) ) . the correlation of the values and the ranks of rjk between the two methods were 0.909 and 0.960 , respectively . interestingly , the values of the correlation coefficients ( profile similarity between samples ) calculated from metagenes spanned a considerably broader range than those from the real gene expression dataset , as apparent in the histograms of the correlation values ( figures 3(c ) , 3(d ) ) . color contrast of the correlation matrix color map ( figure 3(b ) versus 3(a ) ) . thus , it appears that the discriminating power of this technique using metagenes may be increased relative to standard microarray analysis . the differences in the average correlation between sample pairs within the same tissue groups ( intratissue pairs ) and across tissue groups ( intertissue pairs ) were considerably larger when metagenes ( 0.127 , 95% confidence interval : 0.109 to 0.145 ) were used for calculating the correlation , compared to when real genes ( 0.069 , 95% confidence interval : 0.058 to 0.080 ) are used ( figure 3(e ) ) . it remains to be determined statistically in extended data sets whether metagene - based analysis consistently has a greater discriminating power by using larger test sets of tissue samples for patient groups with established diagnosis . in summary , the gedi maps based on metagenes faithfully recapitulate gene expression profiles of the entire gene dataset despite dimension reduction . thus , the visual patterns capture the real similarity relationships among samples with a high fidelity . to further validate how well metapatterns can represent the transcriptome second - level gedi analysis to categorize gedi maps automatically using the ( n = 930 metagenes , we also performed a pca on the original gene data matrix ( with sample columns as the objects and gene rows as the attributes ) . the second - level gedi analysis differed from the first - level gedi analysis performed on the ( n = 12562 real genes m = 25 samples ) matrix in that the objects of clustering were the samples but not genes , and thus a smaller som grid was used . given the discriminatory power of the metagenes , using them as input variables may improve the quality of sample clustering . the 25 samples were assigned to a 5 5 som grid according to their metagene expression profiles . in the resulting second - level gedi map , the tissue samples ( the first - level gedi maps ) of the same diagnosis were grouped within the same neighborhood of the map ( figure 4(a ) ) . the map distances from each tumor - specific sample cluster to that of normal lung ( lung ) were roughly similar , while among the tumors , the carcinoid ( car ) and squamous cell carcinoma ( sq ) samples were most distant from each other , with small - cell lung cancer ( smc ) in between . interestingly , the spatial distribution of these samples in the two - dimensional second - level map was very closely mirrored in the pca in which the samples were projected on the plane spanned by the two first eigenvectors ( figure 4(b ) ) . there was good agreement even with respect to the relative position of the individual samples within each tumor and tissue type ( figure 4(a ) versus 4(b ) ) . importantly , such information revealed by the 2d sample plane , be it the som grid of the second - level gedi or the pca plane , can be directly read from the metapatterns of the gedi maps . visual inspection of the gedi maps readily confirms the notion that sq2 displays significant feature similarity to the smc samples based on the fine structure of the patterns of upregulated genes . specifically , the gedi metapattern showed that sq2 lacked the extension of the red areas ( highly expressed genes ) from the right half into the upper - left quadrant of the gedi map that is characteristic for the other sq samples ( figure 4 ) . interestingly , this group of metagenes that was not expressed in sq2 contained multiple keratin - related genes , consistent with the squamous cell origin of these tumors . without the gedi maps , the samples would be represented by dots in the pca which would be identified solely by their position in the abstract eigenvector space . thus , gedi allows the rapid toggling between gene - centered and sample - centered perspectives , which is an important feature for an integrative yet gene - specific analysis . like small - cell carcinoma , lung carcinoid tumors are also classified as ( low - grade ) neuroendocrine tumors , while squamous carcinoma appears to be unrelated to this group . however , in both hierarchical clustering as well as in pca , smc was closer to sq than to car ( figures 1 and 4 ) , which is consistent with the idea that small - cell lung carcinoma may have an epithelial origin , but competes with the notion of the common neuroendocrine property of smc and car . to examine this dualism we used gedi to analyze the relationship between these three pulmonary tumors and normal lung to compare not only by how much but also how each of these tumors qualitatively differed from normal lung tissue and from each other . the gedi software environment allows the user to easily perform algebraic operations on whole mosaic patterns based on metagene expression values , and for instance to calculate average mosaics from a group of samples with the same diagnosis or difference mosaics to reveal differential expression patterns between two samples ( or averages of two groups ) . ( figure 5(a ) ) by subtracting the averaged gedi maps of normal lung samples from that of smc , car , or sq , respectively . the red areas in the difference maps indicate genes that were upregulated in these tumors compared to normal lung tissue . the outlined areas on the maps represent four islands ( labeled a , b , c , d in figure 5(a ) ) that contain the top 5% differentially expressed genes in smc versus lung . these studies revealed that smc and sq share a set of features , representing a number of genes located within regions a this is consistent with the vicinity of these two tumors in the dendrogram ( figure 1 ) and in the pca sample plane ( figure 4 ) ; it also is in line with the proposed epithelial origin of small - cell lung cancer . c included growth - related genes ( involved in cell proliferation , cell cycle , dna replication , etc ) . such functional enrichment of genes in the gene islands underscores the biological meaning of pattern features in gedi maps . interestingly , the smc samples while globally close to sq samples , shared with the car samples the island d , which contained neuroendocrine - related genes ( involved in synaptic vesicle , neuromuscular physiological process , etc ) , consistent with the neuroendocrine nature of small - cell lung carcinoma . this example illustrates how gedi can extract relationship features that are not revealed by traditional hierarchical clustering or any reduction of sample comparisons to a similarity metric . specifically , while three islands ( a c ) that represent the regions of metagenes upregulated in smc compared to normal lung also were found in sq ( a , b , c ) , they were absent from car . conversely , the island d that was enriched for the neuroendocrine genes was overexpressed in car but not in sq . thus , the gedi analysis exposed a novel facet of relationship between the samples with respect to these signature gene clusters : smc appears to be the union set of the sets of car and sq ( figure 5(b ) ) , sharing the gene cluster d with car and the clusters a , b , and c with sq . with respect to these growth - related gene islands thus , despite the overall higher similarity between smc and sq , when considering the subfeature d with the neuroendocrine genes , smc was closer to car than to sq . such information on a qualitative relationship is lost in conventional clustering dendrograms that reduce relationships to a numerical similarity between two samples . without an a priori hypothesis , such qualitative relationships are almost impossible to identify in the widely used heat maps , but they immediately spring to eye in the differential gedi maps . genome - scale gene expression profiles are not simply high - dimensional sets of variables that provide an opportunity for multivariate statistical analysis . instead , they are the biological manifestation of the constrained dynamics of the underlying complex and hierarchical gene regulatory networks that govern developmental potentials of cells and tissues . tumors arise from mutational rewiring of this molecular network and therefore , display specific , coordinated deviations from the normal transcriptome patterns . to visualize coherent , genome - scale alterations of the transcriptome structure , we used here an integrative visual representation for gene expression profiles . as a test example we analyzed expression profiles of three lung tumor samples as a case study . we show that by delegating the actual process of pattern recognition to human gestalt perception in the format of som - based gedi mosaics , interesting features in the relationship between tumor types can be revealed . specifically , we found that with respect to pathological deviation from normal gene expression , small - cell carcinoma represents the union set of squamous cell carcinoma and carcinoids . such information on higher - order transcriptome changes , which may be useful for understanding developmental relationships and differences in drug responsiveness between tumor types , spring to eye in the gedi maps , but would not have been revealed in conventional algorithms without explicitly asking the appropriate question . microarray - based molecular profiles are increasingly used to capture characteristic high - dimensional molecular most existing methods reduce complex relationships to a numerical value , typically , a distance metric or a visual distance between points in a reduced dimension space . while this is useful for explicitly extracting specific information , these methods may lose potentially useful , unanticipated information inherent in the high - dimensional expression profiles , such as particular higher - order patterns of expression . similarly , even the search for a multigene signatures instead of a single marker gene to improve discrimination between diagnostic groups may miss some of the distributed ( holistic ) information in the profiles . in fact , maximal accuracy of multiclass tumor classification may require that the predictor utilizes all the genes . the gedi visualization software was developed to circumvent the problem of discarding implicit , potentially irreducible information inherent in genome - wide expression profiles in the absence of a specific hypothesis . it provides the opportunity for a holistic , yet molecular exploration of a set of gene expression profiles ( or other high - dimensional data sets ) that can be used to test existing tissue - level biological hypotheses or establish new ones . although gedi uses a som algorithm at its core , it differs fundamentally from the traditional use of som to find biologically meaningful clusters [ 30 , 32 , 33 , 38 ] . the metagenes in gedi are miniclusters that are smaller by an order of magnitude than the explicitly predefined clusters in the conventional cluster analysis , hence they are very tight and of high quality . the identification of biological clusters is not the result of the clustering algorithm per se , but is achieved at a later stage of analysis , namely , by visual inspection and gestalt perception of the metapatterns that emerge from the som - generated metagenes . hence , ambiguities in clustering of samples are not built into the algorithm , but are subject to direct and interactive analysis by the interpreter . gedi provides several technical benefits relative to existing high - dimensional data analysis methods . ( a ) by presenting metapatterns , gedi maps provide a visual engram of each sample 's particular molecular profile , and hence , establish a molecular portrait in the very sense of the word , with a particular visual identity for each sample ( eg , tumor type , patient , treatment condition ) . ( b ) although classification of samples into groups is achieved by human gestalt perception of the metapatterns , it can be supported by an algorithmic approach applied on the metagenes . ( c ) the direct visual monitoring of the portrait of a sample allows gedi to intercept algorithmic idiosyncrasies , such as the dependence of the branching structure of dendrograms on the particular tree - building algorithm used in hierarchical clustering . ( d ) despite a moderate dimension reduction , gedi preserves most of the information richness of entire molecular portraits , allowing detailed , multivariate explorative comparisons between samples . this in turn can help define qualitative differences ( in addition to measuring quantitative dissimilarity between samples ) that may provide additional biological information on the relationships between samples . ( e ) gedi allows the rapid and seamless switching between an integrative , sample - oriented analysis and the more traditional gene - centered analysis . this is facilitated by the interactive user interface that permits retrieval of genes that contribute to metapattern features of interest . ( f ) finally , using gedi to compare the samples and relate them to each other does not require specific knowledge of the underlying algorithm , and thus is an intuitive tool for nonbioinformaticians , such as pathologists and clinicians that will increasingly confront microarray analysis . this is specifically relevant for the explanation of anomalies in cluster analysis , such as outlier samples . the reason for misclassification is usually directly evident in the gedi map and does not require familiarity with the details of the algorithm . although biologists have begun to use gedi maps [ 12 , 13 , 44 ] to ask biological questions , further systematic elucidation of its application , notably , the choice of optimal size of miniclusters is needed . future use of gedi in studies of the genome - scale molecular signature of both normal and disease samples will ultimately help assess the true value of a holistic interpretation of molecular profiles that systems biology is advocating .

genome - wide gene expression profile studies encompass increasingly large number of samples , posing a challenge to their presentation and interpretation without losing the notion that each transcriptome constitutes a complex biological entity . much like pathologists who visually analyze information - rich histological sections as a whole , we propose here an integrative approach . we use a self - organizing maps -based software , the gene expression dynamics inspector ( gedi ) to analyze gene expression profiles of various lung tumors . gedi allows the comparison of tumor profiles based on direct visual detection of transcriptome patterns . such intuitive gestalt perception promotes the discovery of interesting relationships in the absence of an existing hypothesis . we uncovered qualitative relationships between squamous cell tumors , small - cell tumors , and carcinoid tumor that would have escaped existing algorithmic classifications . these results suggest that gedi may be a valuable explorative tool that combines global and gene - centered analyses of molecular profiles from large - scale microarray experiments .

1. INTRODUCTION RESULTS DISCUSSION

in the past decade , mrna expression profiles of tumor tissues have been successfully used to distinguish tumor types or subtypes [ 35 ] . they also appear to hold great promise as a method for predicting clinical outcomes [ 68 ] . for example , gene expression profiles have been used to classify lung adenocarcinoma into subgroups that correlated with the degree of tumor differentiation as well as patient survival . gene expression profile analysis initially emphasized the identification of groups of genes that are differentially regulated in different experimental conditions or patient samples . an approach complementary to this gene - centered view is to take a sample - centered perspective in which one treats the genome - wide profiles of each sample as the entities to be classified with respect to their gene expression patterns . the goal here is to assign samples ( rather than genes ) to groups based on the high - dimensional molecular signature determined by the thousands of individual gene expression values . while the gene - centered perspective is useful for understanding the molecular pathways in which individual genes are involved , the sample - centered view is more relevant for biological and clinical questions , such as in the study of the developmental and pathogenetic relationship between tissues as a whole [ 12 , 13 ] or the identification of prognostic or diagnostic signatures of tumors based on entire gene expression profile portraits [ 4 , 1419 ] . the notion of molecular portraits has gained importance as gene expression profiles for increasingly large numbers of samples or conditions ( eg , experimental variables , patients , treatment groups , etc ) have become available [ 18 , 20 , 21 ] . however , the analysis of large numbers of gene expression profiles as integrated entities poses a challenge in terms of how to best organize and graphically present the high - dimensional data without loss of the notion of an individual profile as an independent entity . it would be desirable to capture the global picture of sample clusters within one visual representation while simultaneously presenting the specific expression pattern within each individual sample , and hence , simultaneously allowing gene - specific analysis . current representations , such as the widely used heat maps in two - way hierarchical clustering [ 22 , 23 ] or coordinate systems in principal component analysis ( pca ) , multidimensional scaling ( mds ) and their variants [ 2426 ] , compress the expression profile information of a sample into a single quantity , such as a scalar value for the distance ( dissimilarity ) between the sample , a branch in a dendrogram , a narrow column in a heat - map , or a point in reduced - dimensional space . such aggregate displays discard possibly relevant information immanent in the complex , higher - order ( system - level ) genome - wide expression pattern . this intrinsic but hidden information reflects the collective behavior of genes orchestrated by genome - scale gene regulatory networks that govern cell behavior . as pathology and radiology teach us , the implicit visual cues present within a complex image ( eg , histological section , radiograph ) can not be reduced to a set of numerical variables without loss of system - level information content . thus , it is possible that some irreducible information contained within high - dimensional gene profiles of patient or experimental samples may be lost in current clustering and representation methods . in the absence of specific questions or hypotheses , it would therefore be desirable to be able to directly compare microarray results of individual tumor samples with their complete feature - richness in the same holistic way as pathologists compare histological tumor samples , namely , based on human gestalt perception . in contrast to histological patterns , the thousands of expression values in a microarray measurement are too dense and irregular to be directly interpreted in a holistic manner . self - organizing maps ( soms ) have the capacity to display information - rich diagrams . in the case of microarray data they can present individual samples as an entity and , at the same time , display high - resolution patterns within the transcriptome . a self - organizing map is a neural network algorithm for unsupervised machine learning with a strong visualization capability . in brief , it assigns a set of n input objects ( eg , genes ) to a number k ( k < n ) rectangular or hexagonal tiles ( som nodes ) , each of which represents a cluster of objects ( genes ) , arranged so as to form a coherent pattern within a two - dimensional mosaic ( som grid ) . the patterns arise because the distances between the tiles on the mosaic are a function of the similarity between the gene clusters that the tiles represent , with most similar clusters being adjacent to each other in the mosaic . early applications of soms for visualization of gene expression profiles emphasized the gene - centered perspective ( clustering of genes ) and used each tile to represent one cluster of genes in order to identify gene clusters with interesting expression patterns or to link them to gene functions . as in k - means clustering , the number of clusters k is chosen in this approach to approximate the number of expected number of gene clusters , for example , k = 12 on a 3 4 = 12 node grid . other studies used som in the sample - centered mode to map individual tumor samples onto the som grid and thereby classify tumor samples into a small number of diagnostic or prognostic groups [ 32 , 33 ] . in both cases , an entire experiment consisting of multiple samples ( expression profiles ) was represented by one single som grid , and the sample - specific visualization capabilities of som were not explored . in another study , soms were used in the gene - centered mode to analyze lymphoma samples , but the number of clusters ( k = 22 14 ) was much larger than the expected number of biological clusters . the characteristic som mosaics contained coherent patterns generated by the colored tiles ordered so as to reflect the clustered gene expression profile of the individual samples . instead , we propose that som displays can be specifically treated as new , complex objects for a next level analysis , namely , visual gestalt recognition . thus , we do not use computer algorithm in the sense of artificial intelligence , but more as intelligence enhancement for the human brain in the holistic comparison of the transcriptomes . to enable such an integrative analysis based on visualization of each tumor sample as a unique and complex molecular portrait , we adapted gedi a som - based tool developed for visualizing the dynamics of genome - wide gene expression profiles to represent static microarray samples as two - dimensional high - resolution som mosaics . using published gene expression profiles from a large set of lung tumor samples , we offer a first assessment of the usefulness of this type of holistic visual analysis of tumor gene expression profiles . these studies reveal that human gestalt perception can lead to discovery of novel biological features without a preconceived hypothesis , and uncover new relationships between lung tumor subtypes that had previously escaped the analysis using conventional algorithmic classification techniques [ 5 , 36 ] . starting from an n m matrix of data from the analysis of n genes across m samples ( mrna expression profiles ) , gedi transforms each sample 's expression profile into a map that contains a visually recognizable color pattern , referred to as a gedi map . these maps are mosaics generated by self - organizing maps ( soms ) ( see materials and methods ) . importantly , in contrast to conventional cluster analysis using k - means or som , where typically k < 30 clusters , here k is many folds higher than the expected or desired number of biologically significant clusters , and hence , each of the k metagenes can be viewed as representing a minicluster of just a few genes ( with a typical median of around 10 genes ) . instead , the som algorithm is used to pixelate the expression profile into k pixels and rearrange them , which is why k is required to be high : typically , k = 100 s to 1000 s [ 12 , 13 ] . based on the characteristic visual metapatterns , gedi maps allow the direct comparison of the biological samples , as well as immediate identification of biologically interesting groups of genes . the genome - wide gene expression profiles of 25 samples of normal lung tissue ( lung ) and different pulmonary tumors , carcinoid ( car ) , squamous cell carcinoma ( sq ) , and small - cell cancer ( smc ) , were visualized as 25 gedi maps , each consisting of a 31-by-30 mosaic , representing 930 miniclusters . each sample exhibits characteristic spatial and color patterns , reflecting genome - wide transcriptional behavior of the respective tissue sample . inspection of gedi maps allows a straightforward classification of the samples into subgroups without the aid of a clustering algorithm , but simply based on the visual differences in the metapatterns . samples grouped together with members of the same category , with the exception of one outlier , a small - cell lung cancer sample , smc6 whose gedi map looked different ( figure 1 ) . therefore , gedi also provides a first - line sample - centered quality control for traditional clustering methods . because gedi maps provide a global view of the gene expression profiles of each sample , they immediately present an explanation for why a particular sample behaves as an outlier ( when sample diagnosis is known ) and which genes account for that behavior . for example , the dramatic difference between the gedi map of an outlier , smc6 ( figure 1 ) , relative to samples within the cluster of nominal small - cell lung cancers immediately reveals that smc6 deviates from the other small - cell carcinomas and the different pattern of tiles explains why . in addition to visually comparing gedi maps as individual entities , one can extract the numerical centroid values yk in sample j of each metagene k to analyze gedi maps quantitatively . to evaluate the fidelity of gedi mosaic patterns in representing the expression profiles established by all the genes , we calculated the correlation coefficients rjk for every pair of samples ( j , k ) using either ( 1 ) the expression data for all of the individual genes or ( 2 ) the metagenes . if the gedi mosaic patterns of metagenes faithfully represent the genome - wide gene expression profiles , the correlation coefficients for all sample pairs calculated in these two ways will be similar . in fact , the gedi patterns preserved the correlation between samples obtained from the real gene expression data ( figures 3(a ) , 3(b ) ) . interestingly , the values of the correlation coefficients ( profile similarity between samples ) calculated from metagenes spanned a considerably broader range than those from the real gene expression dataset , as apparent in the histograms of the correlation values ( figures 3(c ) , 3(d ) ) . in summary , the gedi maps based on metagenes faithfully recapitulate gene expression profiles of the entire gene dataset despite dimension reduction . to further validate how well metapatterns can represent the transcriptome second - level gedi analysis to categorize gedi maps automatically using the ( n = 930 metagenes , we also performed a pca on the original gene data matrix ( with sample columns as the objects and gene rows as the attributes ) . the 25 samples were assigned to a 5 5 som grid according to their metagene expression profiles . in the resulting second - level gedi map , the tissue samples ( the first - level gedi maps ) of the same diagnosis were grouped within the same neighborhood of the map ( figure 4(a ) ) . the map distances from each tumor - specific sample cluster to that of normal lung ( lung ) were roughly similar , while among the tumors , the carcinoid ( car ) and squamous cell carcinoma ( sq ) samples were most distant from each other , with small - cell lung cancer ( smc ) in between . interestingly , the spatial distribution of these samples in the two - dimensional second - level map was very closely mirrored in the pca in which the samples were projected on the plane spanned by the two first eigenvectors ( figure 4(b ) ) . visual inspection of the gedi maps readily confirms the notion that sq2 displays significant feature similarity to the smc samples based on the fine structure of the patterns of upregulated genes . without the gedi maps , the samples would be represented by dots in the pca which would be identified solely by their position in the abstract eigenvector space . thus , gedi allows the rapid toggling between gene - centered and sample - centered perspectives , which is an important feature for an integrative yet gene - specific analysis . like small - cell carcinoma , lung carcinoid tumors are also classified as ( low - grade ) neuroendocrine tumors , while squamous carcinoma appears to be unrelated to this group . however , in both hierarchical clustering as well as in pca , smc was closer to sq than to car ( figures 1 and 4 ) , which is consistent with the idea that small - cell lung carcinoma may have an epithelial origin , but competes with the notion of the common neuroendocrine property of smc and car . the gedi software environment allows the user to easily perform algebraic operations on whole mosaic patterns based on metagene expression values , and for instance to calculate average mosaics from a group of samples with the same diagnosis or difference mosaics to reveal differential expression patterns between two samples ( or averages of two groups ) . the red areas in the difference maps indicate genes that were upregulated in these tumors compared to normal lung tissue . these studies revealed that smc and sq share a set of features , representing a number of genes located within regions a this is consistent with the vicinity of these two tumors in the dendrogram ( figure 1 ) and in the pca sample plane ( figure 4 ) ; it also is in line with the proposed epithelial origin of small - cell lung cancer . such functional enrichment of genes in the gene islands underscores the biological meaning of pattern features in gedi maps . interestingly , the smc samples while globally close to sq samples , shared with the car samples the island d , which contained neuroendocrine - related genes ( involved in synaptic vesicle , neuromuscular physiological process , etc ) , consistent with the neuroendocrine nature of small - cell lung carcinoma . conversely , the island d that was enriched for the neuroendocrine genes was overexpressed in car but not in sq . thus , the gedi analysis exposed a novel facet of relationship between the samples with respect to these signature gene clusters : smc appears to be the union set of the sets of car and sq ( figure 5(b ) ) , sharing the gene cluster d with car and the clusters a , b , and c with sq . without an a priori hypothesis , such qualitative relationships are almost impossible to identify in the widely used heat maps , but they immediately spring to eye in the differential gedi maps . genome - scale gene expression profiles are not simply high - dimensional sets of variables that provide an opportunity for multivariate statistical analysis . to visualize coherent , genome - scale alterations of the transcriptome structure , we used here an integrative visual representation for gene expression profiles . as a test example we analyzed expression profiles of three lung tumor samples as a case study . we show that by delegating the actual process of pattern recognition to human gestalt perception in the format of som - based gedi mosaics , interesting features in the relationship between tumor types can be revealed . specifically , we found that with respect to pathological deviation from normal gene expression , small - cell carcinoma represents the union set of squamous cell carcinoma and carcinoids . such information on higher - order transcriptome changes , which may be useful for understanding developmental relationships and differences in drug responsiveness between tumor types , spring to eye in the gedi maps , but would not have been revealed in conventional algorithms without explicitly asking the appropriate question . while this is useful for explicitly extracting specific information , these methods may lose potentially useful , unanticipated information inherent in the high - dimensional expression profiles , such as particular higher - order patterns of expression . the gedi visualization software was developed to circumvent the problem of discarding implicit , potentially irreducible information inherent in genome - wide expression profiles in the absence of a specific hypothesis . it provides the opportunity for a holistic , yet molecular exploration of a set of gene expression profiles ( or other high - dimensional data sets ) that can be used to test existing tissue - level biological hypotheses or establish new ones . the identification of biological clusters is not the result of the clustering algorithm per se , but is achieved at a later stage of analysis , namely , by visual inspection and gestalt perception of the metapatterns that emerge from the som - generated metagenes . ( a ) by presenting metapatterns , gedi maps provide a visual engram of each sample 's particular molecular profile , and hence , establish a molecular portrait in the very sense of the word , with a particular visual identity for each sample ( eg , tumor type , patient , treatment condition ) . ( b ) although classification of samples into groups is achieved by human gestalt perception of the metapatterns , it can be supported by an algorithmic approach applied on the metagenes . ( e ) gedi allows the rapid and seamless switching between an integrative , sample - oriented analysis and the more traditional gene - centered analysis . future use of gedi in studies of the genome - scale molecular signature of both normal and disease samples will ultimately help assess the true value of a holistic interpretation of molecular profiles that systems biology is advocating .

riboswitches are noncoding structural elements in the leader sequence of select bacterial and eukaryal mrnas that regulate downstream gene expression in a ligand - dependent fashion . the first experimentally verified riboswitch was found in mrnas that code for the expression of a cobalamin ( vitamin b12 ) transport protein ( i.e. , btub in escherichia coli ) . these structured nucleic acids function to maintain intracellular concentrations of vitamin b12 , which is a critical cofactor for methyltransferase and isomerase enzymes associated with the production of s - adenosylmethionine and succinyl - coa , respectively . the cobalamin riboswitch clan ( rfam : cl00101 ) is one of the most abundant and ubiquitous cis - acting mrna regulatory elements throughout the bacterial domain of life . the importance of these rnas and their associated effector molecule is further highlighted by the fact that only certain species of archaea and bacteria can biosynthesize cobalamin , and yet it is essential to many organisms , most notably humans . generally , most riboswitches consist of two interacting structural elements that , together , result in biological functionality . the ( i ) aptamer domain of a riboswitch binds specific target molecules , which commonly promote formation of ( or stabilizes ) an alternative conformation in the ( ii ) regulatory element , or expression platform . single - molecule fluorescence resonance energy transfer(smfret ) is , in principle , ideally suited to observe these ligand - induced conformational transitions . however , only a few investigations have successfully monitored conformational transitions associated with the regulatory element of a riboswitch , and , until now , none have been able to unambiguously link ( i ) ligand binding to ( ii ) conformational transitions within a biologically functional riboswitch . as one major result of the present work , these two processes have been observed both independently and simultaneously using smfret techniques . the novel x - ray crystal structure of a small , entirely biologically functional , cobalamin riboswitch , containing both the aptamer domain and expression platform , revealed that these two structural elements communicate via a tertiary interaction in order to confer biological functionality ; such behavior has also been observed in other more extended cobalamin riboswitches . complementary cell - based experiments conducted using this novel rna construct demonstrated that a tertiary interaction modulates gene expression in a vitamin b12-dependent fashion . specifically , it was shown that ( i ) this riboswitch selectively binds vitamin b12 cofactors with small -axial ligands , like hydroxocobalamin ( hycbl ) , as opposed to the bulkier -axial ligand of adenosylcobalamin , and ( ii ) binding of hycbl , the photolysis product of adenosylcobalamin , facilitates docking between two loops of the rna . it is this tertiary docking interaction that is responsible for regulating gene expression via formation of a kissing loop ( kl ) complex that directly sequesters the shine - dalgarno sequence , which is the ribosome binding site ( rbs ) of many mrnas . importantly , similar regulatory mechanisms have also been identified in other riboswitches like the sam - iii riboswitch . although these recent structural and cell - based ensemble experiments , described above , have unveiled information about the mechanism of action associated with this riboswitch , many detailed questions regarding both ligand binding kinetics and conformational dynamics remain unanswered . to this end , a combination of fluorescence techniques , smfret and cell - based fluorescent - reporter assays , has been used to offer the first , to our knowledge , detailed mechanistic account of a biologically functional riboswitch based on a collection of both cellular and smfret data . the primary sequence ( genbank accession : aacy021350931.1/557456 ) of the env8 hydroxocobalamin riboswitch ( env8hycbl , figure 1a ) used throughout these experiments comes from a comparative genomics analysis of structured noncoding rnas . this rna is a member of the adocbl - variant family ( rfam : rf01689 ) , which is one of three families within the larger cobalamin riboswitch clan ( rfam : cl00101 ) . notably , this rna contains all of the core characteristics associated with the entire cobalamin riboswitch clan , namely , a 4-way junction implicated in ligand recognition and a regulatory kl , making it a highly tractable model system that contains all of the structural elements necessary and sufficient for biological activity in a cellular context . schematic representation of the ( a ) secondary and ( b ) tertiary structure of the env8hycbl riboswitch , complete with the cy3 ( green star ) , cy5 ( red star ) , and biotin ( blue rectangle ) synthetic moieties . the loops , l5 ( red ) and l13 ( green ) , form a regulatory rna kissing - loop ( kl , see inset for sequence ) , which contains the purine - rich ribosome binding site ( shadowed green nucleotides ) . binding of hydroxocobalamin ( bronze spheres , hycbl ) facilitates formation of the kl , which decreases the distance between the cy3 ( donor ) and cy5 ( acceptor ) fluorophores , and thereby results in increased energy transfer efficiency ( efret ) . synthetic modifications to this rna ( figure 1a ) allow smfret experiments to monitor , in real - time and under equilibrium conditions , the docking / undocking of the l5l13 regulatory kl in the presence or absence of ligand ( figure 1b ) . simultaneously , hycbl - dependent quenching of cy3 is used as an orthogonal experimental observable to monitor the kinetics of ligand binding independently of conformation . the results of these smfret experiments provide substantial support for a four - state kinetic model that highlights several key biophysical features associated with this regulatory switch ( figure 2 ) : ( i ) kl docking / undocking occurs in the absence of hycbl and is dependent on mg , ( ii ) hycbl binding predominately occurs when the kl is undocked , ( iii ) kl docking / undocking also occurs while hycbl is bound and is still dependent on mg . additionally , the single - molecule results reveal that , in the presence of mg , hycbl binding preferentially stabilizes the docked conformation . finally , results from complementary cell - based reporter assays are used to relate these biophysical findings to the cellular function of this rna . these comparisons reveal that formation of the regulatory switch ( i.e. , l5l13 kl ) in the single - molecule experiments is strongly correlated with repression of gene expression in the cell - based assay , regardless of whether the ligand is bound to the rna . as a whole , these findings expose valuable new mechanistic and kinetic information about the dynamical relationship between structure and function in cobalamin riboswitches , which can also be applied to other functional rnas . four - state kinetic model for the env8hycbl riboswitch represented ( a ) graphically and ( b ) symbolically . the four macroscopic conformations are linked by three coupled equilibria : ( i ) kl docking in the absence of ligand , ( ii ) ligand binding in the undocked conformation , and ( iii ) kl docking in the presence of ligand . formation of the kl decreases the interdye distance , resulting in more efficient fluorescence resonance energy transfer ( efret ) . ligand binding quenches cy3 , which decreases the total fluorescence of cy3 and cy5 and can therefore be monitored independently of kl docking / undocking events . although it is possible that a fourth ( iv ) equilibrium exists , we observe no experimental evidence for such a process ( section 3.3 ) , indicating that this process is prevented by prohibitively large free energy barriers ( as indicated by the small transparent arrows ) . accordingly , this process is not considered for simplicity and ease of discussion ; its inclusion would not alter the interpretations and conclusions derived from this work . both freely diffusing and surface - immobilized experiments ( see materials and methods ) have been used to characterize the conformational transitions associated with the regulatory kl of the env8hycbl riboswitch . briefly , freely diffusing smfret experiments make use of the subfemtoliter volumes associated with the diffraction - limited focus of a high numerical aperture objective . at 100 pm of cy3cy5 labeled rna , this volume is absent of fluorophores more than 99% of the time . occasionally , an individual molecule will stochastically diffuse through this volume , resulting in a 1 ms burst of fluorescence limited in duration by rna diffusion . the efficiency of fluorescence resonance energy transfer ( efret ) can be calculated for thousands of fluorescent bursts and compiled into a histogram to describe the probability of observing any specific efret value , which therefore reports on the conformational occupancy of the rna . alternatively , individual cy3cy5 labeled rnas can be immobilized to a microscope coverslip using biotin the diffraction - limited focus of the confocal fluorescence microscope can be used to continuously excite the fluorophores , allowing for collection of emitted photons . the collected signal is used to determine the efret for a single rna as a function of time , whereby changes in efret result from discrete conformational transitions . the two single - molecule fluorescence experiments described above are used to rigorously characterize the in vitro behavior of the env8hycbl riboswitch . complementary cell - based fluorescence reporter assays are used to compare and contrast the in vitro and cellular behavior of this rna . specifically , these reporter assays are designed to monitor the regulated expression of a green fluorescent protein variant ( gfpuv ) provided by the env8hycbl riboswitch . as a first step , freely diffusing smfret experiments have been used to explore the equilibrium behavior of the l5l13 kissing loop ( kl ) interaction from the env8hycbl riboswitch in solutions free of hycbl . under standard buffer conditions ( see materials and methods ) , the distribution of efret values associated with thousands of individual fluorescently labeled rna constructs reveals ( figure 3a ) that the riboswitch resides entirely in a single low efret = 0.15(3 ) population . however , addition of mgcl2 is , by itself , sufficient to promote kl docking ( figure 3a ) , as indicated by the mg - dependent increase in the relative probability of observing molecules in a second , higher efret = 0.58(3 ) population . this high efret value corresponds to an inter - dye distance of = 50(3 ) and , given the flexibility of the rna fluorophore linker , is entirely consistent with the 47 prediction based on the recent crystal structure . accordingly , we attribute this high efret value to molecules residing in the docked kl conformation ( figure 2b , dfree ) , with the low efret value corresponding to the undocked species ( figure 2b , ufree ) , where the subscript free the necessity of mg for the formation of this interaction is supported by the x - ray crystal structure ( pdb : 4frn ) , which depicts divalent cations near the l5 and l13 loops . this is a common feature among other kl x - ray crystal structures and likely reflects the importance of mg in the formation of these types of tertiary interactions . freely diffusing smfret experiments reveal the mg - dependent formation of the docked conformation in the ( a ) absence and ( b ) presence of 1000 nm hycbl . note the color scheme in a and b is only intended to represent the increasing [ hycbl ] ; colors do not necessarily correlate one - to - one with [ hycbl ] . ( c ) the presence of saturating hycbl ( 1000 nm , bronze circles ) decreases the concentration of mg required to achieve half - maximal occupancy of the docked conformation ( i.e. , [ mg]1/2 ) by 10-fold . the vertical dashed bar represents near - physiological salt conditions ( see text for details ) . the error bars are smaller than the symbols used for each data point ( materials and methods ) . the residual probability associated with observing the low efret population at high [ mg ] results from molecules that are unable to form the l5l13 kl interaction ( 15% ) , as quantitatively determined in surface immobilized experiments described later ( section 2.5 ) . the abundance of this population is dependent on the conditions associated with synthesis of the construct , perhaps , indicating the existence of misfolded species ( see materials and methods ) . the normalized probability of observing the two efret distributions , when corrected for a 15% nonresponsive population , can be used to characterize the equilibrium behavior of the riboswitch . specifically , the fractional occupancy of molecules in the docked or undocked conformation can be calculated using eqs 1a and 1b , respectively.1a1bthe parameters p(dfree ) and p(ufree ) describe the probability of observing the docked and undocked conformations , respectively , where the subscript free the [ mg ] required to achieve half - maximal occupancy of the docked conformation ( i.e. , [ mg]1/2 ) is obtained by fitting the data to a hill - type binding model . for data collected in the absence of hycbl , such an analysis predicts [ mg]1/2 = 2.2(2 ) mm ( figure 3c ) , which is nearly twice normal physiological concentrations of free mg . although the regulatory kl can form in the absence of hycbl , the fdfree is only 0.22 in solutions that appropriately mimic the ionic environment associated with the cellular milieu ( e.g. , 1 mm mg , 125 mm k , 10 mm na ) , suggesting that the undocked conformation is preferred under these conditions . to confirm that the high efret population results from formation of the l5l13 regulatory switch , a second , doubly labeled rna construct has also been studied this construct , referred to as the l5-mutant env8hycbl riboswitch ( supporting information figure s1 ) , contains a dinucleotide substitution at positions 48 and 49 in l5 ( underlined nucleotides , 5-uacuug-3 5-uacaag-3 ) . the uu to aa mutation prevents formation of two watson crick base pairs that are otherwise present in the wild - type construct and preferentially destabilizes the docked conformation of the kl . as expected , this smfret construct remains in the undocked ( low efret ) conformation even in solutions containing up to 30 mm mgcl2 ( supporting information figure s1 ) , thus confirming that the donor and acceptor fluorophores accurately report on the formation of this regulatory kl interaction . analogous freely diffusing smfret experiments have also been conducted in the presence of ligand in order to interrogate the equilibrium behavior of this interaction in solutions where the hycbl concentration is more than 100 times larger than the previously reported binding affinity of 8(4 ) nm . without any mg in solution , fluorescent molecules reside entirely in a single low efret = 0.12(3 ) population ( figure 3b ) that is experimentally indistinguishable from the low efret = 0.15(3 ) population observed in the absence of ligand ( figure 3a ) . notably , such behavior was also observed in recent smfret investigations of a c - di - gmp riboswitch ; however , complete folding required the presence of ligand , which is unlike the env8hycbl riboswitch in this study . as was the case in the absence of ligand , mg is still required for the formation of the kl interaction , even at saturating [ hycbl ] . specifically , addition of mgcl2 into solutions containing 1000 nm hycbl increases the probability of observing a second , higher efret = 0.61(4 ) population ( figure 3b ) that is also experimentally indistinguishable from the high efret = 0.58(3 ) population noted previously in the absence of ligand ( figure 3a ) . under saturating hycbl conditions , therefore , the above observations support the existence of the l5l13 kl equilibrium associated with the docked ( figure 2b , dhycbl ) and undocked ( figure 2b , uhycbl ) conformations of the rna , where the hycbl subscript now denotes the presence of a bound ligand . notably , bound hycbl reduces [ mg]1/2 by nearly an order of magnitude ( figure 3c ) , which indicates that under physiological free salt conditions ( i.e. , 1 mm mg , 125 mm k , 10 mm na ) these ligand bound riboswitches reside predominantly in the docked conformation ( fdhycbl 0.75 ) . in conjunction with the results of experiments performed in the absence of ligand , it is apparent that hycbl binding increases the stability of the docked conformation in solutions containing mg(e.g . moreover , the increased occupancy of the docked conformation , where the l5l13 kl is formed , demonstrates that ligand binding directly modulates the availability of the ribosome binding site within l13 of this regulatory switch . to directly explore the ligand - binding process , we exploit a unique photophysical property of hydroxocobalamin ( hycbl ) . specifically , the absorbance spectrum of hycbl overlaps with the fluorescence spectrum of cy3 , which allows this cofactor to effectively compete with cy5 for acceptance of energy transfer ( supporting information figure s2 ) . because hycbl is a nonfluorescent acceptor , energy transfer to it results in strongly quenched fluorescence . we can utilize this quenching phenomenon to monitor the ligand binding kinetics of the env8hycbl riboswitch via time - dependent ensemble fluorometry . when hycbl is dissociated from the rna , the average intermolecular distance between it ( acceptor ) and cy3 ( donor ) is large enough to neglect the effects of this quenching energy transfer process ( figure 4a ) . however , upon ligand binding , the intermolecular distance between the two is reduced to 23 , allowing for substantial energy transfer from the excited donor molecule ( cy3 ) to the nonfluorescent acceptor ( hycbl ) . thus , energy transfer to hycbl results in an additional nonradiative ( knrad ) component of the cy3 fluorescence lifetime ( fluor = 1/(krad + knrad ) ) , which leads to a decreased quantum yield ( q.y . = krad/(krad + knrad ) ) . as an attempt to quantify this ligand - dependent quenching , we have measured the fluorescence decay profiles of individual cy3-only env8hycbl rnas in the presence and absence of 1000 nm hycbl ( supporting information figure s2a ) using the time - correlated single - photon counting capabilities associated with the experimental apparatus ( see materials and methods ) . these lifetime measurements ( i.e. , cy3-fluor(free ) = 1.40(2 ) ns and cy3-fluor(hycbl ) = 0.51(6 ) ns ) effectively demonstrate the nonradiative quenching phenomenon associated with energy transfer from cy3 to hycbl . ( a ) hycbl binding quenches cy3 ( see also supporting information figure 2 ) . ( b ) the single - exponential decay of the cy3 fluorescence reports on the sum of the apparent binding ( kbind ) and dissociation ( kdiss ) rate constants associated with the interaction between hycbl and the env8hycbl riboswitch . ( c ) a linear - fit of the equilibration rate constant as a function of [ hycbl ] is used to measure the apparent ligand binding ( kbind ) and dissociation ( kdiss ) rate constants . in the presence of the fluorescent acceptor , cy5 , it is important to stress that the energy transfer process to hycbl competes equally with both the radiative decay process of cy3 and the energy transfer process to cy5 ( supporting information figure s2 ) , resulting in uniform attenuation of both signals . provided that the quenched fluorescence signals remain well above background , the ratiometric efret values associated with the docked ( i.e. , dfree vs dhycbl ) and undocked ( i.e. , ufree vs uhycbl ) populations remain unaltered after ligand binding ( supporting information figure s2b ) , as demonstrated experimentally ( see section 2.2 ) . this photophysical phenomenon is exploited in kinetic ensemble fluorescence experiments to monitor the hycbl - induced reduction in fluorescence intensity of rna constructs labeled with cy3 upon addition of excess ligand ( [ hycbl]/[rna ] > 10 ) . the temporal decay of fluorescence intensity ( figure 4b ) maps out the rate constant responsible for establishing a binding equilibrium ( keq ) . for a single bimolecular process , this rate constant ( keq ) can simply be described as a function of ligand concentration using the ligand - binding ( kbind ) and dissociation ( kdiss ) rate constants ( eq 2).2however , the ligand binding kinetics in the present system are further complicated by the two remaining kl equilibria ( figure 2 ) , which limit the fraction of undocked molecules that can either bind to ( fufree ) or dissociate from hycbl ( fuhycbl ) . the [ hycbl]-dependence of keq is evident in figure 4c and is well described by a straight line . linear regression yields a slope of kbind = 1.0(1 ) 10 m s and an intercept of kdiss = 0.005(3 ) s , where the ratio of these two values characterizes the apparent hycbl dissociation equilibrium constant ( kd = 5(3 ) nm ) . using principles related to michaelis menten enzyme kinetics , we can decouple these apparent kinetic parameters from the two kl docking equilibria by making the following assumption , which is experimentally validated later in surface - immobilized kinetic studies ( sections 2.5 and 2.6 ) . provided the docking / undocking kinetics associated with the kl equilibria are much faster than the ligand binding or dissociation processes , the apparent rate constants can be readily shown to equal true rate constants ( i.e. , kbind and kdiss ) , scaled by the fractional populations of the binding competent ( fufree ) and dissociation competent ( fuhycbl ) species , respectively ( see eqs 3a and 3b).3a3bthe values of fufree 0.78 and fuhycbl 0.25 are obtained from the freely diffusing data depicted in figure 3c at 1 mm mgcl2 . these values are used to calculate the true rate and equilibrium constants , denoted by a lack of the superscript app , that characterize the ligand binding kinetics of the env8hycbl riboswitch at 1 mm mgcl2 ( kbind = 1.3(2 ) 10 m s , kdiss = 0.02(1 ) s , and kd , = 15(8 ) nm ) . the data sets described in the three previous sections ( 2.1 , 2.2 , and 2.3 ) quantitatively characterize all equilibrium constants required to predict the steady - state fractional occupancy of the docked conformation as a function of [ hycbl ] for a given [ mgcl2 ] . to experimentally test these predictions , the fractional occupancy of the docked conformation ( fd ) is determined using freely diffusing smfret experiments wherein hycbl is titrated into solutions at five different fixed mgcl2 concentrations ( 0.0 , 0.5 , 1.0 , 2.5 , 20 mm ) . in solutions lacking mg , the addition of ligand is unable to promote formation of the docked conformation ( figure 5a ) , as mg is required for the formation of the regulatory kl interaction between l5 and l13 . at intermediate concentrations of mgcl2 ( e.g. , 0.5 , 1.0 , and 2.5 mm ) , titration of hycbl into solution facilitates formation of the docked conformation ( figure 5b ) . this observation is certainly consistent with the four - state model ( figure 2 ) , wherein bound hycbl , increases the stability of the regulatory kl interaction ( i.e. , fdfree < fdhycbl ) . titration of hycbl into solutions with saturating concentrations of mg ( e.g. , 20 mm [ mg]1/2 ) reveals that ligand is unable to significantly increase the fd ( figure 5c ) , which nicely demonstrates that mg alone is sufficient to promote complete formation of the regulatory interaction responsible for sequestering the ribosome binding site . freely diffusing single - molecule burst titrations of hydroxocobalamin ( hycbl ) at various concentrations of mgcl2 . ( a ) at 0 mm mg , addition of ligand does not significantly influence the distribution of efret associated with the l5l13 regulatory switch . ( b ) at intermediate concentrations of mg(e.g . , 2.5 mm ) , addition of hycbl does significantly alter the distribution of efret . ( c ) mg ( 20 mm ) is already sufficient to completely form the l5l13 kl interaction , irrespective of [ hycbl ] . together , these results support the notion that docking of the env8hycbl riboswitch is more favorable when the ligand is bound to the rna under near - physiological salt conditions , but that hycbl alone ( i.e. , 0 mm mg ) is insufficient to promote formation of the kl interaction . c is only intended to represent the increasing [ hycbl ] ; colors do not necessarily correlate one - to - one with [ hycbl ] . ( d ) experimental validation of the four - state kinetic model for the env8hycbl riboswitch . the experimentally determined fractional occupancy of the docked conformation ( fd , colored circles ) is well described by the steady - state solution ( dotted line ) to the four - state model ( figure 2 ) over a wide range of mg and hycbl concentrations . figure 5d compares the results of the single - molecule experiments with the model predictions ( colored circles and dashed lines , respectively ) . this comparison demonstrates the remarkable quantitative accuracy achieved by the four - state kinetic model ( figure 2 ) used to describe conformational transitions associated with this biologically functional riboswitch . one noteworthy feature of this model is that the ligand - binding process is mostly insensitive to mg , as indicated by transition midpoints ( i.e. , kd ) near 5 nm for [ mgcl2 ] between 0.5 and 2.5 mm . nucleic acid ligand interactions have previously been shown to be strongly influenced by metal - ion - facilitated conformational rearrangements in many riboswitches , including the lysine , thf , c - di - gmp , sah , and adenine sensing riboswitches . however , in the case of this hycbl - sensing riboswitch , the two coupled kl docking equilibria result in a ligand binding process that is only subtly influenced by mg . specifically , addition of mg promotes docking , which , for ligand - free rnas , decreases the fractional population of the binding competent state ( i.e. , fufree ) . however , this is approximately offset by the corresponding decrease in the fractional population of hycbl - bound species that can readily undergo dissociation ( i.e. , fuhycbl ) . fluorescence trajectories of individual surface - immobilized molecules have been recorded to observe the kl docking dynamics ( figure 6a c ) with 30 ms resolution . in the absence of hycbl ( figure 6a ) , single rnas display strongly anticorrelated fluctuations in donor and acceptor fluorescence , resulting from rapid transitions between the two well - separated fret states . these fret values are in excellent agreement with those measured in the freely diffusing experiments , which supports the conclusion that these time trajectories depict single - molecules transitioning between the docked ( dfree ) and undocked ( ufree ) conformations associated with the l5l13 kl interaction . kinetic analysis of the surface - immobilized data yields hundreds of individual dwell times that are compiled together and fit to single - exponential decays in order to yield the rate constants associated with docking and undocking . these rate constants reveal that , in ligand free solutions at 1 mm mg , kdock , free = 1.2(3 ) s and is slower than kundock , free = 6(2 ) s. together , these rate constants result in fdfree = 0.17(7 ) , which agrees well with results from the freely diffusing experiments ( fdfree 0.22 ) . a titration with respect to mg , analogous to the ones from the freely diffusing smfret experiments , reveals that increasing divalent cation concentration accelerates the docking and decelerates the undocking rate constants , respectively , both of which contribute nearly equally to shifts in the mg - dependent fdfree ( figure 6c ) . as alluded to above , the fact that individual surface - immobilized molecules dock completely ( fdfree 1.0 ) at high [ mgcl2 ] confirms that the residual probability ( 15% ) for observing the low efret population results from inactive molecules that can not form the l5l13 interaction . it is important to emphasize that the results for both freely diffusing and surface immobilized studies are indistinguishable , as indicated by the circles and stars in figure 6d , respectively . such a plot demonstrates convincingly that surface immobilization does not influence the biophysical behavior of this rna . representative ( a ) fluorescence and ( b ) fret time - traces from a surface immobilized env8hycbl riboswitch . orange lines represent the results from a maximum - likelihood 2-state bayesian model that bests describes the data . ( c ) the docking and undocking rate constants , as a function of [ mg ] , are used to calculate the fractional occupancy of the docked conformation ( d ) in the absence of hycbl ( fdfree ) , which reveals that the surface immobilized results ( dark gray stars ) are in quantitative agreement with the freely diffusing experiments ( light gray circles ) . the error bars are often smaller than the symbols used for each data point ( materials and methods ) . to complete the single - molecule kinetic characterization of this riboswitch , fluorescence time - trajectories of individual surface - immobilized molecules are recorded in the solutions containing 2.5 nm hycbl and 1 mm mg ( figure 7 ) . these results further support the proposed kinetic model ( figure 2 ) for ligand - facilitated formation of the regulatory switch . specifically , the time - trajectories in the presence of hycbl display distinct regions with vastly different fluorescence intensities . some regions exhibit normal fluorescence signals for both the donor and the acceptor fluorophores , which are well resolved and anticorrelated ( figure 7b ) . in the other , intervening regions , the total fluorescence signal is significantly quenched ( figure 7c ) . the rate constants associated with the anticorrelated fluorescence fluctuations in the normal regions mimic those from molecules studied in the absence of hycbl ( figure 7b vs 6b ) , as would be expected when the ligand is not bound to the rna . conversely , the quenched regions result from ligand binding events that localize hycbl near cy3 , which introduces additional nonradiative relaxation pathways . indeed , despite the substantially reduced signal in these regions , subtle fluctuations in acceptor intensity in excess of background noise are clearly observable and can be attributed to docking and undocking of the kl interaction during the long ligand - bound episodes . ( a ) representative fluorescence trajectory from a surface immobilized molecule in the presence of 2.5 nm hycbl . for a construct in the undocked conformation , ligand binding ( arrow ) significantly diminishes the fluorescence intensity , which returns back to normal after ligand release . ( b ) when the ligand is not bound , the docking / undocking kinetics of the regulatory kl are experimentally indistinguishable from those in the absence of hycbl ( see also figure 6b ) . ( c ) when the ligand is bound , a maximum - likelihood 2-state model ( black line ) is used to quantify the dwell times associated with fluctuations in the acceptor fluorescence resulting from kl docking and undocking . a comparison between the two kl docking equilibria ( b vs c ) reveals that the effect of hycbl binding significantly decreases the undocking rate constant ( kundock ) . to quantitatively measure the docked and undocked dwell times in the ligand - bound state , a bayesian maximum - likelihood model is used to analyze the two - state fluctuations evident in the quenched regions of the fluorescence trajectories ( figure 7c ) . when the ligand is bound to the env8hycbl riboswitch in solution containing 1 mm mgcl2 , such an analysis reveals that the docking rate decreases only by 2-fold ( kdock , hycbl = 0.64(9 ) vs kdock , free = 1.2(3 ) s ) , whereas the undocking rate exhibits a much more significant hycbl - sensitivity and decreases by 23-fold ( kundock , hycbl = 0.26(8 ) s vs kundock , free = 6(2 ) s ) . the hycbl - induced change to the kl undocking kinetics gives rise to substantially more favorable formation of the l5l13 regulatory switch ( fdfree = 0.17(7 ) vs fdhycbl = 0.7(1 ) ) , as also observed in the corresponding freely diffusing experiments ( fdfree 0.22 vs fdhycbl 0.75 ) . further inspection of the fluorescence trajectories reveals that ( i ) hycbl can only bind and quench fluorescence when the rna is in the undocked ( low efret ) conformation ( supporting information figure s3 ) and ( ii ) ligand - bound dwell times can be longer than 20 s ( figure 7a ) . these bound - state lifetimes are consistent with those predicted from the ensemble time - dependent fluorescence experiments ( figure 4c ) , which yield hycbl = ( 1/koff ) = 200(120 ) s. lastly , the time - traces of surface immobilized molecules clearly verify the previous assumption that the kl kinetics , both in the presence and absence of ligand , are much faster than those associated with establishing a ligand binding equilibrium . in total , the collection of results from the above biophysical fluorescence studies provide strong support for the proposed four - state kinetic model ( figure 2 ) associated with the regulatory conformational transitions of the env8hycbl riboswitch . to correlate the biophysical behavior of this riboswitch with its biological function , cell - based gene expression experiments ( figure 8) have been performed in an e. coli strain lacking cobalamin adenosyl transferase ( btur ) , thereby preventing the subsequent conversion of hycbl to adenosylcobalamin . in these experiments , cells are transformed with vectors containing either the wild type or mutant riboswitches located upstream of a gfpuv reporter gene , termed wt env8hycbl+gfpuv or l5 mutant env8hycbl+gfpuv , respectively , and cultured in liquid media with or without 5000 nm hycbl . the corresponding l5-mutant env8hycbl+gfpuv vector contains a riboswitch with the exact same dinucleotide substitution used in the smfret experiments ( supporting information figure s1 ) . the results of our biophysical experiments suggest that this l5-mutant riboswitch should be incapable of regulating gene expression because it is unable to from the regulatory kl interaction that sequesters the ribosome - binding site . provided that such a conservative , two nucleotide substitution mutation does not significantly alter the mrna abundance associated with the l5-mutant env8hycbl+gfpuv vector , the cells transformed with this vector can be used to provide a reasonable assessment of the maximum amount of gfpuv expression resulting from completely undocked riboswitches . ( a ) schematic diagram relating the four - state kinetic model to the regulation of gene expression via inhibition of translation initiation . ( b ) histograms comparing the normalized fluorescence and relative gfpuv expression of cell cultures grown in the presence ( bronze bars ) or absence ( gray bars ) of 5000 nm hycbl . as expected , in growth media lacking hycbl , cells transformed with wt env8hycbl+gfpuv have normalized fluorescence values ( materials and methods ) lower than cells transformed with the l5-mutant env8hycbl+gfpuv plasmid ( gray bars , figure 8b ) . this compares nicely with the single - molecule observation that the wt env8hycbl fret construct is mostly undocked ( fufree 0.78 ) at 1 mm mg , whereas the l5-mutant is completely undocked ( fufree 1.0 ) . additionally , when cultured in media containing 5000 nm hycbl , only cells harboring wt env8hycbl+gfpuv show a substantial ligand - dependent reduction in gfpuv expression ( bronze bars , figure 8b ) , indicating that hycbl decreases the expression of gfpuv , presumably via sequestration of the ribosome binding site within l13 of the env8hycbl riboswitch . the pronounced similarities between the results of the in vitro and cell - based experiments suggests that ( i ) the fractional occupancy of the undocked conformation correlates nicely with the level of gene expression , regardless of whether or not ligand is bound and ( ii ) formation of the docked conformation , resulting from a long - range kl interaction between l5 and l13 , sequesters the ribosome binding site within l13 and prevents initiation of translation . ligand - induced conformational transitions are central to the function of many riboswitches . a significant body of structural work has revealed the numerous ways in which riboswitches can specifically interact with their cognate ligands . however , much less is known about how molecular recognition in the riboswitch aptamer domain translates into alternative conformations in the expression platform . the results of these single - molecule experiments mechanistically link : ( i ) ligand binding to ( ii ) conformational transitions in the expression platform of a fully functional riboswitch . specifically , the fluorescently labeled rna constructs examined in this work provide information about the equilibrium and kinetic behavior of a regulatory kissing loop ( kl ) interaction . furthermore , the novel use of hydroxocobalamin - induced fluorescence quenching as an orthogonal experimental observable offers additional insights into the ligand binding process . together , these results highlight several key findings about the functional mechanism of gene regulation for the env8hycbl riboswitch , which can serve as a model system for the entire cobalamin riboswitch clan . both the freely diffusing and surface immobilized experiments clearly identify the l5l13 kissing loop ( kl ) interaction as the structural motif that directly regulates biological function ( i.e. , gene expression ) . additionally , this motif is also partly responsible for organizing the global conformation of the rna . many other functional nucleic acids make use of similar types of tertiary interactions to accomplish these same tasks . for example , kl interactions are important for organizing the three - dimensional structure of self - cleaving ribozymes and regulate a variety of biological processes , such as dimerization of retroviral genomes and plasmid replication . one characteristic of both intra- and intermolecular kl interactions is the formation of canonical watson crick base pairs between the two loops that dock together . in addition to five complementary base pairs , the l5l13 interaction contains a g a mismatch adjacent to a dinucleotide ( ag ) bulge . presumably , these structural defects serve to destabilize this interaction . in fact , recent kinetic studies of an intermolecular 6 bp rna kl interaction associated with hiv-1 genome dimerization have reported rate constants for kl undocking ( kundock 1.00.1 min ) that are significantly slower than what is observed in the present study . the comparatively fast kundock for env8hycbl in the absence of ligand likely results from the structural defects associated with the l5l13 kl interaction . notably , the presence of hycbl partially compensates for these defects by slowing down the kundock , which results in ligand - dependent sequestration of the ribosome binding site . this nicely demonstrates how nucleotide sequence can be used to tune the stability of a rna kl interaction . other examples exist where localization of mg modulates the stability of these types of interactions , as is the case with intermolecular hiv ( human immunodeficiency virus ) genome dimerization . in the env8hycbl riboswitch , indeed , the x - ray crystal structure of this rna depicts substantial electron density associated with ba ( the more strongly x - ray scattering mimetic of mg ) near the interacting loops . it is important to mention that , for the env8hycbl riboswitch , the presence of a bound ligand not only lowers the [ mg ] required for half - maximal docking ( [ mg]1/2 ) , but that it also decreases the number of ions that condense onto the rna during the conformational transition ( figure 3c ) . together , these two observations indicate that hycbl binding complements cation uptake , thus attenuating the mg requirements associated with this regulatory kl interaction . while divalent cations and nucleotide sequence represent two effective means to adjust the stability of rna kissing loops ( kls ) , there is also another pathway to accomplish such a task . in addition to distinctly identifying the existence of a kl docking equilibrium in the absence of ligand , the above experimental results reveal that kl docking and undocking also occurs when hycbl is bound . specifically , the presence of a bound ligand alters the energetics of this process to substantially favor the docked conformation . this is yet another example of how kl stability can be tuned to achieve a particular biological function . careful inspection of the hycbl - bound crystal structure reveals that most of the intermolecular contacts between the rna and the ligand are van der waals interactions governed by shape complementarity . these interactions significantly stabilize the docked conformation , as evidenced by the 23-fold slower kundock . this represents one example of how a specific macromolecular cosolute can stabilize the formation of a kl interaction directly responsible for a specific biological function . interestingly , a similar mechanism is employed in the rop - rna i - rna ii plasmid replication system . specifically , the rop protein acts as a molecular scaffold that stabilizes the intermolecular kl interaction between the loops of rna i and rna ii and regulates formation of rna primers required for dna plasmid replication . in this system , the presence of rop substantially reduces the rate constant describing rna i dissociation from the ternary complex , similar to how the presence of hycbl reduces the rate constant for kl undocking in the env8hycbl riboswitch . interestingly , the many ethanamide and propanamide functional groups in the corrin ring of hycbl chemically recapitulate the amino acid side chains of asparagine and glutamine , respectively . this might suggest that the protein - like chemical functionality of hycbl allows such a cofactor to imitate the stabilizing properties of rop . moreover , structural models of rop revealed that these amino acids are localized to the proposed interface between the protein and the two kissing loops of rna i and ii , while binding studies have demonstrated that these amino acids are essential for recognizing the rna kl . the functional similarities between the rop - rna i - rna ii system and the env8hycbl riboswitch demonstrate how kl formation can be facilitated by a macromolecular binding partner ( e.g. , protein or ligand ) in order to regulate a biologically relevant process ( e.g. , plasmid replication or gene expression ) . a long - standing question in nucleic acid recognition of small molecule ligands is whether : ( i ) an induced - fit or ( ii ) a conformational - capture model most appropriately describes these aptamer the induced - fit model demands that ligand binding occurs in open ( e.g. , undocked ) conformations of the aptamer , which then induces a structural transition that encapsulates the bound molecule . this is in contrast to the conformational - capture model , whereby ligand recognition occurs in transient , highly organized folded structures , similarly resulting in stabilization of the bound ( e.g. , docked ) conformation ; such a mechanism was recently identified via smfret investigations of a c - di - gmp riboswitch . for the env8hycbl riboswitch , the experimental validation of the four - state model ( figure 5d ) and the surface - immobilized time - traces in the presence of hycbl ( figure 7a , supporting information figure s3 ) strongly suggest that ligand binding ( i.e. , fluorescence quenching ) primarily occurs in the undocked conformation , thus favoring the induced - fit mechanism . this notion is further supported by existing structural data , which reveal that the env8hycbl riboswitch conceals a majority ( 60% ) of the solvent accessible surface area of the ligand when the kl interaction is formed . these steric constraints would make it difficult for the approximately spherical hycbl to enter , or exit , the binding pocket when the l5l13 interaction is formed , resulting in negligibly slow ligand association / dissociation from the docked conformation ( figure 2 ) . as additional support for predominant ligand recognition in the undocked state , previous isothermal titration calorimetry experiments demonstrate that riboswitches lacking l13 can still effectively bind hycbl , implying that ligand binding does not require the rna to be in the docked conformation . together , these observations indicate that ligand - recognition follows the induced - fit model , which reinforces the notion that this behavior is commonly associated with many small - molecule binding rnas . the detailed kinetic characterization of this riboswitch enables comparisons with other biologically related processes . to do so , we draw on the following principles of chemical kinetics:(i ) the rate constant describing the approach to equilibrium ( keq ) is given by the sum of the forward / reverse rate constants and ( ii ) the reciprocal of a first - order rate - constant represents the characteristic 1/e time - scale for that particular process . the ligand binding kinetics experiments reveal that the bimolecular kbind is fast ( 10 m s ) , relative to many other rna ligand interactions . however , the relatively slow ( 0.01 s ) kdiss mandates that , at hycbl concentrations near kd , the time - scale associated with ligand binding ( i.e. , [ kon kd ] 200 s ) is much longer than that required to establish the two kl docking equilibria ( i.e. , [ kdock , free + kundock , free ] 0.14 s and [ kdock , hycbl + kundock , hycbl ] 1.1 s ) . therefore , at [ hycbl ] < 1000 nm ( i.e. , concentrations where ligand binding and kl docking time scales are comparable ) , ligand binding represents the rate limiting process in the four - state kinetic model ( figure 2 ) . the above conclusion raises a significant question with regard to genetic regulation : does the riboswitch - containing mrna have enough time , after being transcribed , to bind the ligand before being degraded in the cell ? specifically , at [ hycbl ] near kd , the time - scale associated with ligand binding ( i.e. , 200 s ) is indeed comparable to that of mrna decay ( 300 s ) . such a comparison suggests that ligand binding may be temporally limited , and thus that regulation of gene expression provided by the env8hycbl riboswitch may not be completely thermodynamically controlled ; further quantitative investigations focusing on both mrna abundance and decay will be required to resolve such issues definitively . lastly , we make use of qualitative comparisons between the in vitro and cell - based results in order to obtain information about the relationship between the physical mechanism of the env8hycbl riboswitch and the biologically relevant ligand - dependent modulation of gene expression . provided that cells transformed with the l5-mutant env8hycbl+gfpuv vectors do not suffer from any unforeseen changes in mrna abundance , they can be used to define the maximum amount of gene expression ( figure 8b ) due to their complete inability to form the regulatory kl . the normalized fluorescence ( materials and methods ) associated with maximal gene expression is used to approximate the relative gene expression associated with the wt env8hycbl riboswitch in the presence or absence of 5000 nm ligand . the approximate quantitation of the cell - based experiments reveals that , in the absence of hycbl , the env8hycbl riboswitch encoded upstream of a gfpuv fluorescence reporter results in a relative gene expression value of 0.90(8 ) . this cell - based value is quite similar to the in vitro values of fufree = 0.83(7 ) and fufree 0.78 ( at 1 mm mg ) for surface - immobilized and freely diffusing smfret experiments , respectively . the similarity of these values seems to suggest that gene expression is well correlated with fufree . furthermore , the relative gene expression for the wt env8hycbl in the presence of 5000 nm hycbl drops to a value of 0.10(3 ) . again , the hycbl - dependent decrease in relative gene expression ( 0.90 0.10 ) observed in the cell - based experiments is comparable to the decrease in the fractional occupancy of the undocked conformation ( fu ) as determined via smfret experiments ( 0.83 0.25 ) . together , the in vitro and cell - based experiments demonstrate that ( i ) the fractional occupancy of the undocked conformation at 1 mm mg is strongly correlated with the hycbl - dependent expression of gfpuv and ( ii ) the l5-l13 kl interactions is certainly the physical switch associated with regulation of gene expression . this work represents the first quantitative single - molecule investigations of a fully functional riboswitch , complete with complementary cell - based assays . the results of these experiments clearly identify the conformational mechanism responsible for regulation of gene expression by the env8hycbl riboswitch , which serves as a model system for the entire clan of cobalamin riboswitches . specifically , ligand binding primarily occurs when the l5l13 kissing loop is absent and alters the energetics associated with this regulatory kissing - loop ( kl ) , resulting in preferential stabilization of the docked state . in this conformation , these base pairs conceal this region of the mrna from the cellular machinery responsible for translation initiation , thus repressing expression of the downstream gene . time - resolved ligand - binding experiments indicate that although this process is fast relative to other riboswitches , it is still much slower than the equilibration time - scale for the kl interaction . the high degree of correlation between cell - based gfpuv reporter assays and biophysical fluorescence experiments further supports the notion that formation of the kl interaction is directly responsible for repression of gene expression and identifies this region of the rna as the physical regulatory switch . this kl interaction shares a number of biochemical features with other functional kissing loop systems , for example , the hiv genome dimerization system and the rop - rna i - rna ii plasmid replication system . together , these similarities highlight the importance of ( i ) nucleotide sequence , ( ii ) divalent cations , and ( iii ) macromolecular binding partners in tuning the stability of this structural motif . one interesting direction for further study would be to more rigorously explore the importance of structural defects within the kl and their ability to confer ligand - facilitated docking . most importantly , this work presents a detailed kinetic characterization of a ligand - facilitated conformational transition that serves as a point - of - comparison for other such studies of similar rnas . the env8 riboswitch constructs used in this work are based on a sequence originally identified from comparative metagenomic analyses of functional noncoding rnas . the various constructs designed for single - molecule fluorescence resonance energy transfer ( smfret ) experiments are prepared via nonsplinted ligation of two custom synthetically modified rna oligonucleotides ( integrated dna technologies ) . the wild - type env8 hycbl smfret construct is assembled from the following two rnas : wt strand 1 ( 5-biotin - aaa aaa aag gcc uaa aag cgu agu ggg aaa g[dt*]g acg uga aau ucg ucc aga uua c-3 ) and wt strand 2 ( 5-phosphate - uug aua cgg uua uac ucc gaa ugc cac cua ggc cau aca acg agc aag gag acu c - cy33 ) . according to the x - ray crystal structure of env8hycbl , nucleotide u24 is completely solvent exposed and lacks intramolecular contacts with other functional groups of the rna . by way of additional confirmation , shape chemical probing experiments reveal this nucleotide to be hyper - reactive , as is often the case with highly solvent exposed nucleotides . in conjunction with the x - ray crystal structure , this suggests that u24 is well suited for synthetic modification . accordingly , this nucleotide is replaced by an amino - modified deoxynucleotide ( dt * ) , which is subsequently chemically labeled with an nhs - functionalized cy5 fluorophore ( ge healthcare ) following the manufacturer s suggested protocol . microcentrifuge size - exclusion columns ( thermoscientific ) are used to remove the excess unreacted fluorophores from the labeling reaction . the wt strands 1 and 2 are annealed together by slowly cooling from 85 c to room temperature in annealing buffer ( 50 mm hepes , 800 m hydroxocobalamin ( sigma - aldrich ) , 100 mm kcl , ph 7.5 ) prior to enzymatic ligation via t4 rna ligase i ( new england biolabs ) note , the slow cooling and lack of mg was chosen to maximize the abundance of rna constructs that were in a ligand - responsive conformation ( 85% ) . isolation of full - length doubly labeled rna construct is achieved through the use of a reverse phase high - performance liquid chromatography ( hplc ) column ( agilent ) . preparation of the l5-mutant construct is carried out using the same procedures , except that l5-mutant strand 2 ( 5-phosphate - aag aua cgg uua uac ucc gaa ugc cac cua ggc cau aca acg agc aag gag acu c - cy3 - 3 ) is used instead of wt strand 2 , with the underlined nucleotides corresponding to the location of the desired mutations . synthesis of the cy3-only env8hycbl construct used in the ensemble fluorometry experiments is performed using in vitro transcription by t7 rna polymerase , as described previously . the resulting transcripts are exposed to naio4 and nabh3cn for selective oxidation of the 3-ribose and reacted with hydrazide - functionalized cy3 to covalently attach the fluorophore to this position . removal of excess dye and hplc purification is performed , as described above , in order to isolate full - length fluorescently labeled rna constructs . freely diffusing smfret experiments are performed on a home - build inverted confocal fluorescence microscope , described previously , with a 1.2 n.a . when the concentration of fluorescently labeled rna is sufficiently low ( 125 pm ) , individual molecules will transiently diffuse into the overlapping foci of two ( 532 nm , time - bandwidth products and 635 nm , picoquant gmbh ) alternating laser excitation ( alex ) sources with 100 w average power . this yields a short ( < 1 ms ) burst of fluorescence photons dictated by the time the rna spends within the overlapping excitation and detection volumes . the fret efficiency ( efret ) resulting from 532 nm excitation is calculated for each fluorescent burst , where the use of alex techniques ensures that only dually labeled , nonphotophysically damaged constructs are considered for analysis . all experiments are performed at ambient temperatures ( 2022 c ) in standard buffer ( 50 mm hepes , 25 mm koh , 10 mm naoh , 100 mm kcl , 2 mm trolox , 100 nm pcd and 5 mm pca , ph 7.7 ) consisting of the well - characterized enzymatic oxygen scavenging system , pca / pcd , and various concentrations of mgcl2 and hydroxocobalamin ( hycbl ) . error bars for freely diffusing experiments are often smaller than the associated data points and represent the propagated uncertainty associated with the fractional occupancy of the high fret state as determined via a nonlinear least - squares ( nlls ) fit to a sum of two - gaussian distributions . hycbl - dependent cy3 quenching ensemble experiments are performed on a fluorimeter ( jobin yvon ) with 500 pm fluorescent rna in standard buffer containing 1 mm mgcl2 and 1050 nm hydroxocobalamin ( figure 4 ) . the excitation and emission filters are set to 532 1 and 570 7 nm , respectively , with each data point representing one second of integrated fluorescence . the narrow , off - peak excitation filter ensures that photobleaching of cy3 represents a negligible contribution to the time - dependent reduction in the fluorescence intensity . error bars for the ensemble fluorometry experiments represent the standard deviation associated with triplicate measurements of each data point . surface - immobilzed smfret experiments are performed on the same inverted confocal fluorescence microscope used for the freely diffusing experiments . streptavidin chemistry , resulting in a surface coverage of < 1 molecule per m . average laser powers are 2 w in order to reduce the effects of photobleaching and prolong the observation of surface immobilized molecules . docking and undocking rate constants are determined using a previously described thresholding routine and a bayesian maximum - likelihood model . by way of validating each of these approaches , the two analyses result in rate constants that are experimentally indistinguishable when applied to the same data set . error bars for surface immobilized experiments are often smaller than the associated data points . they represent the standard deviation derived from nlls fits to decaying exponentials for three unique cumulative dwell time distributions , each containing approximately the same number of dwell times ( 100500 each ) . riboswitch constructs are amplified via recombinant overlapping pcr and cloned into a pbr327 derivative between nsii and hindiii restriction sites . for the cell - based assays , plasmids are transformed into e. coli keio strain btur ( keio collection(61 ) jw1262 ) . the cells are plated onto lb agar plates supplemented with 100 g / ml ampicillin , and incubated at 37 c for 1416 h to facilitate colony formation . three colonies are picked for each construct and grown to saturation in a rich , chemically defined medium ( csb media : nah2po4 ( 4.6 mg / ml ) , k2hpo4 ( 11.7 mg / ml ) , and ( nh4)2so4 ( 2.0 mg / ml ) , d - glucose ( 0.6% ) , sodium citrate ( 5 mm ) , and mgso4 ( 492 m ) , fecl36h2o ( 109 m ) , zncl24h2o ( 28.3 m ) , cocl26h2o ( 24.8 m ) , na2moo4 ( 13.8 m ) , cacl22h2o ( 20.1 m ) , cucl2 ( 21.9 m ) , mncl2 ( 23.4 m ) , and h3bo3 ( 23.9 m ) ) supplemented with 100 g / ml ampicillin to maintain plasmid selection . to measure cellular fluorescence resulting from gfpuv expression , 5 l of saturated overnight culture has been added to 5 ml of csb medium supplemented with 100 g / ml ampicillin and 5 m hydoxocobalamin , and grown to mid - log phase via incubation at 37 c in a rotating drum . for fluorescence measurements , 300 l of cells from each biological replicate is added to the wells of a greiner 96 well , half - area microplate . gfpuv expression is monitored at an excitation wavelength of 395 nm and a 510 nm emission wavelength using a tecan infinite m200 pro plate - reader . the average and standard deviation for the fluorescence of each individual well is determined using three biological replicates measured in triplicate and normalized to cell density as determined by the od600 . normalized fluorescence values for each construct are background corrected via subtraction of cell - density normalized fluorescence from a pbr327 empty vector control . error bars represent the propagated uncertainty in the relative gene expression as determined by the standard deviations of the normalized fluorescence values .

riboswitches represent a family of highly structured regulatory elements found primarily in the leader sequences of bacterial mrnas . they function as molecular switches capable of altering gene expression ; commonly , this occurs via a conformational change in a regulatory element of a riboswitch that results from ligand binding in the aptamer domain . numerous studies have investigated the ligand binding process , but little is known about the structural changes in the regulatory element . a mechanistic description of both processes is essential for deeply understanding how riboswitches modulate gene expression . this task is greatly facilitated by studying all aspects of riboswitch structure / dynamics / function in the same model system . to this end , single - molecule fluorescence resonance energy transfer ( smfret ) techniques have been used to directly observe the conformational dynamics of a hydroxocobalamin ( hycbl ) binding riboswitch ( env8hycbl ) with a known crystallographic structure.1 the single - molecule rna construct studied in this work is unique in that it contains all of the structural elements both necessary and sufficient for regulation of gene expression in a biological context . the results of this investigation reveal that the undocking rate constant associated with the disruption of a long - range kissing - loop ( kl ) interaction is substantially decreased when the ligand is bound to the rna , resulting in a preferential stabilization of the docked conformation . notably , the formation of this tertiary kl interaction directly sequesters the shine - dalgarno sequence ( i.e. , the ribosome binding site ) via base - pairing , thus preventing translation initiation . these results reveal that the conformational dynamics of this regulatory switch are quantitatively described by a four - state kinetic model , whereby ligand binding promotes formation of the kl interaction . the results of complementary cell - based gene expression experiments conducted in escherichia coli are highly correlated with the smfret results , suggesting that kl formation is directly responsible for regulating gene expression .

Introduction Results Discussion Conclusions Materials and Methods

riboswitches are noncoding structural elements in the leader sequence of select bacterial and eukaryal mrnas that regulate downstream gene expression in a ligand - dependent fashion . the ( i ) aptamer domain of a riboswitch binds specific target molecules , which commonly promote formation of ( or stabilizes ) an alternative conformation in the ( ii ) regulatory element , or expression platform . however , only a few investigations have successfully monitored conformational transitions associated with the regulatory element of a riboswitch , and , until now , none have been able to unambiguously link ( i ) ligand binding to ( ii ) conformational transitions within a biologically functional riboswitch . complementary cell - based experiments conducted using this novel rna construct demonstrated that a tertiary interaction modulates gene expression in a vitamin b12-dependent fashion . specifically , it was shown that ( i ) this riboswitch selectively binds vitamin b12 cofactors with small -axial ligands , like hydroxocobalamin ( hycbl ) , as opposed to the bulkier -axial ligand of adenosylcobalamin , and ( ii ) binding of hycbl , the photolysis product of adenosylcobalamin , facilitates docking between two loops of the rna . it is this tertiary docking interaction that is responsible for regulating gene expression via formation of a kissing loop ( kl ) complex that directly sequesters the shine - dalgarno sequence , which is the ribosome binding site ( rbs ) of many mrnas . to this end , a combination of fluorescence techniques , smfret and cell - based fluorescent - reporter assays , has been used to offer the first , to our knowledge , detailed mechanistic account of a biologically functional riboswitch based on a collection of both cellular and smfret data . notably , this rna contains all of the core characteristics associated with the entire cobalamin riboswitch clan , namely , a 4-way junction implicated in ligand recognition and a regulatory kl , making it a highly tractable model system that contains all of the structural elements necessary and sufficient for biological activity in a cellular context . the loops , l5 ( red ) and l13 ( green ) , form a regulatory rna kissing - loop ( kl , see inset for sequence ) , which contains the purine - rich ribosome binding site ( shadowed green nucleotides ) . the results of these smfret experiments provide substantial support for a four - state kinetic model that highlights several key biophysical features associated with this regulatory switch ( figure 2 ) : ( i ) kl docking / undocking occurs in the absence of hycbl and is dependent on mg , ( ii ) hycbl binding predominately occurs when the kl is undocked , ( iii ) kl docking / undocking also occurs while hycbl is bound and is still dependent on mg . additionally , the single - molecule results reveal that , in the presence of mg , hycbl binding preferentially stabilizes the docked conformation . finally , results from complementary cell - based reporter assays are used to relate these biophysical findings to the cellular function of this rna . these comparisons reveal that formation of the regulatory switch ( i.e. , l5l13 kl ) in the single - molecule experiments is strongly correlated with repression of gene expression in the cell - based assay , regardless of whether the ligand is bound to the rna . formation of the kl decreases the interdye distance , resulting in more efficient fluorescence resonance energy transfer ( efret ) . both freely diffusing and surface - immobilized experiments ( see materials and methods ) have been used to characterize the conformational transitions associated with the regulatory kl of the env8hycbl riboswitch . the efficiency of fluorescence resonance energy transfer ( efret ) can be calculated for thousands of fluorescent bursts and compiled into a histogram to describe the probability of observing any specific efret value , which therefore reports on the conformational occupancy of the rna . as a first step , freely diffusing smfret experiments have been used to explore the equilibrium behavior of the l5l13 kissing loop ( kl ) interaction from the env8hycbl riboswitch in solutions free of hycbl . accordingly , we attribute this high efret value to molecules residing in the docked kl conformation ( figure 2b , dfree ) , with the low efret value corresponding to the undocked species ( figure 2b , ufree ) , where the subscript free the necessity of mg for the formation of this interaction is supported by the x - ray crystal structure ( pdb : 4frn ) , which depicts divalent cations near the l5 and l13 loops . specifically , the fractional occupancy of molecules in the docked or undocked conformation can be calculated using eqs 1a and 1b , respectively.1a1bthe parameters p(dfree ) and p(ufree ) describe the probability of observing the docked and undocked conformations , respectively , where the subscript free the [ mg ] required to achieve half - maximal occupancy of the docked conformation ( i.e. to confirm that the high efret population results from formation of the l5l13 regulatory switch , a second , doubly labeled rna construct has also been studied this construct , referred to as the l5-mutant env8hycbl riboswitch ( supporting information figure s1 ) , contains a dinucleotide substitution at positions 48 and 49 in l5 ( underlined nucleotides , 5-uacuug-3 5-uacaag-3 ) . as expected , this smfret construct remains in the undocked ( low efret ) conformation even in solutions containing up to 30 mm mgcl2 ( supporting information figure s1 ) , thus confirming that the donor and acceptor fluorophores accurately report on the formation of this regulatory kl interaction . under saturating hycbl conditions , therefore , the above observations support the existence of the l5l13 kl equilibrium associated with the docked ( figure 2b , dhycbl ) and undocked ( figure 2b , uhycbl ) conformations of the rna , where the hycbl subscript now denotes the presence of a bound ligand . in conjunction with the results of experiments performed in the absence of ligand , it is apparent that hycbl binding increases the stability of the docked conformation in solutions containing mg(e.g . moreover , the increased occupancy of the docked conformation , where the l5l13 kl is formed , demonstrates that ligand binding directly modulates the availability of the ribosome binding site within l13 of this regulatory switch . to directly explore the ligand - binding process , we exploit a unique photophysical property of hydroxocobalamin ( hycbl ) . in the presence of the fluorescent acceptor , cy5 , it is important to stress that the energy transfer process to hycbl competes equally with both the radiative decay process of cy3 and the energy transfer process to cy5 ( supporting information figure s2 ) , resulting in uniform attenuation of both signals . provided that the quenched fluorescence signals remain well above background , the ratiometric efret values associated with the docked ( i.e. for a single bimolecular process , this rate constant ( keq ) can simply be described as a function of ligand concentration using the ligand - binding ( kbind ) and dissociation ( kdiss ) rate constants ( eq 2).2however , the ligand binding kinetics in the present system are further complicated by the two remaining kl equilibria ( figure 2 ) , which limit the fraction of undocked molecules that can either bind to ( fufree ) or dissociate from hycbl ( fuhycbl ) . provided the docking / undocking kinetics associated with the kl equilibria are much faster than the ligand binding or dissociation processes , the apparent rate constants can be readily shown to equal true rate constants ( i.e. in solutions lacking mg , the addition of ligand is unable to promote formation of the docked conformation ( figure 5a ) , as mg is required for the formation of the regulatory kl interaction between l5 and l13 . this observation is certainly consistent with the four - state model ( figure 2 ) , wherein bound hycbl , increases the stability of the regulatory kl interaction ( i.e. , 20 mm [ mg]1/2 ) reveals that ligand is unable to significantly increase the fd ( figure 5c ) , which nicely demonstrates that mg alone is sufficient to promote complete formation of the regulatory interaction responsible for sequestering the ribosome binding site . together , these results support the notion that docking of the env8hycbl riboswitch is more favorable when the ligand is bound to the rna under near - physiological salt conditions , but that hycbl alone ( i.e. the experimentally determined fractional occupancy of the docked conformation ( fd , colored circles ) is well described by the steady - state solution ( dotted line ) to the four - state model ( figure 2 ) over a wide range of mg and hycbl concentrations . figure 5d compares the results of the single - molecule experiments with the model predictions ( colored circles and dashed lines , respectively ) . ( c ) the docking and undocking rate constants , as a function of [ mg ] , are used to calculate the fractional occupancy of the docked conformation ( d ) in the absence of hycbl ( fdfree ) , which reveals that the surface immobilized results ( dark gray stars ) are in quantitative agreement with the freely diffusing experiments ( light gray circles ) . the rate constants associated with the anticorrelated fluorescence fluctuations in the normal regions mimic those from molecules studied in the absence of hycbl ( figure 7b vs 6b ) , as would be expected when the ligand is not bound to the rna . ( b ) when the ligand is not bound , the docking / undocking kinetics of the regulatory kl are experimentally indistinguishable from those in the absence of hycbl ( see also figure 6b ) . ( c ) when the ligand is bound , a maximum - likelihood 2-state model ( black line ) is used to quantify the dwell times associated with fluctuations in the acceptor fluorescence resulting from kl docking and undocking . when the ligand is bound to the env8hycbl riboswitch in solution containing 1 mm mgcl2 , such an analysis reveals that the docking rate decreases only by 2-fold ( kdock , hycbl = 0.64(9 ) vs kdock , free = 1.2(3 ) s ) , whereas the undocking rate exhibits a much more significant hycbl - sensitivity and decreases by 23-fold ( kundock , hycbl = 0.26(8 ) s vs kundock , free = 6(2 ) s ) . these bound - state lifetimes are consistent with those predicted from the ensemble time - dependent fluorescence experiments ( figure 4c ) , which yield hycbl = ( 1/koff ) = 200(120 ) s. lastly , the time - traces of surface immobilized molecules clearly verify the previous assumption that the kl kinetics , both in the presence and absence of ligand , are much faster than those associated with establishing a ligand binding equilibrium . in total , the collection of results from the above biophysical fluorescence studies provide strong support for the proposed four - state kinetic model ( figure 2 ) associated with the regulatory conformational transitions of the env8hycbl riboswitch . to correlate the biophysical behavior of this riboswitch with its biological function , cell - based gene expression experiments ( figure 8) have been performed in an e. coli strain lacking cobalamin adenosyl transferase ( btur ) , thereby preventing the subsequent conversion of hycbl to adenosylcobalamin . the results of our biophysical experiments suggest that this l5-mutant riboswitch should be incapable of regulating gene expression because it is unable to from the regulatory kl interaction that sequesters the ribosome - binding site . ( a ) schematic diagram relating the four - state kinetic model to the regulation of gene expression via inhibition of translation initiation . the pronounced similarities between the results of the in vitro and cell - based experiments suggests that ( i ) the fractional occupancy of the undocked conformation correlates nicely with the level of gene expression , regardless of whether or not ligand is bound and ( ii ) formation of the docked conformation , resulting from a long - range kl interaction between l5 and l13 , sequesters the ribosome binding site within l13 and prevents initiation of translation . the results of these single - molecule experiments mechanistically link : ( i ) ligand binding to ( ii ) conformational transitions in the expression platform of a fully functional riboswitch . specifically , the fluorescently labeled rna constructs examined in this work provide information about the equilibrium and kinetic behavior of a regulatory kissing loop ( kl ) interaction . both the freely diffusing and surface immobilized experiments clearly identify the l5l13 kissing loop ( kl ) interaction as the structural motif that directly regulates biological function ( i.e. it is important to mention that , for the env8hycbl riboswitch , the presence of a bound ligand not only lowers the [ mg ] required for half - maximal docking ( [ mg]1/2 ) , but that it also decreases the number of ions that condense onto the rna during the conformational transition ( figure 3c ) . in addition to distinctly identifying the existence of a kl docking equilibrium in the absence of ligand , the above experimental results reveal that kl docking and undocking also occurs when hycbl is bound . this represents one example of how a specific macromolecular cosolute can stabilize the formation of a kl interaction directly responsible for a specific biological function . this is in contrast to the conformational - capture model , whereby ligand recognition occurs in transient , highly organized folded structures , similarly resulting in stabilization of the bound ( e.g. for the env8hycbl riboswitch , the experimental validation of the four - state model ( figure 5d ) and the surface - immobilized time - traces in the presence of hycbl ( figure 7a , supporting information figure s3 ) strongly suggest that ligand binding ( i.e. this notion is further supported by existing structural data , which reveal that the env8hycbl riboswitch conceals a majority ( 60% ) of the solvent accessible surface area of the ligand when the kl interaction is formed . these steric constraints would make it difficult for the approximately spherical hycbl to enter , or exit , the binding pocket when the l5l13 interaction is formed , resulting in negligibly slow ligand association / dissociation from the docked conformation ( figure 2 ) . provided that cells transformed with the l5-mutant env8hycbl+gfpuv vectors do not suffer from any unforeseen changes in mrna abundance , they can be used to define the maximum amount of gene expression ( figure 8b ) due to their complete inability to form the regulatory kl . the approximate quantitation of the cell - based experiments reveals that , in the absence of hycbl , the env8hycbl riboswitch encoded upstream of a gfpuv fluorescence reporter results in a relative gene expression value of 0.90(8 ) . again , the hycbl - dependent decrease in relative gene expression ( 0.90 0.10 ) observed in the cell - based experiments is comparable to the decrease in the fractional occupancy of the undocked conformation ( fu ) as determined via smfret experiments ( 0.83 0.25 ) . together , the in vitro and cell - based experiments demonstrate that ( i ) the fractional occupancy of the undocked conformation at 1 mm mg is strongly correlated with the hycbl - dependent expression of gfpuv and ( ii ) the l5-l13 kl interactions is certainly the physical switch associated with regulation of gene expression . this work represents the first quantitative single - molecule investigations of a fully functional riboswitch , complete with complementary cell - based assays . the results of these experiments clearly identify the conformational mechanism responsible for regulation of gene expression by the env8hycbl riboswitch , which serves as a model system for the entire clan of cobalamin riboswitches . specifically , ligand binding primarily occurs when the l5l13 kissing loop is absent and alters the energetics associated with this regulatory kissing - loop ( kl ) , resulting in preferential stabilization of the docked state . the high degree of correlation between cell - based gfpuv reporter assays and biophysical fluorescence experiments further supports the notion that formation of the kl interaction is directly responsible for repression of gene expression and identifies this region of the rna as the physical regulatory switch . the various constructs designed for single - molecule fluorescence resonance energy transfer ( smfret ) experiments are prepared via nonsplinted ligation of two custom synthetically modified rna oligonucleotides ( integrated dna technologies ) .

open radical hysterectomy has been the standard treatment for early stage cervical cancer for decades . recent advances in laparoscopic instrumentation , however , have made it possible to safely perform radical hysterectomy laparoscopically . the first total laparoscopic radical hysterectomy ( tlrh ) with pelvic and paraaortic lymphadenectomy was performed by nezhat et al in june of 1989 . since then , tlrh with pelvic or paraaortic lymph node dissection , or both , has gained acceptance as a feasible alternative to an open radical hysterectomy . numerous authors have published their experience with tlrh , firmly establishing its safety and feasibility . though no randomized trials have been performed , existing data suggest that the cure rate for laparoscopic radical hysterectomy is similar to that seen for the open procedure . despite the advantages of conventional laparoscopy over laparotomy ( shorter hospitalization , faster bowel function recovery , less postoperative pain , decreased overall cost ) , it is not without drawbacks . first , the surgeon operates in an awkward and uncomfortable position at the operating table , using a flat , 2-dimensional image . second , the majority of the instruments used are nonarticulating with an ergonomically inadequate handle design , which makes performance of fine movements exceedingly difficult . third , though not clearly defined for laparoscopic radical hysterectomy , advanced laparoscopic surgery is associated with a significant learning curve , mostly due to the counterintuitive nature of the operation . recently , computer enhanced technology ( robotics ) has been introduced into laparoscopic surgical practice . the advantages offered by this new technology include a 3-dimensional magnified field , tremor filtration , and 5 or 6 degrees of instrument mobility inside the body , thus significantly reducing the ergonomic problems associated with the conventional laparoscopic approach . there is convincing evidence that the intuitive nature of the robotic system provides an additional advantage in terms of the learning curve . the initial development of the robotic system was intended for remote surgery as a telesurgical mentoring system that would allow an experienced surgeon to provide guidance during a procedure taking place in a different location . the early robotic systems used for telementoring or telesurgery , however , had technical difficulties associated with signal latency and network instability . with advances in signal transmission technology , the earlier problems associated with remote communications are being resolved , expanding the role of robotics in telementoring and allowing inexperienced surgeons to adopt minimally invasive techniques safely while minimizing the risk of serious complications during their learning curve . clinical applications for robotic systems have been evolving rapidly and are now used widely in various surgical fields . comparative studies of robotically assisted and standard laparoscopic prostatectomies , colon resections , and nissen fundoplications show that the robotic technique is feasible , safe , and improves the surgeon 's dexterity and flexibility without compromising patient care . in this study , we conducted a comparative analysis of our data from early cervical cancer patients who underwent tlrh versus those who had rrh with respect to intraoperative , pathologic , and postoperative outcomes . this was a prospective nonrandomized analysis of all cases of rrh performed for cervical cancer at mount sinai medical center , new york . the collected data were compared with a set of 30 cases of tlrh performed for cervical cancer at the same institution from august 2000 to june 2006 . starting in 2006 , the robotic - assistance approach was offered to all patients for whom a laparoscopic approach was deemed appropriate . selection criteria for tlrh and rrh were the same and included all women with newly diagnosed invasive cervical cancer stages ia1 to iia who desired a laparoscopic approach , after providing informed consent . patients were not considered for the laparoscopic approach if they had metastatic disease beyond the uterus , a cervical lesion of more than 4 cm in size , a uterus size of more than 12 cm , inadequate bone marrow , or compromised renal and hepatic function , or if they were pregnant . this was a prospective nonrandomized analysis of all cases of rrh performed for cervical cancer at mount sinai medical center , new york . the collected data were compared with a set of 30 cases of tlrh performed for cervical cancer at the same institution from august 2000 to june 2006 . starting in 2006 , the robotic - assistance approach was offered to all patients for whom a laparoscopic approach was deemed appropriate . selection criteria for tlrh and rrh were the same and included all women with newly diagnosed invasive cervical cancer stages ia1 to iia who desired a laparoscopic approach , after providing informed consent . patients were not considered for the laparoscopic approach if they had metastatic disease beyond the uterus , a cervical lesion of more than 4 cm in size , a uterus size of more than 12 cm , inadequate bone marrow , or compromised renal and hepatic function , or if they were pregnant . clinical data for both the laparoscopic groups and the robotic groups were analyzed by a review of patients ' medical records and operation reports . all patients were staged according to the international federation of gynecology and obstetrics ( figo ) criteria . all patients had a computerized tomography ( ct ) scan of the abdomen and pelvis done preoperatively to evaluate lymph node status and potential extrapelvic and extraabdominal disease . intraoperative and postoperative data including patient characteristics , operation details , histological data , and follow - up information were recorded . duration of surgery was defined from the time of skin incision to the closure of the skin incision . robotic docking time was recorded as the time to attach the robotic arms to the trocars and insertion of robotic instruments . postoperative complications included those occurring during the same hospitalization or within 30 days following discharge . perioperative workup and postoperative care were provided by a gynecologic oncology service run by a fellow . all surgeries were performed by a gynecologic oncology fellow and a mentoring gynecologic oncology attending physician with experience in advanced laparoscopy . the fellow at the beginning of his or her training participated as first assistant . as the fellow became more experienced and skilled , the fellow 's role transitioned to a primary surgeon with an attending surgeon or junior fellow acting as a first assistant under the attending physician 's supervision . the da vinci robot is an integrated computer based system , consisting of 3 interactive robotic arms , a camera arm and a remote control console with 3-dimensional visual capabilities . the motions of the surgeon at the console unit are replicated by the robotic arms placed within the patient . during robotic surgery , an assistant is available at the operating table . the assistant performs robot - related tasks , including alignment and exchange of robotic instruments , operative maneuvers with conventional instruments such as organ manipulation , tissue countertraction , suction and irrigation , and any necessary alterations in the position of the intrauterine manipulator . the presence of the scrubbed assistant is also crucial in the event that an emergency conversion to a laparotomy is required . the technique of total laparoscopic hysterectomy and bilateral pelvic lymphadenectomy using harmonic shears has been previously described and served as the basis for the robotic procedure . after patients received appropriate preoperative counseling and gave their written informed consent , a standard outpatient mechanical bowel preparation was prescribed . the procedure was performed with the patient under general endotracheal anesthesia in the dorsal lithotomy position with adjustable allen stirrups and lower extremity compression devices for deep venous thrombosis prophylaxis . betadine solution was applied topically , and sterile drapes were placed in the usual sterile fashion . a foley catheter was placed into the bladder before the procedure was started ; the catheter was drained by gravity for the duration of the surgery . traditional diagnostic laparoscopy was performed first to assess for feasibility of the intended procedure , as well as to detect intraabdominal metastatic disease . the procedure was terminated if metastatic disease was detected , and confirmed by frozen section evaluation , outside of the pelvis ( eg , in the omentum , bladder , liver , or bowel ) , in the uterine adnexa , or if the tumor extruded through the uterine wall into the peritoneal cavity . if none of the above were found , the laparoscopic equipment was removed . a standard 12-mm trocar placed at the umbilicus was used for camera placement , 2 working robotic arms were attached to 8-mm reusable trocars placed bilaterally , and ancillary 10-mm trocars were placed in the suprapubic region and the left or right upper quadrant . the robotic ports were placed 1 cm to 2 cm below and 8 cm to 10 cm lateral to the intraumbilical trocar , so as to enable optimal movement of the robotic arm and to minimize the risk of collision ( figure 1 ) . a 12-mm camera trocar is placed at the umbilicus , 2 working robotic arms are attached to 8-mm reusable trocars placed bilaterally , and additional ancillary 5-mm to 10-mm trocars are placed in the suprapubic region and the left upper quadrant . the camera port and each working robotic port are placed in a way that allows for optimal robot arm movement and minimizes risk of collisions . the whole procedure is performed using the robotic monopolar electrosurgical scissors placed through the right port , and the fenestrated bipolar forceps placed through the left robotic port . dorsey smokevac suction irrigator pump , probe ( bard inc , murray hill , nj ) , grasping forceps and bipolar forceps as needed , as well as harmonic shears ( ethicon endo - surgery inc , blue ash , oh ) or a ligasure vessel sealing device ( valleylab , boulder , co ) . adhesions were lysed first to restore normal anatomy , and the undersurfaces of the diaphragm , liver , gallbladder , stomach , omentum , and large and small bowel were examined visually , when possible . the paraaortic lymph nodes were inspected , followed by the pelvic lymph nodes . proceeding with a radical hysterectomy requires that 6 avascular pelvic spaces be developed and that the bladder and rectum be mobilized . traditionally , we start out by dissecting the rectovaginal space . the uterus is sharply anteverted by using an intrauterine manipulator , and the peritoneum between the uterosacral ligaments is incised by using monopolar scissors ; the rectum can then be brought down gently away from the vagina . a moistened sponge on a sponge - forceps is placed in the posterior vaginal fornix to facilitate visualization and development of this surgical plane ( figure 2 ) . the posterior vaginal fornix is placed on tension ( marked by the arrow ) , and a moistened sponge on sponge - forceps is placed in the vagina to facilitate delineation of the tissue planes . after round ligaments on either side of the uterus were desiccated and cut with the monopolar scissors , the anterior leaf of the broad ligament was opened bilaterally . the bladder was gradually dissected away from the cervix and vagina with a moistened sponge on a sponge - forceps placed in the anterior vaginal fornix to facilitate development of the vesicovaginal space ( figure 3 ) . the uterus ( b ) is pushed cephalad into the abdominal cavity to facilitate visualization . the posterior leaf of the broad ligament was opened using monopolar scissors and forceps and the paravesical and pararectal spaces were developed using gentle blunt dissection . in cases where ovarian preservation was indicated or desired , the uteroovarian ligament and the proximal portion of the fallopian tube were coagulated and divided . if the adnexa were to be removed , the infundibulopelvic ligament was isolated , desiccated and divided using the bipolar forceps and scissors . the paravesical space was developed by placing tension on the umbilical ligaments with sharp , blunt dissection performed with scissors , forceps , and suction irrigator . the dissection was continued inferiorly to the iliac vessels , after which the obturator space was developed . the structures surrounding the obturator space , including the obturator internus muscle and pubic bone , were examined visually and care was taken to avoid injury to the obturator nerve and vessels that traverse this area . after development of the paravesical and pararectal spaces , the pelvic lymphadenectomy can be performed . pelvic lymphadenectomy involves removal of the lymph node packets from the common iliac vessels and external iliac vessels down to the level of the deep circumflex iliac veins ( figure 4 ) . the obturator nerve was identified , and the obturator fossa nodes and the hypogastric lymph nodes were completely removed and sent for pathological examination . at this point , the medial umbilical ligament was suspended with upward tension , and the origin of the uterine artery from the hypogastric artery was identified ( figure 5 ) . the uterine artery was desiccated and divided at its origin with bipolar forceps and monopolar scissors as shown in figure 6 . the uterine vessels were placed on medial tension , and the ureter was unroofed using the curved tip of the monopolar scissor out of the tunnel ( figure 7 ) , and then the surrounding tissues were coagulated and divided ( figure 8) . the uterosacral ligaments , cardinal ligaments , and a portion of the paracolpos were then divided with the bipolar forceps and scissors , enabling complete mobilization of the uterus . a circumferential incision was made into the vagina using monopolar scissors to ensure an adequate margin ( figure 9 ) . the uterus was separated completely from the vagina and removed while attached to the uterine manipulator . in some cases , the specimen removal was done vaginally to allow for a superior visualization and delineation of the vaginal margins . the vaginal cuff was closed with interrupted or running 0 vicryl suture tied intracorporeally or vaginally ( figure 10 ) . lymph node packets ( f ) are removed from the left common external iliac artery ( a ) and vein ( b ) . the left obturator nerve ( c ) , the left obliterated umbilical artery ( d ) , and the left ureter ( e ) are identified . the uterine artery ( a ) is identified and dissected from the point of its origin at the hypogastric artery ( b ) traversing over the ureter ( c ) . the right uterine artery ( a ) is coagulated and divided at its origin by using bipolar forceps and monopolar scissors . the right pararectal ( b ) and paravesical ( c ) spaces are fully developed ; and the right ureter ( d ) , right umbilical ( e ) , and right external iliac artery ( f ) are visible . the paravesical space ( b ) and right obliterated umbilical artery ( c ) are identified . the right ureter ( b ) , right obliterated umbilical ( c ) , and right external iliac arteries ( d ) are seen . using monopolar scissors , a circumferential incision is made into the vagina assuring adequate margin . vaginal cuff closure with intracorporeal tying . after removal of the specimen and closure of the vaginal cuff , the pelvic cavity was thoroughly evaluated . both the pelvic and abdominal cavities were irrigated copiously with normal saline ( figure 11 ) . once the surgeon had ensured hemostasis , indigo carmine was administered intravenously to assess ureteral and bladder integrity . the rectum was then insufflated with air and evaluated intraabdominally under saline to rule out any bowel injuries . the bladder was either distended with saline , or cystoscopy was performed to further ensure its integrity . upon completion of the procedure , the da vinci system was undocked , all of the instruments were removed , and the trocar sites were closed using a figure - of - eight 0 vicryl suture and 40 vicryl in a subcuticular fashion . both ureters ( a and b ) have been dissected to the level of the bladder . we routinely use subcutaneous heparin , low molecular weight heparin , or pneumatic compression devices until patients are fully ambulatory . the patients were discharged home on the second or third postoperative day with a foley catheter in place . comparative analysis was performed using statview software ( sas , north carolina ) . the outcomes from the laparoscopic radical and robotic - assisted groups were compared using fisher 's exact test and the chi - squared test for categorical variables and 2 sample student t tests for continuous variables . a total of forty seven patients met our inclusion criteria and had either tlrh or rrh with pelvic lymphadenectomy performed . three of these 4 patients had only a portion of the entire procedure ( pelvic lymphadenectomy , bilateral ureterolysis or part of the parametrial dissection ) performed robotically with the majority of the procedure performed laparoscopically . the remaining 13 cases comprised our rrh with bilateral pelvic lymphadenectomy cohort performed between april 2006 and january 2008 . there were no differences in clinical tumor characteristics , such as stage , histology , and lympho - vascular space involvement between the two groups ( table 1 ) . two patients in the rrh group underwent neoadjuvant chemotherapy , while only one patient in the tlrh group did so . patient characteristics as shown in table 2 , mean operative time , estimated blood loss , and length of postoperative stay were similar between the 2 patient groups ( p > 0.05 ) . the mean operative time for tlrh with pelvic lymphadenectomy leveled off at 318 minutes and did not significantly decrease over time . mean operative time for rrh changed little over the study period as well ( figure 12 ) . in contrast , the mean time for robotic docking was 12 minutes ( range 430 min ) , and decreased as the surgeon gained experience ( figure 13 ) . clinical variables and outcomes three patients in the rrh group and 6 in the tlrh group underwent paraaortic lymphadenectomy before the hysterectomy due to paraaortic lymph node enlargement detected on preoperative imaging , or cervical lesions with a diameter greater than 2 cm . seven patients in the tlrh group and one patient in the rrh group had ovarysparing procedures . in addition , 2 patients in the tlrh group underwent umbilical hernia repair and one patient had a bartholin 's abscess excised . the robotic group had 2 intraoperative incidental cystotomies , which occurred at the time of vaginal transection before specimen removal . one of these patients had extensive endometriosis at the anterior vaginal margin , and the other had vaginal tumor extension with significantly fore - shortened anterior vaginal fornix . in the tlrh group , 2 patients underwent inadvertent cystotomies at the time of laparoscopic bladder dissection of the anterior vagina . both patients had bilateral jj ureteral stents placed intraoperatively and had an otherwise uncomplicated recovery . outpatient cystoscopy , cystogram , and stent removal was performed on both patients 10 days later with no long - term sequela . postoperative complications in the rrh group included one case of postoperative ileus , prolonged urinary retention , vaginal lymph drainage , and c. difficile colitis . the tlrh group had complications including two cases of deep vein thrombosis pulmonary embolism and c. difficile colitis and cases of ileus , and prolonged urinary retention ( table 3 ) . the mean yield of the pelvic lymph nodes was 24.7 in the rrh group and 31 in the tlrh group ( table 2 ) . there were no recurrences in either group with a mean follow - up time of 12 months in the rrh group and 29 months in the tlrh group . we did not note a significant difference between rrh and tlrh with respect to operative time , operative blood loss , length of hospital stay , or complication profile . the only intraoperative complication observed in both groups was cystotomy . along with ureteral injury , these are the most common reported intraoperative complications associated with laparoscopic radical hysterectomy . bladder injuries in the tlrh group were neither related to radical parametrial resection nor to lymph node dissection . similarly , in the rrh group , cystotomies were not related to the use of the robot system and took place at the time of the vaginal specimen removal . none of the patients had a recurrence with a mean follow - up of 12 months in the robotic group and 29 months in the laparoscopic group . several recent publications strongly demonstrated that computer - assisted surgical approaches are becoming increasingly feasible . in fact , our evidence , as well as the evidence of others , supports robotic surgery as a more attractive option , both for the surgeon and the patient . however , questions remain , including whether the robot provides any additional benefits to a surgeon who is an experienced laparoscopist and comfortable performing the most advanced gynecologic procedures using traditional laparoscopy , whether there is an advantage for an inexperienced laparoscopic surgeon to use robotic technology compared with traditional laparoscopic instrumentation , and what the learning curve is with either approach . several gynecologic surgeons have reported their experiences performing tubal reanastomosis , salpingo - oophorectomy , and hysterectomy using an earlier robotic system . most recently , nezhat and colleagues and koh and colleagues reported their experiences performing various advanced gynecologic procedures using the current generation of the da vinci system . the largest experience using robotic systems for the surgical treatment of gynecologic cancers was reported by j. magrina of the mayo clinic ( scottsdale , az ) . it comprised 142 patients treated surgically with the da vinci robotic system for various primary and recurrent gynecologic malignancies . the mean operating time was 218 minutes , estimated blood loss was 176 ml , and the hospitalization time was 1.9 days . the authors concluded that robotic surgery is preferable to conventional laparoscopy for gynecologic oncology procedures because it provides improved dexterity , 3-dimensional viewing , surgical precision with tremor filtration , a comfortable fatigue - reducing console , and greater motion freedom allowed by the robotic instruments , which significantly reduced ergonomic problems associated with conventional laparoscopic equipment . the author performed 13 rrh procedures that were compared with 48 historic abdominal radical hysterectomies . lymph node yield was significantly higher in the robotic group ( 33 vs. 22 ) , and operative time was similar between the groups . blood loss as well as transfusion requirements in the robotic group were significantly less than that in the abdominal group . all patients who underwent robotic rrh were discharged the day after surgery with significantly lower pain medication requirements than patients who underwent an open procedure . the gynecologic oncology community has been appropriately cautious in accepting laparoscopic procedures , including tlrh , as a standard of care due to a lack of oncological outcome data . data on specimen size , margin adequacy , and parametria appear to be equivalent . with the follow - up data in some of the studies approaching or exceeding 5 years , does the recurrence rate in the laparoscopically managed patients exceed that of the patients who underwent an open procedure . as was the case with laparoscopy 10 years ago abdominal radical hysterectomy continues to be the most common surgical approach in treatment of an early stage carcinoma of the cervix . the role of laparoscopy in this setting is to offer all of the benefits of a minimally invasive approach , namely faster recovery , decreased blood loss and transfusion rates , and decreased postoperative pain , while maintaining the excellent oncological outcomes of an open approach . while all gynecologic surgeons taking care of patients with early cervical cancer are trained to perform abdominal radical hysterectomy , only a few are comfortable performing the procedure laparoscopically . tlrh is one of the most challenging laparoscopic procedures in gynecologic oncology , requiring significant technical expertise and experience . because this is a relatively new technique , the number of cases required to obtain proficiency is not known . as more centers perform these procedures , report their experiences , and the technique itself is developed , standardized , and taught systematically , we will better understand the learning curve required for both tlrh and rrh . the available urological data suggest that the intuitive nature of the robotic approach may provide a significant advantage in terms of its learning curve especially to surgeons with little or no advanced laparoscopic experience . ahlering and colleagues reported the initial experience of performing robotic - assisted radical prostatectomy by an experienced abdominal surgeon without any laparoscopic experience . it required only 12 cases to achieve proficiency in performing radical prostatectomy with robotic assistance . robotic - assisted prostatectomy outcomes were comparable to those achieved by a skilled laparoscopic surgeon after 100 cases of laparoscopic radical prostatectomies . as the number of early cervical cancer cases is decreasing , fast acquisition of advanced endoscopic skills is paramount . therefore , the robotic interface , which allows for significant shortening of the learning curve , may make a minimally invasive approach possible even in centers with very few cases of early cervical cancer . the robotic systems have their own drawbacks ; most commonly mentioned are the absence of tensile feedback , the complexity of the system , the size of the system , and the cost . robotic technology is developing rapidly , and new instruments , smaller arms , the addition of a fourth arm and tactile feedback are already becoming available . currently , operations performed with a robot are expensive , but the widespread use of this technology , combined with the shorter hospital stay , hopefully , will lead to an overall , and substantial , decrease in cost . though robotic technology has revolutionized urologists ' treatment of prostate cancer , its use in the treatment of cervical cancer by gynecologic oncologists is still in development . we have found that the substantial magnification , dexterity , and flexibility offered by the robotic system significantly simplify the most difficult stages of radical hysterectomy and pelvic lymphadenectomy , which would enable a greater number of surgeons to perform this procedure laparoscopically . as we continue to develop new surgical techniques , we can not compromise the patient 's safety or oncological outcome , so we should subject these newer approaches to thorough evaluation before they become the standard .

background and objectives : to compare intraoperative , pathologic and postoperative outcomes of robotic radical hysterectomy ( rrh ) to total laparoscopic radical hysterectomy ( tlrh ) in patients with early stage cervical carcinoma.methods:we prospectively analyzed cases of tlrh or rrh with pelvic lymphadenectomy performed for treatment of early cervical cancer between 2000 and 2008.results:thirty patients underwent tlrh and pelvic lymph - adenectomy for cervical cancer from august 2000 to june 2006 . thirteen patients underwent rrh and pelvic lymph - adenectomy for cervical cancer from april 2006 to january 2008 . there were no differences between groups for age , tumor histology , stage , lymphovascular space involvement or nodal status . no statistical differences were observed regarding operative time ( 323 vs 318 min ) , estimated blood loss ( 157 vs 200 ml ) , or hospital stay ( 2.7 vs 3.8 days ) . mean pelvic lymph node count was similar in the two groups ( 25 vs 31 ) . none of the robotic or laparoscopic procedures required conversion to laparotomy . the differences in major operative and postoperative complications between the two groups were not significant . all patients in both groups are alive and free of disease at the time of last follow up.conclusion:based on our experience , robotic radical hysterectomy appears to be equivalent to total laparoscopic radical hysterectomy with respect to operative time , blood loss , hospital stay , and oncological outcome . we feel the intuitive nature of the robotic approach , magnification , dexterity , and flexibility combined with significant reduction in surgeon 's fatigue offered by the robotic system will allow more surgeons to use a minimally invasive approach to radical hysterectomy .

INTRODUCTION MATERIALS AND METHODS Study Design Patient Population Data Collection Surgeons Robotic Laparoscopic Radical Hysterectomy Surgical Technique Postoperative Care Statistical Analysis RESULTS DISCUSSION CONCLUSION

open radical hysterectomy has been the standard treatment for early stage cervical cancer for decades . the first total laparoscopic radical hysterectomy ( tlrh ) with pelvic and paraaortic lymphadenectomy was performed by nezhat et al in june of 1989 . since then , tlrh with pelvic or paraaortic lymph node dissection , or both , has gained acceptance as a feasible alternative to an open radical hysterectomy . though no randomized trials have been performed , existing data suggest that the cure rate for laparoscopic radical hysterectomy is similar to that seen for the open procedure . despite the advantages of conventional laparoscopy over laparotomy ( shorter hospitalization , faster bowel function recovery , less postoperative pain , decreased overall cost ) , it is not without drawbacks . second , the majority of the instruments used are nonarticulating with an ergonomically inadequate handle design , which makes performance of fine movements exceedingly difficult . third , though not clearly defined for laparoscopic radical hysterectomy , advanced laparoscopic surgery is associated with a significant learning curve , mostly due to the counterintuitive nature of the operation . the advantages offered by this new technology include a 3-dimensional magnified field , tremor filtration , and 5 or 6 degrees of instrument mobility inside the body , thus significantly reducing the ergonomic problems associated with the conventional laparoscopic approach . there is convincing evidence that the intuitive nature of the robotic system provides an additional advantage in terms of the learning curve . the initial development of the robotic system was intended for remote surgery as a telesurgical mentoring system that would allow an experienced surgeon to provide guidance during a procedure taking place in a different location . with advances in signal transmission technology , the earlier problems associated with remote communications are being resolved , expanding the role of robotics in telementoring and allowing inexperienced surgeons to adopt minimally invasive techniques safely while minimizing the risk of serious complications during their learning curve . comparative studies of robotically assisted and standard laparoscopic prostatectomies , colon resections , and nissen fundoplications show that the robotic technique is feasible , safe , and improves the surgeon 's dexterity and flexibility without compromising patient care . in this study , we conducted a comparative analysis of our data from early cervical cancer patients who underwent tlrh versus those who had rrh with respect to intraoperative , pathologic , and postoperative outcomes . this was a prospective nonrandomized analysis of all cases of rrh performed for cervical cancer at mount sinai medical center , new york . the collected data were compared with a set of 30 cases of tlrh performed for cervical cancer at the same institution from august 2000 to june 2006 . starting in 2006 , the robotic - assistance approach was offered to all patients for whom a laparoscopic approach was deemed appropriate . selection criteria for tlrh and rrh were the same and included all women with newly diagnosed invasive cervical cancer stages ia1 to iia who desired a laparoscopic approach , after providing informed consent . patients were not considered for the laparoscopic approach if they had metastatic disease beyond the uterus , a cervical lesion of more than 4 cm in size , a uterus size of more than 12 cm , inadequate bone marrow , or compromised renal and hepatic function , or if they were pregnant . this was a prospective nonrandomized analysis of all cases of rrh performed for cervical cancer at mount sinai medical center , new york . the collected data were compared with a set of 30 cases of tlrh performed for cervical cancer at the same institution from august 2000 to june 2006 . starting in 2006 , the robotic - assistance approach was offered to all patients for whom a laparoscopic approach was deemed appropriate . selection criteria for tlrh and rrh were the same and included all women with newly diagnosed invasive cervical cancer stages ia1 to iia who desired a laparoscopic approach , after providing informed consent . patients were not considered for the laparoscopic approach if they had metastatic disease beyond the uterus , a cervical lesion of more than 4 cm in size , a uterus size of more than 12 cm , inadequate bone marrow , or compromised renal and hepatic function , or if they were pregnant . clinical data for both the laparoscopic groups and the robotic groups were analyzed by a review of patients ' medical records and operation reports . all patients had a computerized tomography ( ct ) scan of the abdomen and pelvis done preoperatively to evaluate lymph node status and potential extrapelvic and extraabdominal disease . intraoperative and postoperative data including patient characteristics , operation details , histological data , and follow - up information were recorded . duration of surgery was defined from the time of skin incision to the closure of the skin incision . robotic docking time was recorded as the time to attach the robotic arms to the trocars and insertion of robotic instruments . perioperative workup and postoperative care were provided by a gynecologic oncology service run by a fellow . the motions of the surgeon at the console unit are replicated by the robotic arms placed within the patient . during robotic surgery , an assistant is available at the operating table . the assistant performs robot - related tasks , including alignment and exchange of robotic instruments , operative maneuvers with conventional instruments such as organ manipulation , tissue countertraction , suction and irrigation , and any necessary alterations in the position of the intrauterine manipulator . the presence of the scrubbed assistant is also crucial in the event that an emergency conversion to a laparotomy is required . the technique of total laparoscopic hysterectomy and bilateral pelvic lymphadenectomy using harmonic shears has been previously described and served as the basis for the robotic procedure . the procedure was performed with the patient under general endotracheal anesthesia in the dorsal lithotomy position with adjustable allen stirrups and lower extremity compression devices for deep venous thrombosis prophylaxis . betadine solution was applied topically , and sterile drapes were placed in the usual sterile fashion . the procedure was terminated if metastatic disease was detected , and confirmed by frozen section evaluation , outside of the pelvis ( eg , in the omentum , bladder , liver , or bowel ) , in the uterine adnexa , or if the tumor extruded through the uterine wall into the peritoneal cavity . if none of the above were found , the laparoscopic equipment was removed . a standard 12-mm trocar placed at the umbilicus was used for camera placement , 2 working robotic arms were attached to 8-mm reusable trocars placed bilaterally , and ancillary 10-mm trocars were placed in the suprapubic region and the left or right upper quadrant . the robotic ports were placed 1 cm to 2 cm below and 8 cm to 10 cm lateral to the intraumbilical trocar , so as to enable optimal movement of the robotic arm and to minimize the risk of collision ( figure 1 ) . a 12-mm camera trocar is placed at the umbilicus , 2 working robotic arms are attached to 8-mm reusable trocars placed bilaterally , and additional ancillary 5-mm to 10-mm trocars are placed in the suprapubic region and the left upper quadrant . the whole procedure is performed using the robotic monopolar electrosurgical scissors placed through the right port , and the fenestrated bipolar forceps placed through the left robotic port . dorsey smokevac suction irrigator pump , probe ( bard inc , murray hill , nj ) , grasping forceps and bipolar forceps as needed , as well as harmonic shears ( ethicon endo - surgery inc , blue ash , oh ) or a ligasure vessel sealing device ( valleylab , boulder , co ) . adhesions were lysed first to restore normal anatomy , and the undersurfaces of the diaphragm , liver , gallbladder , stomach , omentum , and large and small bowel were examined visually , when possible . the paraaortic lymph nodes were inspected , followed by the pelvic lymph nodes . proceeding with a radical hysterectomy requires that 6 avascular pelvic spaces be developed and that the bladder and rectum be mobilized . the uterus is sharply anteverted by using an intrauterine manipulator , and the peritoneum between the uterosacral ligaments is incised by using monopolar scissors ; the rectum can then be brought down gently away from the vagina . the posterior vaginal fornix is placed on tension ( marked by the arrow ) , and a moistened sponge on sponge - forceps is placed in the vagina to facilitate delineation of the tissue planes . after round ligaments on either side of the uterus were desiccated and cut with the monopolar scissors , the anterior leaf of the broad ligament was opened bilaterally . the bladder was gradually dissected away from the cervix and vagina with a moistened sponge on a sponge - forceps placed in the anterior vaginal fornix to facilitate development of the vesicovaginal space ( figure 3 ) . after development of the paravesical and pararectal spaces , the pelvic lymphadenectomy can be performed . pelvic lymphadenectomy involves removal of the lymph node packets from the common iliac vessels and external iliac vessels down to the level of the deep circumflex iliac veins ( figure 4 ) . at this point , the medial umbilical ligament was suspended with upward tension , and the origin of the uterine artery from the hypogastric artery was identified ( figure 5 ) . the uterine vessels were placed on medial tension , and the ureter was unroofed using the curved tip of the monopolar scissor out of the tunnel ( figure 7 ) , and then the surrounding tissues were coagulated and divided ( figure 8) . the uterosacral ligaments , cardinal ligaments , and a portion of the paracolpos were then divided with the bipolar forceps and scissors , enabling complete mobilization of the uterus . in some cases , the specimen removal was done vaginally to allow for a superior visualization and delineation of the vaginal margins . the left obturator nerve ( c ) , the left obliterated umbilical artery ( d ) , and the left ureter ( e ) are identified . the right pararectal ( b ) and paravesical ( c ) spaces are fully developed ; and the right ureter ( d ) , right umbilical ( e ) , and right external iliac artery ( f ) are visible . the right ureter ( b ) , right obliterated umbilical ( c ) , and right external iliac arteries ( d ) are seen . the bladder was either distended with saline , or cystoscopy was performed to further ensure its integrity . upon completion of the procedure , the da vinci system was undocked , all of the instruments were removed , and the trocar sites were closed using a figure - of - eight 0 vicryl suture and 40 vicryl in a subcuticular fashion . the outcomes from the laparoscopic radical and robotic - assisted groups were compared using fisher 's exact test and the chi - squared test for categorical variables and 2 sample student t tests for continuous variables . a total of forty seven patients met our inclusion criteria and had either tlrh or rrh with pelvic lymphadenectomy performed . three of these 4 patients had only a portion of the entire procedure ( pelvic lymphadenectomy , bilateral ureterolysis or part of the parametrial dissection ) performed robotically with the majority of the procedure performed laparoscopically . the remaining 13 cases comprised our rrh with bilateral pelvic lymphadenectomy cohort performed between april 2006 and january 2008 . there were no differences in clinical tumor characteristics , such as stage , histology , and lympho - vascular space involvement between the two groups ( table 1 ) . two patients in the rrh group underwent neoadjuvant chemotherapy , while only one patient in the tlrh group did so . patient characteristics as shown in table 2 , mean operative time , estimated blood loss , and length of postoperative stay were similar between the 2 patient groups ( p > 0.05 ) . the mean operative time for tlrh with pelvic lymphadenectomy leveled off at 318 minutes and did not significantly decrease over time . mean operative time for rrh changed little over the study period as well ( figure 12 ) . in contrast , the mean time for robotic docking was 12 minutes ( range 430 min ) , and decreased as the surgeon gained experience ( figure 13 ) . clinical variables and outcomes three patients in the rrh group and 6 in the tlrh group underwent paraaortic lymphadenectomy before the hysterectomy due to paraaortic lymph node enlargement detected on preoperative imaging , or cervical lesions with a diameter greater than 2 cm . seven patients in the tlrh group and one patient in the rrh group had ovarysparing procedures . in addition , 2 patients in the tlrh group underwent umbilical hernia repair and one patient had a bartholin 's abscess excised . the robotic group had 2 intraoperative incidental cystotomies , which occurred at the time of vaginal transection before specimen removal . one of these patients had extensive endometriosis at the anterior vaginal margin , and the other had vaginal tumor extension with significantly fore - shortened anterior vaginal fornix . in the tlrh group , 2 patients underwent inadvertent cystotomies at the time of laparoscopic bladder dissection of the anterior vagina . outpatient cystoscopy , cystogram , and stent removal was performed on both patients 10 days later with no long - term sequela . postoperative complications in the rrh group included one case of postoperative ileus , prolonged urinary retention , vaginal lymph drainage , and c. difficile colitis . the tlrh group had complications including two cases of deep vein thrombosis pulmonary embolism and c. difficile colitis and cases of ileus , and prolonged urinary retention ( table 3 ) . the mean yield of the pelvic lymph nodes was 24.7 in the rrh group and 31 in the tlrh group ( table 2 ) . there were no recurrences in either group with a mean follow - up time of 12 months in the rrh group and 29 months in the tlrh group . we did not note a significant difference between rrh and tlrh with respect to operative time , operative blood loss , length of hospital stay , or complication profile . the only intraoperative complication observed in both groups was cystotomy . along with ureteral injury , these are the most common reported intraoperative complications associated with laparoscopic radical hysterectomy . bladder injuries in the tlrh group were neither related to radical parametrial resection nor to lymph node dissection . similarly , in the rrh group , cystotomies were not related to the use of the robot system and took place at the time of the vaginal specimen removal . none of the patients had a recurrence with a mean follow - up of 12 months in the robotic group and 29 months in the laparoscopic group . however , questions remain , including whether the robot provides any additional benefits to a surgeon who is an experienced laparoscopist and comfortable performing the most advanced gynecologic procedures using traditional laparoscopy , whether there is an advantage for an inexperienced laparoscopic surgeon to use robotic technology compared with traditional laparoscopic instrumentation , and what the learning curve is with either approach . several gynecologic surgeons have reported their experiences performing tubal reanastomosis , salpingo - oophorectomy , and hysterectomy using an earlier robotic system . the largest experience using robotic systems for the surgical treatment of gynecologic cancers was reported by j. magrina of the mayo clinic ( scottsdale , az ) . it comprised 142 patients treated surgically with the da vinci robotic system for various primary and recurrent gynecologic malignancies . the mean operating time was 218 minutes , estimated blood loss was 176 ml , and the hospitalization time was 1.9 days . the authors concluded that robotic surgery is preferable to conventional laparoscopy for gynecologic oncology procedures because it provides improved dexterity , 3-dimensional viewing , surgical precision with tremor filtration , a comfortable fatigue - reducing console , and greater motion freedom allowed by the robotic instruments , which significantly reduced ergonomic problems associated with conventional laparoscopic equipment . lymph node yield was significantly higher in the robotic group ( 33 vs. 22 ) , and operative time was similar between the groups . blood loss as well as transfusion requirements in the robotic group were significantly less than that in the abdominal group . the gynecologic oncology community has been appropriately cautious in accepting laparoscopic procedures , including tlrh , as a standard of care due to a lack of oncological outcome data . data on specimen size , margin adequacy , and parametria appear to be equivalent . with the follow - up data in some of the studies approaching or exceeding 5 years , does the recurrence rate in the laparoscopically managed patients exceed that of the patients who underwent an open procedure . as was the case with laparoscopy 10 years ago abdominal radical hysterectomy continues to be the most common surgical approach in treatment of an early stage carcinoma of the cervix . the role of laparoscopy in this setting is to offer all of the benefits of a minimally invasive approach , namely faster recovery , decreased blood loss and transfusion rates , and decreased postoperative pain , while maintaining the excellent oncological outcomes of an open approach . while all gynecologic surgeons taking care of patients with early cervical cancer are trained to perform abdominal radical hysterectomy , only a few are comfortable performing the procedure laparoscopically . tlrh is one of the most challenging laparoscopic procedures in gynecologic oncology , requiring significant technical expertise and experience . as more centers perform these procedures , report their experiences , and the technique itself is developed , standardized , and taught systematically , we will better understand the learning curve required for both tlrh and rrh . the available urological data suggest that the intuitive nature of the robotic approach may provide a significant advantage in terms of its learning curve especially to surgeons with little or no advanced laparoscopic experience . robotic - assisted prostatectomy outcomes were comparable to those achieved by a skilled laparoscopic surgeon after 100 cases of laparoscopic radical prostatectomies . as the number of early cervical cancer cases is decreasing , fast acquisition of advanced endoscopic skills is paramount . therefore , the robotic interface , which allows for significant shortening of the learning curve , may make a minimally invasive approach possible even in centers with very few cases of early cervical cancer . the robotic systems have their own drawbacks ; most commonly mentioned are the absence of tensile feedback , the complexity of the system , the size of the system , and the cost . currently , operations performed with a robot are expensive , but the widespread use of this technology , combined with the shorter hospital stay , hopefully , will lead to an overall , and substantial , decrease in cost . though robotic technology has revolutionized urologists ' treatment of prostate cancer , its use in the treatment of cervical cancer by gynecologic oncologists is still in development . we have found that the substantial magnification , dexterity , and flexibility offered by the robotic system significantly simplify the most difficult stages of radical hysterectomy and pelvic lymphadenectomy , which would enable a greater number of surgeons to perform this procedure laparoscopically .

while it is remarkable to consider the sheer wealth and architectural diversity of natural products that can be produced from a relatively small set of starting materials , equally striking is the number of structures within that collection that can be envisioned to arise via the union of a secondary metabolite with itself . indeed , by some estimates , between 15 and 20% of all natural products likely include a dimerization process at some point in their biogenesis . this analysis includes materials with obvious symmetry , such as sceptrin ( 1 , scheme 1 ) , compounds with equivalent halves but non - symmetric unions , such as complanadine a ( 2 ) , and materials whose symmetry has been partially erased through subsequent structural modifications like oxidation or decarboxylation , such as cp-225,917 ( 3 ) . given the significant energy invested in the creation of any natural product , the ubiquity of such dimers is logical , since dimerization enables rapid access to additional molecular scaffolds without invoking entirely new biosynthetic pathways . indeed , with their distinct three - dimensional shapes and functional group presentations , these new materials may well afford evolutionary advantages to the producing species . dimer linkages are colored in purple . considering only those dimeric natural products that possess obvious monomer symmetry ( i.e. , those that have not undergone extensive modifications , such as 3 ) , nature appears to deploy two general strategies to access such materials . the first and most typical approach forges the dimer in a final synthetic operation from fully functionalized monomers . such processes can range from the simple and direct construction of a single connecting bond to far more complex bond - forming unions , such as that postulated for the conversion of sorbicillin ( 4 ) into trichodimerol ( 5 ) through a series of michael reactions and ketalizations . in the second dimerization approach , monomer union occurs at an earlier stage , with subsequent tandem modifications of each half leading to the final structure ( as in 6 7 8 and 10 11 12 ) . during the past several decades , synthetic chemists have become particularly skilled at utilizing both of these bio - inspired strategies to access dimeric materials from monomers . the first strategy is the most appealing from a retrosynthetic standpoint , especially if it directly replicates nature s synthesis . in practice , though , it often requires extensive screening of conditions to achieve success and sometimes affords only modest yields of final product . the second approach has provided the opportunity for further creativity , as dimers distinct from those deployed by nature can also be elaborated in tandem sequences to the final target . this concept was , to the best of our knowledge , first demonstrated by stork in the synthesis of -onocerin ( 8) in four steps from 9 , and used more recently to great effect by a number of groups , including boger in his approach to 12 . it also arguably constitutes the only general solution for dimer synthesis when direct , final - step dimerization can not be achieved , whether due to challenges in target patterning , monomer reactivity , and/or the absence of suitable enzymes to achieve the needed bond constructions . yet , despite their respective advantages , these two approaches also share a potential limitation : the key step linking the monomeric materials typically possesses a single reactive course . thus , if the needed bond(s ) and/or stereocenter(s ) are improperly established in this operation , it is exceedingly difficult to overcome such results . such issues arose during our efforts to synthesize the coccinellid family of alkaloids , materials secreted by numerous species of ladybugs as defensive compounds when provoked and viewed by some as potential commercial insecticides , particularly for the control of aphid pest populations . figure 1 provides the structures of eight of the nine monomers within this class ( 1421 ) , tricyclic architectures differing in ring junction stereochemistry , oxidation state , and olefin placement . structures of the coccinellid class of alkaloids : unique monomeric and dimeric frameworks . in addition to these materials , several larger and more complex compounds are known wherein half of their framework possesses these monomer cores , such as chilocorine a ( 22 ) , as well as two other species that structurally reflect the dimeric combination of these cores in the form of psylloborine a ( 23 ) and isopsylloborine a ( 24 ) . the sole distinction between these latter two natural products is ring - junction enamine isomerism within their fused , highly congested , and stereochemically rich frameworks . to date , the monomeric members have elicited significant synthetic interest , with several approaches based on both classical and modern bond constructions affording every tricycle drawn in figure 1 . intriguingly , however , it is only within the past year that the first asymmetric synthesis of any of these members has been accomplished . equally surprising , no work toward any higher - order structure has been reported , nor has any mechanistic hypothesis been advanced to account for dimer formation in nature . to date , that work has identified a single common synthetic intermediate capable of rapidly affording every monomer drawn in figure 1 . it has also led to a biosynthetic proposal for the formation of both psylloborine a ( 23 ) and isopsylloborine a ( 24 ) , one that , when reduced to practice , resulted in a non - natural , regioisomeric dimer . this unexpected result , coupled with observations from other instances where incorrect dimeric unions have occurred in biomimetic constructions , has led to the development of a unique , non - biomimetic strategy for complex dimer synthesis . as will be described in the ensuing sections , this alternate strategy has afforded rapid syntheses of both psylloborine a ( 23 ) and isopsylloborine a ( 24 ) through sequences involving some of the most complex condensation / michael / mannich cascade chemistry yet reported . significantly , this approach can likely be applied to other challenging dimerizations and may , in certain cases , be as efficient and powerful as an overall synthetic design . given the absence of any proposal for how either psylloborine a ( 23 ) or isopsylloborine a ( 24 ) might arise in nature , we began by pondering mechanistic pathways that could account for their formation from the known tricyclic coccinellid alkaloids . the idea that ultimately proved the most attractive is shown in scheme 2 and was inspired by a key structural observation among the monomers depicted in figure 1 . namely , although three of those monomers ( 1416 ) have a stable n - oxide counterpart ( 1921 , drawn below its respective precursor ) , propyleine and isopropyleine ( 17 and 18 , a 1:3 equilibrating mixture in solution , respectively ) do not . thus , perhaps the n - oxides of 17 and/or 18 are unstable and , if generated ( 25 ) , convert to a reactive electrophilic species such as 26 . if this material was formed in the presence of a molecule of propyleine ( 17 ) , then perhaps it could undergo the sequence of events shown in scheme 2 , involving a vinylogous mannich reaction , proton transfer , mannich reaction , and terminating proton loss to generate dimers 23 and/or 24 . in total , this proposed direct , final - stage dimerization sequence would form two new c the main assumption of this analysis , at least in terms of a successful laboratory execution , is that pre - existing chirality within the monomers could dictate the facial presentation of the reacting partners ( i.e. , enzymatic intervention would not be required ) . equally critical , but unclear , were ( 1 ) whether one or both dimeric products would result from such a pathway , and ( 2 ) whether only propyleine ( 17 ) would be the active nucleophile , or if its equilibrating and more dominant enamine isomer , isopropyleine ( 18 ) , could participate in addition to , or instead of , 17 . despite these concerns , the overall attractiveness of such a dimerization process prompted us to test its viability . thus , we set out to develop synthetic pathways to access 17/18 as well as 26 . scheme 3 provides a retrosynthetic approach that we hoped could ultimately address the synthetic challenges posed by the nucleophilic and electrophilic partners needed to test our proposed dimerization sequence . as indicated , our goal was to prepare bicyclic intermediate 30 through an intramolecular condensation between the deprotected amine variant of 31 and its neighboring carbonyl . then , if its thermodynamically favored trisubstituted enamine could be isomerized to its less stable , exocyclic counterpart and intramolecularly displace a suitably disposed leaving group , propyleine ( 17 ) would result alongside its equilibrating enamine isomer , isopropyleine ( 18 ) . although isomerization to the exocyclic enamine isomer is clearly disfavored , we believed the final ring formation would be an energetically downhill and essentially irreversible process that would enable the reaction to be driven to completion . worth noting is that this sequence , while not fully biomimetic , is certainly bio - inspired as a disubstituted piperidine undergoing enamine condensation and subsequent attack on an intramolecular electrophile has been proposed to account for the biosynthetic formation of several monomeric alkaloids in this class . indeed , as shown in the lower half of scheme 3 , braekman and co - workers demonstrated that polyketo - myristic acid ( formed from stearic acid through enzymatic oxidations ) is the biosynthetic precursor to coccinelline ( 19 ) via the proposed intermediacy of 33 , a compound containing a 10-membered ring ; the intervening steps listed above each arrow are one proposal for how these net transformations might specifically occur . our proposed synthesis of 17/18 is based on a similar , but differently ordered , set of chemical events involving no individual ring size greater than six , in which a disubstituted piperidine ( 34 ) becomes the tricyclic system through an enamine condensation and a terminating c c bond formation driven by nucleophilic attack of the enamine moiety . assuming success in our proposed synthetic operations leading to 17/18 , attempts to generate electrophile 26 in situ through n - oxide formation would then begin . as an alternative approach to access 26 , we also considered a de novo preparation from 32 , again proposing enamine equilibration and subsequent c c bond formation to close the final ring of its tricyclic framework . given that this proposed key starting material ( 31 ) possesses three reactive domains ( highlighted in blue , green , and orange ) with sufficiently distinct and tunable electrophilic and nucleophilic properties , we also questioned whether every other monomer configuration ( i.e. , 1416 and 1921 ) within the class could be accessed from the same entry point . if so , then a near - universal , family - level solution for the coccinellid alkaloids would exist , with enantioselective syntheses of all monomeric members possessing optical activity being achieved , some for the first time . based on this plan , our first objective was to devise an efficient synthesis of our key , common building block ( 31 ) . as shown in scheme 4 , we ultimately developed two different routes to accomplish that goal , both starting from the commercially available n - boc-(s)-()-piperidine-2-ethanol ( 36 ) . this material was chosen because it possessed a key chiral center that we anticipated would readily encode the remaining stereochemical information into the final target . our first route began with silylation of the alcohol and was followed by a copper - mediated allylation with branched bromide 37 , itself available in two steps from propargyl alcohol ( see supporting information for synthesis ) . these operations led to trans - disposed intermediate 38 in 84% overall yield and with > 19:1 diastereoselectivity based on h nmr analysis . with all of the carbons of the final target already in place , only functional group manipulations and redox adjustments remained . these events began by conversion of 38 into enone 39 through a modestly yielding and sometimes capricious wacker oxidation ( 41% yield ) promoted by pdcl2(phcn)2 in dmf / h2o at 60 c , followed by a nearly quantitative base - induced ( naoh ) olefin isomerization ; these transformations afforded the new alkene with 4:1 selectivity which we assume to have the structure shown , though we never explicitly confirmed the process as being e - dominant . next , we hoped to set the methyl stereocenter of the target through a diastereoselective reduction . this step ultimately proved to be the most challenging of the sequence , with repeated attempts at substrate - directed hydrogenation using wilkinson s or crabtree s catalysts affording either no diastereoselection or preference for the undesired methyl epimer on 39 or its deconjugated enone precursor . fortunately , use of yamamoto s bulky lewis acid , aluminum tris(2,6-diphenylphenoxide ) ( atph ) , in tandem with l - selectride afforded the desired stereocenter with a 15:1 diastereomeric ratio ( dr ) and in 75% yield . a final desilylation using tbaf in thf at 25 c then delivered the desired building block ( 31 ) in a sequence that was just seven steps overall ( including the two - step preparation of 37 ) . reagents and conditions : ( a ) tbdpscl ( 1.05 equiv ) , imidazole ( 2.0 equiv ) , ch2cl2 , 25 c , 19 h ; ( b ) s - buli ( 1.5 equiv ) , tmeda ( 1.6 equiv ) , et2o , 78 to 45 c , 1 h ; cucn2licl ( 1.5 equiv ) , 78 c , 1 h ; 37 ( 3.0 equiv ) , 78 to 25 c , 4 h , 84% over two steps ; ( c ) pdcl2(phcn)2 ( 0.1 equiv ) , cucl ( 1.0 equiv ) , dmf / h2o ( 10/1 ) , o2 , 60 c , 6 h , 41% ; ( d ) naoh ( 2.0 equiv ) , i - proh , 25 c , 1 h , 100% ; ( e ) atph ( 1.5 equiv ) , ch2cl2 , 0 c , 20 min ; l - selectride ( 2.0 equiv ) , 78 c , 1 h , 75% , 15:1 dr ; ( f ) tbaf ( 2.0 equiv ) , thf , 25 c , 2 h , 91% ; ( g ) tbscl ( 1.1 equiv ) , imidazole ( 2.0 equiv ) , ch2cl2 , 25 c , 19 h ; ( h ) same as step b but with 40 ( 5.0 equiv ) instead of 37 , 92% ; ( i ) k2oso42h2o ( 0.005 equiv ) , naio4 ( 4.0 equiv ) , 2,6-lutidine ( 2.0 equiv ) , 1,4-dioxane / h2o ( 3/1 ) , 25 c , 24 h ; ( j ) 42 ( 2.2 equiv ) , thf , 78 c , 2.5 h ; ( k ) k2oso42h2o ( 0.005 equiv ) , naio4 ( 4.0 equiv ) , 2,6-lutidine ( 2.0 equiv ) , 1,4-dioxane / h2o ( 3/1 ) , 25 c , 16 h , 95% ; ( l ) tfaa ( 5.0 equiv ) , pyridine ( 15 equiv ) , 4-dmap ( 0.1 equiv ) , ch2cl2 , 0 c , 1.5 h ; ( m ) dbu ( 1.5 equiv ) , ch2cl2 , 0 to 25 c , 1.5 h , 80% ( n ) atph ( 1.5 equiv ) , ch2cl2 , 0 c , 30 min ; l - selectride ( 2.0 equiv ) , 78 c , 1 h ; 1 m hcl ( 10 equiv ) , meoh , 25 c , 1 h , 79% . concurrent with these efforts , especially in light of the low - yielding wacker oxidation step , we developed an additional sequence to 31 . though slightly longer at eight steps , it proved to be operationally simpler and higher yielding overall . it began similarly from 36 , with a two - step sequence of silyl protection of the alcohol ( this time as a tbs ether ) and a copper - mediated addition using allylic bromide 40 that proceeded in 92% overall yield . subsequent oxidative cleavage of the new alkene , mediated by k2oso4 and naio4 , afforded methyl ketone 41 . the remaining carbon atoms of the target were then installed via nucleophilic addition of grignard reagent 42 , with a second oxidative cleavage then generating the ketone within 43 in 95% yield over three steps . formation of the desired enone was accomplished via trifluoroacetate formation and dbu - induced elimination in 80% yield over two steps . finally , this transformation was followed by the same facially selective hydride delivery , with a terminating in situ deprotection upon quenching of the -reduction product with hcl , completing the synthesis of 31 in 79% yield and in 8:1 dr . worth noting is that both routes contributed substantively to the delivery of material for subsequent studies , with each being performed on relatively large scales . indeed , the first route was able to deliver 2.68 g of 31 in a single campaign , while the second afforded 3.02 g of this key intermediate . with compound 31 in hand , our efforts to synthesize the monomeric members of the coccinellid class began in earnest . starting with propyleine and isopropyleine ( 17 and 18 , scheme 5 ) , we first converted the alcohol within 31 to a reactive bromide ( 44 ) through the intermediacy of its n - deprotected tfa salt ( tfa ; pbr3 , one - pot operation ) . we then dissolved this material ( 44 ) in i - proh and treated it with et3n in hopes that its enamine could be isomerized to its less stable , exocyclic counterpart ( 45 ) as noted earlier , thereby inducing a terminating cyclization . pleasingly , this conjecture proved true , affording ( )-propyleine ( 17 ) and ( )-isopropyleine ( 18 ) as an equilibrating 1:3 mixture in 43% yield . this nine - step sequence ( using the step count of the shorter route to 31 ) is the first asymmetric solution for these targets and the shortest route to date . in addition , the levorotatory rotation of the final synthetic materials matched that of the natural isolates , confirming the absolute configuration of these compounds for the first time as based on the initial assignment of 36 ( cf . reagents and conditions : ( a ) tfa / ch2cl2 ( 1:1 ) , 0 c , 1 h ; solvent removal , pbr3 ( 5.0 equiv ) , et2o , 70 c , 5 h ; ( b ) et3n ( 1.0 equiv ) , i - proh ( cat . ) , ch2cl2 , 25 c , 13 h , 43% yield over two steps ; ( c ) nabh(oac)3 ( 5.0 equiv ) , ch2cl2 , 0 c , 3 h , 80% , 3.7:1 dr ; ( d ) bzonhmehcl ( 1.0 equiv ) , dmso , 25 c , 2 d , 62% ; ( e ) tfa / ch2cl2 ( 1:1 ) , 0 c , 1 h ; concentrate , pbr3 ( 5.0 equiv ) , et2o , 70 c , 5 h ; ( f ) et3n ( 0.77 equiv ) , i - proh ( 0.91 equiv ) , ch2cl2 , 40 c , 4 h ; concentrate , aq naoh ( 10 equiv ) , meoh , 65 c , 6 h , 46% over two steps , 1:1.2 dr , recyclable . from here , subsequent reduction of a portion of these natural products with nabh(oac)3 led to a 3.7:1 mixture of precoccinelline ( 14 ) and hippodamine ( 15 , scheme 5 ) . due to the trans - ring fusions of these materials , it was difficult to predict the outcome of this reaction a priori . however , given the observed facial bias , we presume that if the top ring ( as drawn in the two - dimensional depiction of 46 ) were to adopt a twist - boat orientation , as indicated by the three - dimensional representation of imine 46 in scheme 5 , then hydride delivery from the bottom face would appear to be preferred , forming precoccinelline ( 14 ) . if true , then hippodamine ( 15 ) would have resulted from hydride delivery onto the other side of the structure . presumably , delivery from the top face , forming hippodamine , forces the methyl group into an unfavorable 1,3-diaxial relationship with the incoming hydride in the transition state , whereas delivery from the opposite face produces no such interaction . from the standpoint of synthesis , however , as precoccinelline ( 14 ) has previously been readily converted into its n - oxide congener coccinelline ( 19 , cf . figure 1 ) , its preparation allowed us to claim a formal synthesis of this oxidized monomer as well . alternatively , -oxidation of common intermediate 31 with bzonhmehcl , followed by the same general steps already described ( tfa ; pbr3 in one pot then et3n , i - proh ; naoh ) generated oxidized skeleton 48 by way of 47 as a mixture of recyclable diastereomers about the new , highlighted chiral center . this compound ( 48 ) has previously been advanced by mueller to hippodamine and hippocasine ( 15 and 16 , respectively ) as well as their n - oxides ( 20 and 21 , cf . figure 1 ) , thus completing total and/or formal syntheses of all eight monomers drawn in scheme 1 , all starting from a single starting material ( i.e. , 31 ) . with syntheses of our targeted monomers complete , our attention now turned to dimerization . although our proposed nucleophilic partner was already available from the synthesis of propyleine and isopropyleine ( 17 and 18 ) , efforts to generate a reactive electrophile directly from these materials through n - oxidation were unsuccessful . as such , we sought an alternate and potentially more controlled path to the needed electrophilic dimerization precursor in the form of cross - conjugated diene 49 ( scheme 6 ) . our hope was that , upon exposure to an appropriate proton source , iminium electrophile 26 ( schemes 2 and 7 ) could be generated in situ , and then we could expose that species to the appropriate nucleophilic partners . our initial route to access this compound sought to dehydrate 51 , itself readily prepared from 31 in just two steps via oxidation and a one - pot tfa - promoted deprotection / condensation / mannich reaction sequence . unfortunately , no conditions were found that could reliably afford 49 from this intermediate . as such , an alternate , slightly longer four - step pathway was developed as shown in the lower half of scheme 6 . the key operation was the final step involving dibal - h - mediated reduction of vinylogous amide 53 . this step proceeded in 45% conversion ( based on crude h nmr of samples accounting for full mass recovery ) to afford 49 along with the -reduced counterpart of 53 . as this critical compound proved unstable and difficult to purify , it was carried forward directly into dimerization studies once formed . reagents and conditions : ( a ) ( cocl)2 ( 1.6 equiv ) , dmso ( 3.2 equiv ) , et3n ( 6.0 equiv ) , ch2cl2 , 78 to 0 c , 90 min , 94% ; ( b ) tfa / ch2cl2 ( 1/1 ) , 0 c , 1 h , carried forward crude ; ( c ) ruo2xh2o ( 0.10 equiv ) , naio4 ( 4.0 equiv ) , acetone / h2o ( 1:1 ) , 0 c , 90 min , 77% ; ( d ) p - nitrophenol ( 1.2 equiv ) , dcc ( 1.2 equiv ) , 4-dmap ( 0.10 equiv ) , 25 c , 20 h ; filter , solvent removal , tfa / ch2cl2 ( 1:1 ) , 0 c , 1 h ; ( e ) i - proh ( cat . ) , ch2cl2 , 40 c , 18 h , 31% over two steps ; ( f ) dibal - h ( 2.5 equiv ) , thf/1,4-dioxane ( 4:1 ) , 25 c , 4 min . following extensive experimentation with acid source and solvent , we found that electrophile 26 ( scheme 7 ) could be obtained when 49 was taken up in cd2cl2 ( to enable close monitoring by nmr analysis ) and exposed to tfa at 25 c . when propyleine and isopropyleine ( 17 and 18 ) were then added as a cd2cl2 solution to this electrophile 2 min later , a new dimeric material was formed over the course of 2 h in 21% overall yield from 53 . unfortunately , this material did not match the spectral data for either 23 or 24 . extensive nmr analysis ( h , c , cosy , tocsy , noesy , hsqc , and hmbc ; see supporting information for full details ) ultimately revealed that this dimer was non - natural , resulting from incorrect regiocontrol in the union of the two building blocks , as noted by the highlighted carbons within their frameworks . this result meant that isopropyleine ( 18 ) , not propyleine ( 17 ) , served as the nucleophilic partner . we have elected to give this new dimeric material , compound 54 , the name psylloborine b , should it ever prove to be a natural isolate . reagents and conditions : ( a ) tfa ( 1.0 equiv based on vinylogous amide 53 ) , 25 c , 2 min ; 17 and 18 ( 1:3 , 1.07 equiv combined based on vinylogous amide 53 ) , 25 c , 2 h , 21% over two steps . as indicated in figure 2 , extensive molecular modeling employing hand - held model kits has provided a rationale as to why this outcome may have occurred . critically , we believe it is the result of kinetic control based on the stereoelectronic and conformational demands of the lone desymmetrizing methyl group within nucleophiles 17 and 18 , not the ratio of these two components in solution . in theory , there are a total of four possible reactive pathways : either propyleine or isopropyleine serving as a nucleophile ( with the nucleophilic carbons colored to match that of scheme 7 ) , with approach of the electrophile occurring from either the top or bottom face . these possibilities are drawn in figure 2 as transition states a d , all viewed from the perspective of looking down the c n bond of the enamine , with the nitrogen atom located behind the circle of the newman projection . possible basis for the observed dimerization result based on transition - state models for electrophile addition ( i.e. , 26 ) using either the propyleine ( 17 ) or isopropyleine ( 18 ) enamines as nucleophile . based on the analysis provided earlier in the context of scheme 2 , the requisite pathway to psylloborine a ( 23 ) or isopsylloborine a ( 24 ) would require nucleophilic attack by propyleine ( transition states a and/or b ) . in transition state a , a favorable pseudochair - like transition state can be achieved , but it incurs a significant steric penalty by requiring the incoming electrophile to approach syn to the pendant methyl group . in transition state b , addition from the bottom face is now anti to the pendant methyl group , but it seems to require a pseudoboat - like orientation to proceed , with a number of destabilizing flagpole and eclipsing interactions resulting as indicated . by contrast , when isopropyleine ( 18 ) is the enamine nucleophile ( transition states c and d ) , there are no proximal substituents that can destabilize the approach of the electrophile as in pathway a or b. therefore , the lowest energy pathway is likely to proceed through these transition states , and thus 54 results . given this experimental outcome , a new synthetic approach was needed for psylloborine a ( 23 ) and/or isopsylloborine a ( 24 ) . ultimately , it was careful consideration of this , as well as other cases where direct dimerization had also failed , which led to the revised retrosynthesis as drawn in scheme 8 . this analysis is based on a conceptually different approach for dimer synthesis from those posited within the confines of scheme 1 and centered on the long - established principle that intramolecular linking can potentially overcome those factors governing intermolecular reactivity . specifically , rather than combine advanced materials in a final step or merge simpler materials earlier and then elaborate in tandem , instead ( 1 ) link two simpler precursors at an appropriate site to ensure proper regiocontrol and ( 2 ) embed enough chiral information and reactivity within the overall structure to establish the remaining rings and stereocenters as well as forge any remaining bonds between the two halves . intramolecular dimerization , and compound 59 was designed for the coccinellid alkaloids on the basis of its general concepts , outfitted with one of the requisite dimer linkages ( highlighted in purple in scheme 8) that could not be forged from direct , advanced monomer coupling . from here , two cascades were envisioned to forge the remaining rings , stereocenters , and final dimeric linkage needed to complete the targets . initiation of the first cascade required the ability to differentiate selectively between the protecting groups on the two piperidine rings in 59 to reach 58 via a condensation and michael closure . the second cascade would utilize an added electron - withdrawing group ( ewg , colored in blue ) on one of the pendant methyl groups of the final target to dictate the correct order of bond constructions through condensation , enamine - based michael attack to form the tricyclic ring system , and a terminating mannich ring - closure to complete the carbon framework . collectively , these two cascades would forge five new bonds , five new rings , and four stereogenic centers , assuming again that pre - existing chirality within 59 could govern the incorporation of the remaining chiral elements . if successful , then a terminating excision of the ewg in cascade product 57 would complete the synthesis of 23 and/or 24 . on initial inspection , this approach appears contrary to the general tenets of retrosynthetic analysis , since an arguably more complex precursor and set of terminating events are required than those needed for the two dimerization strategies presented in scheme 1 . however , given the failure of direct dimerization and the likely inapplicability of tandem elaboration to a non - symmetric dimer , we required a distinct strategy . one potential and logical benefit of this new approach is that the final stitching operations appear to take advantage of biosynthetic efficiency through the use of cascades that resemble nature s synthesis of the monomeric frameworks . indeed , apart from the linkage within 59 , the portions of this material colored in scheme 8 match very closely the analogous portions of structures 55 and 56 , moving only one bond colored in black and changing the positioning and identity of the functional groups colored in blue . moreover , we anticipated that this synthetic sequence would not be much longer than the failed direct dimerization strategy . indeed , key test substrate 59 was expected to readily arise from a horner wadsworth emmons coupling between phosphonate 60 , a material we anticipated could be readily synthesized , and aldehyde 50 , the oxidized version of our key common intermediate for monomer synthesis which was already available on gram scale ( cf . , the incorporation of this group would be attempted once most of 59 had been assembled to afford opportunities to probe different variants as needed to successfully induce the designed cascades . as shown in scheme 9 , the key elements of this new dimerization precursor were indeed synthesized quite readily , starting once again from piperidine 36 , the same material used earlier to commence our monomer syntheses . its core elements closely mirror the synthetic pathways described earlier in the context of scheme 4 , differing only in terms of the fragments coupled , and thus will not be discussed in detail ( scheme 9 ) . pleasingly , after phosphonate 60 was accessed in just six steps from 36 , the masamune roush variant of the horner wadsworth emmons reaction ( licl , i - pr2net in ch3cn at 25 c ) coupled it with aldehyde 50 to afford 65 , with the previously inaccessible intermolecular dimerization linkage now in place ( highlighted in purple in scheme 9 ) . reagents and conditions : ( a ) tbscl ( 1.1 equiv ) , imidazole ( 2.0 equiv ) , ch2cl2 , 25 c , 19 h ; ( b ) sec - buli ( 1.5 equiv ) , tmeda ( 1.6 equiv ) , et2o , 78 to 45 c , 1 h ; cucn2licl ( 1.5 equiv ) , 78 c , 1 h ; allyl bromide ( 5.0 equiv ) , 78 to 25 c , 2 h , 91% over two steps ; ( c ) k2oso42h2o ( 0.005 equiv ) , naio4 ( 4.0 equiv ) , 2,6-lutidine ( 2.0 equiv ) , 1,4-dioxane / h2o ( 3/1 ) , 25 c , 2.5 h ; ( d ) 62 ( 1.22 equiv ) , ch2cl2 , 25 c , 14 h , 84% over two steps ; ( e ) pd / c ( 10% , 0.06 equiv ) , h2 , meoh / etoac ( 3/1 ) , 78 to 25 c , 20 h , 97% ; ( f ) 64 ( 2.0 equiv ) , thf , 78 to 45 c , 1.5 h , 87% ; ( g ) licl ( 4.0 equiv ) , i - pr2net ( 2.0 equiv ) , 25 c , 30 min ; 50 ( 1.0 equiv ) , 25 c , 4 h ; hcl ( 6.0 equiv ) , meoh , 0 c , 25 min , 79% . from here , treatment of 65 ( scheme 10 ) with tfa at 78 c differentiated the two boc - protected piperidine ring systems by taking advantage of neighboring group participation , selectively transforming the upper ring boc group ( as drawn ) into a base - labile carbamate through cyclization onto the enone while leaving the lower ring boc group intact . although this operation afforded no stereocontrol at the highlighted center within 66 , that outcome was of no consequence , as this chiral center would be subsequently destroyed . the synthesis of the key precursor ( in protected form as 66 ) was then completed by oxidizing the alcohol and performing a horner wadsworth emmons coupling with an aryl sulfone - containing phosphonate [ either ph- , 3,5-(cf3)2ph- , or 4-no2ph- , vide infra ] . pleasingly , treatment of all three of these variants of 66 with 1,1,3,3-tetramethylguanidine ( tmg ) in a 9:1 mixture of toluene / i - proh effected the desired reaction sequence of carbamate cleavage , enone regeneration , condensation , enamine equilibration to the exocyclic isomer , and a terminating michael addition . however , despite the high control in bond construction events , the highlighted chiral center within 68 was generated in a 1:1.2 dr favoring the undesired , undrawn epimer . exploration of various conditions revealed that this outcome could not be improved , with several alternatives affording inferior stereoselection . while not optimal , it was certainly an improvement on the direct dimerization approach where that center could not be forged correctly to any degree . reagents and conditions : ( a ) 10% v / v tfa in ch2cl2 ( 10 equiv ) , ch2cl2 , 78 c , 2 h , 89% , 2:1 dr ; ( b ) oxalyl chloride ( 1.6 equiv ) , dmso ( 3.2 equiv ) , et3n ( 6.0 equiv ) , 78 c , 30 min ; 0 c , 1 h ; ( c ) phosphonate ( ar = 3,5-(cf3)2c6h3 , 1.0 equiv ) , licl ( 2.0 equiv ) , i - pr2net ( 2.0 equiv ) , ch3cn , 25 c , 30 min ; substrate ( 1.0 equiv ) , ch3cn , 25 c , 2 h , 67% over two steps ( all ensuing yields are for when ar = 3,5-(cf3)2c6h3 ) ; ( d ) tmg ( 1.0 equiv ) , toluene / i - proh ( 9:1 ) , 25 c , 5.5 h , 1:1.2 dr ; ( e ) tfa / ch2cl2 , 0 c , 1 h ; ( f ) c6d6 , 65 c , 3 h , 15% yield over three steps ( 79% yield per transformation ) ; ( g ) 5 wt % na / hg ( 276 equiv ) , i - proh , 25 c , 30 min , 46% ; ( h ) tfa ( 2.0 equiv ) , clch2ch2cl , 75 c , 30 min , 75% . pressing forward with both diastereomers ( as they could not be separated at this stage when the ewg was an aryl sulfone ) , treatment of 68 with tfa in ch2cl2 at 0 c for 1 h removed the remaining boc group to unveil the free amine needed to initiate the second cascade . subsequent dissolution in benzene - d6 ( to monitor the reaction ) and heating at 65 c then initiated that cascade sequence : condensation to 69 , michael closure to 70 , and a terminating mannich reaction to complete the synthesis of 72 . as long as the aryl sulfone was sufficiently electron - deficient [ i.e. , ar = 3,5-(cf3)2ph- or 4-no2ph- ] , these operations proceeded in the order designed however , when the unsubstituted phenyl sulfone was used , a large portion of the material appeared to undergo mannich reaction prior to michael addition to afford materials believed to have structure 71 . despite various attempts , these compounds thus , the more electron - withdrawing aryl sulfones seem to have enabled the sequence to succeed by ensuring that michael reaction preceded mannich closure . taken together , these two cascade events arguably constitute the most complex use of condensation / michael / mannich chemistry yet described , given that they involve a total of seven distinct chemical events that forged three c n bonds , five rings , and four stereocenters ; the overall yield obtained for the 3,5-(cf3)2ph variant , at 15% , reflects a throughput of 79% per chemical transformation . finally , exposure of the 3,5-(cf3)2ph derivative of 72 to na / hg amalgam transformed it into psylloborine a ( 23 ) , a material identical in all respects to the natural isolate , thereby completing the first total synthesis of this molecule as well as that of any dimeric coccinellid alkaloid . as a final experiment , heating this material with tfa in clch2ch2cl at 75 c afforded isopsylloborine a ( 24 ) , completing the first total synthesis of this dimer as well as establishing 23 as a viable biosynthetic precursor to 24 . in total , the route to 23 required 16 linear steps , only four steps more than the original direct dimerization approach that failed to deliver the target , thus highlighting the efficiency of this alternate dimerization strategy . finally , it is worth noting that while only the 3,5-(cf3)2phso2- group afforded complete success for the entire sequence , ewgs other than sulfones were also probed for the final cascades . as shown in scheme 11 , use of the same sequence with a simple methyl ketone ( i.e. , 73 , x = me ) proceeded in similar overall yield ( 79% per transformation for the steps shown ; see supporting information ) to generate the full heptacyclic core of the dimeric coccinellid alkaloids ( 74 ) . however , no approach could be discovered to convert the methyl ketone to the final pendant methyl of the target natural products . by contrast , use of a simple methyl ester ( i.e. , 73 , x = ome ) , a far easier group to potentially remove , arrested at compound 75 with mannich closure preceding michael addition in the second cascade , just as a simple vinyl phenyl sulfone had putatively done ( 69 71 , ar = ph ) . reagents and conditions : ( a ) tfa / ch2cl2 , 0 c , 1 h ; ( b ) tmg ( 2.0 equiv ) , i - proh , 25 c , 1 h ; ( c ) i - proh , 60 c , 2.5 h , 38% yield over three steps ; ( d ) tfa / ch2cl2 , 0 c , 1 h ; ( e ) i - proh , 80 c , 2 h , 56% yield over two steps . scheme 12 provides a possible graphical explanation for sequence arrest , with steric clashing likely being the basis for the failed terminating ring - closure . collectively , these findings reveal overall that , while there is some flexibility in the groups that can allow the key elements of this cascade chemistry to proceed , careful control of electronics is required to fully orchestrate the designed sequences . in summary , a concise synthesis of ten members of the coccinellid family of alkaloids has been accomplished in both total and formal format , all starting from a single , common intermediate . key components of the developed chemistry include the use of several cascade - based bond constructions involving finely tuned and highly reactive intermediates coupled with a new synthetic logic for the formation of dimeric natural products where biomimetic , direct dimerization approaches have failed to control regio- and stereoselectivity . we anticipate that this unique design for dimerization is applicable to a number of other natural product compound classes , foremost of which may be the myrmicarin alkaloids for which available approaches have failed . work is ongoing to verify that assertion , as are biochemical studies of the synthesized materials and efforts to prepare other molecules in the class .

although dimeric natural products can often be synthesized in the laboratory by directly merging advanced monomers , these approaches sometimes fail , leading instead to non - natural architectures via incorrect unions . such a situation arose during our studies of the coccinellid alkaloids , when attempts to directly dimerize nature s presumed monomeric precursors in a putative biomimetic sequence afforded only a non - natural analogue through improper regiocontrol . herein , we outline a unique strategy for dimer formation that obviates these difficulties , one which rapidly constructs the coccinellid dimers psylloborine a and isopsylloborine a through a terminating sequence of two reaction cascades that generate five bonds , five rings , and four stereocenters . in addition , a common synthetic intermediate is identified which allows for the rapid , asymmetric formal or complete total syntheses of eight monomeric members of the class .

Introduction Results and Discussion Conclusion

this analysis includes materials with obvious symmetry , such as sceptrin ( 1 , scheme 1 ) , compounds with equivalent halves but non - symmetric unions , such as complanadine a ( 2 ) , and materials whose symmetry has been partially erased through subsequent structural modifications like oxidation or decarboxylation , such as cp-225,917 ( 3 ) . given the significant energy invested in the creation of any natural product , the ubiquity of such dimers is logical , since dimerization enables rapid access to additional molecular scaffolds without invoking entirely new biosynthetic pathways . considering only those dimeric natural products that possess obvious monomer symmetry ( i.e. the first and most typical approach forges the dimer in a final synthetic operation from fully functionalized monomers . such processes can range from the simple and direct construction of a single connecting bond to far more complex bond - forming unions , such as that postulated for the conversion of sorbicillin ( 4 ) into trichodimerol ( 5 ) through a series of michael reactions and ketalizations . in the second dimerization approach , monomer union occurs at an earlier stage , with subsequent tandem modifications of each half leading to the final structure ( as in 6 7 8 and 10 11 12 ) . this concept was , to the best of our knowledge , first demonstrated by stork in the synthesis of -onocerin ( 8) in four steps from 9 , and used more recently to great effect by a number of groups , including boger in his approach to 12 . such issues arose during our efforts to synthesize the coccinellid family of alkaloids , materials secreted by numerous species of ladybugs as defensive compounds when provoked and viewed by some as potential commercial insecticides , particularly for the control of aphid pest populations . figure 1 provides the structures of eight of the nine monomers within this class ( 1421 ) , tricyclic architectures differing in ring junction stereochemistry , oxidation state , and olefin placement . structures of the coccinellid class of alkaloids : unique monomeric and dimeric frameworks . in addition to these materials , several larger and more complex compounds are known wherein half of their framework possesses these monomer cores , such as chilocorine a ( 22 ) , as well as two other species that structurally reflect the dimeric combination of these cores in the form of psylloborine a ( 23 ) and isopsylloborine a ( 24 ) . the sole distinction between these latter two natural products is ring - junction enamine isomerism within their fused , highly congested , and stereochemically rich frameworks . equally surprising , no work toward any higher - order structure has been reported , nor has any mechanistic hypothesis been advanced to account for dimer formation in nature . to date , that work has identified a single common synthetic intermediate capable of rapidly affording every monomer drawn in figure 1 . it has also led to a biosynthetic proposal for the formation of both psylloborine a ( 23 ) and isopsylloborine a ( 24 ) , one that , when reduced to practice , resulted in a non - natural , regioisomeric dimer . this unexpected result , coupled with observations from other instances where incorrect dimeric unions have occurred in biomimetic constructions , has led to the development of a unique , non - biomimetic strategy for complex dimer synthesis . as will be described in the ensuing sections , this alternate strategy has afforded rapid syntheses of both psylloborine a ( 23 ) and isopsylloborine a ( 24 ) through sequences involving some of the most complex condensation / michael / mannich cascade chemistry yet reported . given the absence of any proposal for how either psylloborine a ( 23 ) or isopsylloborine a ( 24 ) might arise in nature , we began by pondering mechanistic pathways that could account for their formation from the known tricyclic coccinellid alkaloids . if this material was formed in the presence of a molecule of propyleine ( 17 ) , then perhaps it could undergo the sequence of events shown in scheme 2 , involving a vinylogous mannich reaction , proton transfer , mannich reaction , and terminating proton loss to generate dimers 23 and/or 24 . equally critical , but unclear , were ( 1 ) whether one or both dimeric products would result from such a pathway , and ( 2 ) whether only propyleine ( 17 ) would be the active nucleophile , or if its equilibrating and more dominant enamine isomer , isopropyleine ( 18 ) , could participate in addition to , or instead of , 17 . indeed , as shown in the lower half of scheme 3 , braekman and co - workers demonstrated that polyketo - myristic acid ( formed from stearic acid through enzymatic oxidations ) is the biosynthetic precursor to coccinelline ( 19 ) via the proposed intermediacy of 33 , a compound containing a 10-membered ring ; the intervening steps listed above each arrow are one proposal for how these net transformations might specifically occur . our proposed synthesis of 17/18 is based on a similar , but differently ordered , set of chemical events involving no individual ring size greater than six , in which a disubstituted piperidine ( 34 ) becomes the tricyclic system through an enamine condensation and a terminating c c bond formation driven by nucleophilic attack of the enamine moiety . assuming success in our proposed synthetic operations leading to 17/18 , attempts to generate electrophile 26 in situ through n - oxide formation would then begin . given that this proposed key starting material ( 31 ) possesses three reactive domains ( highlighted in blue , green , and orange ) with sufficiently distinct and tunable electrophilic and nucleophilic properties , we also questioned whether every other monomer configuration ( i.e. if so , then a near - universal , family - level solution for the coccinellid alkaloids would exist , with enantioselective syntheses of all monomeric members possessing optical activity being achieved , some for the first time . as shown in scheme 4 , we ultimately developed two different routes to accomplish that goal , both starting from the commercially available n - boc-(s)-()-piperidine-2-ethanol ( 36 ) . next , we hoped to set the methyl stereocenter of the target through a diastereoselective reduction . this step ultimately proved to be the most challenging of the sequence , with repeated attempts at substrate - directed hydrogenation using wilkinson s or crabtree s catalysts affording either no diastereoselection or preference for the undesired methyl epimer on 39 or its deconjugated enone precursor . concurrent with these efforts , especially in light of the low - yielding wacker oxidation step , we developed an additional sequence to 31 . it began similarly from 36 , with a two - step sequence of silyl protection of the alcohol ( this time as a tbs ether ) and a copper - mediated addition using allylic bromide 40 that proceeded in 92% overall yield . the remaining carbon atoms of the target were then installed via nucleophilic addition of grignard reagent 42 , with a second oxidative cleavage then generating the ketone within 43 in 95% yield over three steps . finally , this transformation was followed by the same facially selective hydride delivery , with a terminating in situ deprotection upon quenching of the -reduction product with hcl , completing the synthesis of 31 in 79% yield and in 8:1 dr . indeed , the first route was able to deliver 2.68 g of 31 in a single campaign , while the second afforded 3.02 g of this key intermediate . with compound 31 in hand , our efforts to synthesize the monomeric members of the coccinellid class began in earnest . starting with propyleine and isopropyleine ( 17 and 18 , scheme 5 ) , we first converted the alcohol within 31 to a reactive bromide ( 44 ) through the intermediacy of its n - deprotected tfa salt ( tfa ; pbr3 , one - pot operation ) . in addition , the levorotatory rotation of the final synthetic materials matched that of the natural isolates , confirming the absolute configuration of these compounds for the first time as based on the initial assignment of 36 ( cf . from here , subsequent reduction of a portion of these natural products with nabh(oac)3 led to a 3.7:1 mixture of precoccinelline ( 14 ) and hippodamine ( 15 , scheme 5 ) . however , given the observed facial bias , we presume that if the top ring ( as drawn in the two - dimensional depiction of 46 ) were to adopt a twist - boat orientation , as indicated by the three - dimensional representation of imine 46 in scheme 5 , then hydride delivery from the bottom face would appear to be preferred , forming precoccinelline ( 14 ) . as such , we sought an alternate and potentially more controlled path to the needed electrophilic dimerization precursor in the form of cross - conjugated diene 49 ( scheme 6 ) . following extensive experimentation with acid source and solvent , we found that electrophile 26 ( scheme 7 ) could be obtained when 49 was taken up in cd2cl2 ( to enable close monitoring by nmr analysis ) and exposed to tfa at 25 c . when propyleine and isopropyleine ( 17 and 18 ) were then added as a cd2cl2 solution to this electrophile 2 min later , a new dimeric material was formed over the course of 2 h in 21% overall yield from 53 . extensive nmr analysis ( h , c , cosy , tocsy , noesy , hsqc , and hmbc ; see supporting information for full details ) ultimately revealed that this dimer was non - natural , resulting from incorrect regiocontrol in the union of the two building blocks , as noted by the highlighted carbons within their frameworks . critically , we believe it is the result of kinetic control based on the stereoelectronic and conformational demands of the lone desymmetrizing methyl group within nucleophiles 17 and 18 , not the ratio of these two components in solution . based on the analysis provided earlier in the context of scheme 2 , the requisite pathway to psylloborine a ( 23 ) or isopsylloborine a ( 24 ) would require nucleophilic attack by propyleine ( transition states a and/or b ) . in transition state a , a favorable pseudochair - like transition state can be achieved , but it incurs a significant steric penalty by requiring the incoming electrophile to approach syn to the pendant methyl group . by contrast , when isopropyleine ( 18 ) is the enamine nucleophile ( transition states c and d ) , there are no proximal substituents that can destabilize the approach of the electrophile as in pathway a or b. therefore , the lowest energy pathway is likely to proceed through these transition states , and thus 54 results . given this experimental outcome , a new synthetic approach was needed for psylloborine a ( 23 ) and/or isopsylloborine a ( 24 ) . specifically , rather than combine advanced materials in a final step or merge simpler materials earlier and then elaborate in tandem , instead ( 1 ) link two simpler precursors at an appropriate site to ensure proper regiocontrol and ( 2 ) embed enough chiral information and reactivity within the overall structure to establish the remaining rings and stereocenters as well as forge any remaining bonds between the two halves . intramolecular dimerization , and compound 59 was designed for the coccinellid alkaloids on the basis of its general concepts , outfitted with one of the requisite dimer linkages ( highlighted in purple in scheme 8) that could not be forged from direct , advanced monomer coupling . from here , two cascades were envisioned to forge the remaining rings , stereocenters , and final dimeric linkage needed to complete the targets . the second cascade would utilize an added electron - withdrawing group ( ewg , colored in blue ) on one of the pendant methyl groups of the final target to dictate the correct order of bond constructions through condensation , enamine - based michael attack to form the tricyclic ring system , and a terminating mannich ring - closure to complete the carbon framework . collectively , these two cascades would forge five new bonds , five new rings , and four stereogenic centers , assuming again that pre - existing chirality within 59 could govern the incorporation of the remaining chiral elements . if successful , then a terminating excision of the ewg in cascade product 57 would complete the synthesis of 23 and/or 24 . however , given the failure of direct dimerization and the likely inapplicability of tandem elaboration to a non - symmetric dimer , we required a distinct strategy . one potential and logical benefit of this new approach is that the final stitching operations appear to take advantage of biosynthetic efficiency through the use of cascades that resemble nature s synthesis of the monomeric frameworks . moreover , we anticipated that this synthetic sequence would not be much longer than the failed direct dimerization strategy . indeed , key test substrate 59 was expected to readily arise from a horner wadsworth emmons coupling between phosphonate 60 , a material we anticipated could be readily synthesized , and aldehyde 50 , the oxidized version of our key common intermediate for monomer synthesis which was already available on gram scale ( cf . its core elements closely mirror the synthetic pathways described earlier in the context of scheme 4 , differing only in terms of the fragments coupled , and thus will not be discussed in detail ( scheme 9 ) . pleasingly , treatment of all three of these variants of 66 with 1,1,3,3-tetramethylguanidine ( tmg ) in a 9:1 mixture of toluene / i - proh effected the desired reaction sequence of carbamate cleavage , enone regeneration , condensation , enamine equilibration to the exocyclic isomer , and a terminating michael addition . subsequent dissolution in benzene - d6 ( to monitor the reaction ) and heating at 65 c then initiated that cascade sequence : condensation to 69 , michael closure to 70 , and a terminating mannich reaction to complete the synthesis of 72 . , ar = 3,5-(cf3)2ph- or 4-no2ph- ] , these operations proceeded in the order designed however , when the unsubstituted phenyl sulfone was used , a large portion of the material appeared to undergo mannich reaction prior to michael addition to afford materials believed to have structure 71 . despite various attempts , these compounds thus , the more electron - withdrawing aryl sulfones seem to have enabled the sequence to succeed by ensuring that michael reaction preceded mannich closure . taken together , these two cascade events arguably constitute the most complex use of condensation / michael / mannich chemistry yet described , given that they involve a total of seven distinct chemical events that forged three c n bonds , five rings , and four stereocenters ; the overall yield obtained for the 3,5-(cf3)2ph variant , at 15% , reflects a throughput of 79% per chemical transformation . finally , exposure of the 3,5-(cf3)2ph derivative of 72 to na / hg amalgam transformed it into psylloborine a ( 23 ) , a material identical in all respects to the natural isolate , thereby completing the first total synthesis of this molecule as well as that of any dimeric coccinellid alkaloid . as shown in scheme 11 , use of the same sequence with a simple methyl ketone ( i.e. , 73 , x = me ) proceeded in similar overall yield ( 79% per transformation for the steps shown ; see supporting information ) to generate the full heptacyclic core of the dimeric coccinellid alkaloids ( 74 ) . however , no approach could be discovered to convert the methyl ketone to the final pendant methyl of the target natural products . , 73 , x = ome ) , a far easier group to potentially remove , arrested at compound 75 with mannich closure preceding michael addition in the second cascade , just as a simple vinyl phenyl sulfone had putatively done ( 69 71 , ar = ph ) . collectively , these findings reveal overall that , while there is some flexibility in the groups that can allow the key elements of this cascade chemistry to proceed , careful control of electronics is required to fully orchestrate the designed sequences . in summary , a concise synthesis of ten members of the coccinellid family of alkaloids has been accomplished in both total and formal format , all starting from a single , common intermediate . key components of the developed chemistry include the use of several cascade - based bond constructions involving finely tuned and highly reactive intermediates coupled with a new synthetic logic for the formation of dimeric natural products where biomimetic , direct dimerization approaches have failed to control regio- and stereoselectivity . work is ongoing to verify that assertion , as are biochemical studies of the synthesized materials and efforts to prepare other molecules in the class .

epithelia are sheets of cells that constitute the lining of most of the organs of the body such as the skin , the digestive and respiratory tracts , and the urogenital system and separate the inside of the body from the outside ( blanpain et al . , 2007 ) . an epithelium can be composed of one ( simple epithelium ) or multiple layers of cells ( stratified epithelium ) , and forms the majority of the glands . a variety of specialized differentiated cells present in these epithelia ensure the important diversity of physiological functions they control . the skin epidermis acts as a barrier that keeps fluids in and protects the animal from the aggressions of the external environment . the urologic system allows the evacuation of ions , water , and toxic byproducts of metabolism . some epithelia such as the skin or the intestine have a very high cellular turnover to replace the cells that are continuously lost ( barker et al . , 2010a ) . other epithelia , such as those of the airway tracts , renew much more slowly under physiological conditions but nevertheless can rapidly and extensively proliferate to repair tissue upon damage or injuries ( rock and hogan , 2011 ) . stem cells ( scs ) located in these different adult tissues are essential to sustain tissue turnover and repair these different epithelia upon injuries . scs can renew throughout life and can differentiate into the different cell lineages of their tissue of origin . the balance between sc proliferation and differentiation must be precisely controlled , as deregulation of this process may lead to tissue atrophy and cancer formation . carcinoma , which are tumors arising from epithelium , are by far the most common cancers in humans and lead to millions of death per year throughout the world . inspired by the field of hematopoietic scs , the most common assay to assess the renewal and differentiation of putative epithelial scs is their transplantation into immunodeficient animals ( blanpain et al . , 2007 ) . although transplantation assays are very informative about the differentiation potential of scs , they do not necessarily reflect physiological conditions because scs are dissociated from their normal environment and very often transplanted into a heterotopic site , such as into the renal capsule ( the fibrous layer surrounding the kidney ) or the dermis , with or without their normal underlying mesenchyme . lineage tracing has now become the method of choice to study the renewal and the differentiation potential of scs in intact tissue , as this approach avoids the many drawbacks of transplantation experiments . in this technique , a particular cell lineage is labeled and the fate of the labeled cells and their progeny is analyzed over time . these experiments are usually performed in mice coexpressing two different transgenes : a cre recombinase expressed under a lineage - specific promoter , and a reporter gene only expressed when the cre removes a stop cassette that precedes the reporter gene . upon cre excision of the stop cassette , the reporter transgene is permanently expressed in these cells and all their future progeny . two different cre genes can be used to perform such experiments : a constitutive cre , which is naturally active , and an inducible cre , which is usually fused to a mutated nuclear hormone receptor such as the estrogen receptor ( er ) called creer or progesterone receptor ( pr ) called crepr . in the absence of their synthetic ligands ( such as tamoxifen for the creer or ru486 for the crepr ) , the creer is maintained inactive in the cytoplasm . administration of the synthetic ligand induces activation of the inducible cre recombinase , which in turn induces the expression of the reporter gene in the cells expressing the cre and all their subsequent progeny , allowing the fate of labeled cells to be followed over time . the limitation of the inducible lineage tracing experiments is the identification of a specific promoter that targets the sc of interest and the mosaic expression of the reporter gene within the sc population . the latter precludes one from drawing any firm conclusions about the fate of the nonlabeled cells . in these studies , it is generally assumed that the labeled cells are representative of the whole sc population . because there is an important heterogeneity within epithelial scs , one should always leave open the possibility of the existence of another population of epithelial scs not labeled by this approach . the frequency of labeled cells over time is a good indication of whether these cells are in equilibrium with other sc populations ; if nonlabeled scs replace labeled cells , the frequency of labeled cells should decrease over time . , it is necessary to mark individual cells sufficiently distant from each other to follow the fate of individual clones . this can be achieved by lowering the dose of the tam until it induces creer activity in isolated cells . lineage - tracing experiments provide important information about the cellular hierarchy that governs epithelium development and homeostasis , and uncover the diversity of epithelial scs , their origin , tissue location , renewal , migration , and differentiation potential , as well as their ability to contribute to tissue repair and tumor initiation . different methods to perform lineage tracing in vertebrates and invertebrates have been recently thoroughly reviewed elsewhere ( buckingham and meilhac , 2011 ; kretzschmar and watt , 2012 ) . in this review , we discuss the novel insights into epithelial scs and their lineages that have been gathered thanks to the use of this technique in mice . the skin epidermis is composed of the interfollicular epidermis , which forms the skin barrier , and its various appendages such as the hair follicle ( hf ) , the sebaceous gland , and the sweat gland ( fig . a group of cells that present an elongated morphology called the placode progress to form a bud called the hair germ , which keeps growing and differentiates into the seven concentric cell lineages that form the mature hf ( blanpain and fuchs , 2006 ) . 2005 ) or sox9cre ( nowak et al . , 2008 ) , which are specifically expressed in the hair germ , demonstrated that all hf lineages including adult hf scs and the sebaceous gland arise from embryonic progenitors expressing these two markers during hf morphogenesis . similar long - term labeling of the hf lineages has been obtained using lgr6creer lineage tracing at e17.5 , which is also expressed in embryonic hair germ , although its expression is broader and included cells of the interfollicular epidermis ( snippert et al . , 2010a ) . 2005 ) and sox9cre ( nowak et al . , 2008 ) also revealed that hfs were entirely labeled whereas the interfollicular epidermis was not labeled , demonstrating that the interfollicular epidermis and the hf lineages arise from two independent sources of cells that are maintained independently in adult animals . ( a ) schematic representation of the skin epidermis and the different epidermal scs : the interfollicular epidermis ( ife ) scs , the isthmus scs , and the bulge scs . ( b and c ) lineage tracing of bulge stem cells ( green ) using k19creer - rosayfp mice induced with 10 mg tamoxifen between d 21 and 25 , and analyzed 1 wk ( b ) or 5 wk ( c ) after induction . immunostaining of cd34 ( b ) or integrin 4 ( c ) and yfp show the initial labeling of cd34 + bulge sc ( b ) and the presence of yfp cells in the newly formed hair follicle 5 wk after ( c ) . ( d ) lineage tracing of basal cells ( green ) from the interfollicular epidermis using k14creer - rosayfp mice induced with 1 mg tamoxifen and analyzed 5 wk later . immunostaining of k14 ( red ) and yfp ( green ) shows the presence of a column of yfp - marked cells spanning from the basal layer ( red ) to the cornified layer , corresponding to a unit of interfollicular epidermis maintained by a single sc . ( e and f ) migration of h2b - gfp label - retaining bulge scs to the interfollicular epidermis after wounding , showing the early contribution of bulge sc to the wound repair . adapted from tumbar et al . ( g ) lineage tracing using lgr6-gfp - irescreer / rosa - lacz mice induced at d 20 and analyzed 1 yr later , showing the labeling of the sebaceous gland , and demonstrating the existence of long - lived sc of the sebaceous gland . adapted from snippert et al . bu , bulge ; dp , dermal papilla ; hg , hair germ ; ife , interfollicular epidermis ; sg , sebaceous gland ; in , infudibulum ; ep , epidermis ; w , wound . in addition to ensuring the barrier function and thermoregulation , the skin epidermis is also a sensory organ that perceives all kinds of sensory stimuli . merkel cells are neuroendocrine cells scattered in the epidermis that are responsible for the fine touch sensation ( maricich et al . , 2009 ; they are excitable cells that express pro - neural transcription factors , possess many components of the presynaptic machinery , and contact the end of sensory nerves ( haeberle et al . , 2004 ) . due to their resemblance with neuronal cells , there has been a long - standing debate whether merkel cells originate from the neural crest derivatives or from epidermal cells . two independent groups have recently demonstrated using lineage - tracing experiments that merkel cells do not originate from neural crest cells , but rather from epidermal progenitors by a mechanism depending on the expression of the proneural transcription factor atoh1 in epidermal progenitors ( morrison et al . transplantation experiments suggested that the permanent portion of the hf called the bulge contains multipotent scs able to differentiate into all epidermal lineages including hf , sebaceous gland , and interfollicular epidermis upon transplantation with embryonic skin mesenchyme into immunodeficient mice ( rochat et al . , 1994 ; oshima et al . , 2001 ; blanpain et al . , 2004 ; morris et al . , 2004 2004 ) , shhcre ( levy et al . , 2005 ) , sox9cre ( nowak et al . , 2008 ) , lgr5creer ( jaks et al . , 2008 ) , and k19creer ( youssef et al . , 2010 ) markers confirmed that bulge scs are indeed multipotent scs responsible for the maintenance of all hf lineages under physiological conditions ( fig . 1 , b and c ) . clonal analysis of bulge scs indicated that some bulge scs can differentiate into all hf lineages , whereas others are committed to either the outer or the inner cell layers ( legu et al . , 2010 ; zhang et al . , 2010 ) . these results suggest that the heterogeneity in the differentiation potential of bulge scs is either related to an intrinsic heterogeneity of bulge cells containing multipotent and unipotent scs or through a regulation of the lineage differentiation potential of multipotent scs . similarly to the embryonic hf lineage tracing , lineage tracing of adult bulge scs also revealed that the hf lineages do not contribute to the homeostasis the interfollicular epidermis under physiological conditions ( morris et al . , 2004 ; jaks et al . , 2008 ; youssef et al . , 2010 ) , consistent with the presence of two separate pools of scs that separately maintain hf and interfollicular epidermis turnover ( fig . however , upon wounding , bulge scs are rapidly activated and actively participate in the repair of the damaged interfollicular epidermis ( fig . 1 , e and f ; tumbar et al . , 2004 ; ito et al . , 2005 ; these experiments suggested that the multipotency of bulge scs observed in transplantation assays represents the fate of bulge scs in a wounding or regenerative environment . lineage tracing of hf matrix cells demonstrated that these cells are transient amplifying cells that differentiate into one of the six lineages depending on their spatial position within the matrix ( legu and nicolas , 2005 ; youssef et al . , 2010 ) . sebaceous gland scs arise from multipotent embryonic hf progenitors expressing shh , lgr5 , sox9 , and lgr6 ( levy et al . , 2005 ; nowak et al . , 2008 ; within the next few days , blimp1-expressing progenitors are specified along the hf and give rise to the cells of the adult sebaceous gland including sebaceous gland scs ( horsley et al . the long - term labeling of the sebaceous gland observed after lgr6 lineage tracing in adult skin demonstrates the presence of resident lgr6-expressing sebaceous gland scs ( fig . similar to bulge scs , lgr6-expressing isthmus scs are also recruited toward the interfollicular epidermis upon wounding and contribute to the repair of the damaged epidermis ( snippert et al . , 2010a ) . a recent study using new k15creer transgenic mice suggests that a fraction of bulge scs migrate upward toward the isthmus region and replenish the sebaceous gland under homeostatic and physiopathological conditions ( petersson et al . , 2011 ) . further studies will be required to better understand the respective contribution of bulge versus resident lgr6 + scs in maintaining the homeostasis of the sebaceous gland and defining the mechanisms leading to the recruitment and the migration of bulge scs to this gland . as previously mentioned , the interfollicular epidermis is maintained by a source of scs that is distinct from the hf lineages during postnatal physiological conditions . many small units of proliferation called epidermal proliferative units ( epus ) scattered all along the interfollicular epidermis ensure the constant turnover of the epidermis and its proper differentiation . based on morphological and proliferation studies , it has been proposed that the interfollicular epidermis is maintained by hexagonal shaped epus containing one sc and 10 transit - amplifying cells ( potten , 1974 , 1981 ) . retroviral fate mapping indeed demonstrated the presence of columns of labeled cells spanning from the basal layer to the top of the cornified layer a long time after the clonal marking ( ghazizadeh and taichman , 2001 ) , consistent with the presence of long - lived scs ensuring the long - term homeostasis of an epu . however , genetic lineage tracing showed that the epu was not hexagonal in shape as suggested by histological analysis and was sometimes bigger than the 10 basal cells expected from the model based on morphological observations ( ro and rannala , 2004 , 2005 ) . these data were interpreted as the consequence of sc migration from one epu to another . recently , clonal analysis studies labeling basal epidermal cells of the tail and the ear epidermis using a nontissue - specific drug inducible creer ( ahcreer ) showed that the majority of labeled cells are not maintained long - term and are lost over time . however , the surviving clones seemed to expand continuously , in contrast to what would be expected if these cells were maintaining a discrete and predetermined unit of the epidermis . mathematical modeling of these data suggests that the epidermis could be maintained by one type of progenitor , without the presence of transit - amplifying cells , and these progenitors undergo population asymmetric renewal by balancing asymmetric and symmetric cell division in a stochastic manner ( clayton et al . although this model is appealing in its simplicity , it relies on the assumption that all basal cells of the epidermis behave like the basal cells labeled by the ahcreer . the model also does not easily explain the ability of the tissue to respond rapidly to increased cell demand , such as during wound healing . importantly , these data can not rule out the presence of a small proportion of more quiescent scs that would exist in equilibrium with more committed basal cells , as it has been previously suggested . new studies combining clonal analysis with different epidermal - specific creer and proliferation kinetic data will be helpful to address these open questions . in summary , lineage - tracing experiments in the skin epidermis indicate the existence of multiple populations of stem cells that ensure the maintenance of different compartments of the epidermis and play distinct roles during homeostasis and repair . the mammary gland of the mother plays an essential role during the early postnatal life of young mammalian offspring by providing them nutrients , water , and electrolytes , and immune defense until they reach the size and maturity to survive independently . mammary glands are epidermal appendages that are specified along the ventral epidermis around e12 during mouse embryonic development . these glands are initially visible as placode - like structures that progressively invade the underlying mesenchyme , called the mammary fat pad . at puberty , the mammary gland expands considerably to form a highly branched tubular structure that progressively fills the entire fat pad . the epithelial part of the mammary gland comprises two main cell types , the basal myoepithelial cells and the luminal cells , which can differentiate either into ductal cells or milk - producing alveolar cells ( fig . further expands and differentiates into milk - producing cells . after each cycle of pregnancy , the mammary gland involutes through a massive apoptosis of alveolar cells after which the gland returns to a similar morphology as before pregnancy ( watson and khaled , 2008 ) . transplantation studies using different populations of mammary epithelial cells isolated by fluorescence - activated cell sorting have demonstrated that rare single cells are able to reconstitute an entire functional mammary gland ( shackleton et al . , 2006 ; stingl et al . , these results suggest the presence of rare multipotent mammary scs at the top of the cellular hierarchy of the breast epithelium ( visvader , 2009 ) . the whey acidic protein ( wap ) promoter is active in luminal cells during pregnancy and lactation . lineage - tracing experiments using the wap - cre , which labeled luminal cells during pregnancy , identified luminal cells capable of resisting apoptosis during involution and which clonally expand upon the succeeding pregnancy to give rise to luminal and alveolar cells . these cells were therefore described as alveolar progenitors and called parity - induced cells ( wagner et al . , 2002 ) . more recently , lineage tracing of the mammary gland using inducible cre expressed either in myoepithelial cells ( k14- or k5-expressing cells ) or in luminal cells ( k8- or k18-expressing cells ) demonstrated that the mammary gland initially develops from multipotent embryonic k14-expressing progenitors , which give rise to both myoepithelial cells and luminal cells . however , postnatal mammary gland development that occurred during puberty , as well as mammary gland expansion that accompanied pregnancies , are ensured by the presence of two types of long - lived scs . k14/k5-expressing myoepithelial and k8/18-expressing luminal unipotent scs are able to differentiate at the clonal level into myoepithelial or luminal lineages , respectively , rather than being maintained by rare multipotent scs ( fig . decreasing the ratio of luminal to myoepithelial scs stimulates the luminal differentiation of myoepithelial scs in transplantation assay ( van keymeulen et al . , 2011 ) . further studies will be necessary to define the mechanisms that restrict the differentiation potential of myoepithelial scs under physiological conditions , and whether pathophysiological conditions can expand the differentiation potential of scs in the intact mammary gland . also , it would be interesting to determine whether other glandular epithelia such as the prostate or sweat glands are also maintained by the presence of distinct classes of scs during postnatal development and adult homeostasis . ( a and b ) schematic representation of the main epithelial cell types ( myoepithelial , luminal , and alveolar cells ) of the breast during post - natal development ( a ) and pregnancy ( b ) . ( c e ) lineage tracing of myoepithelial cells ( green ) using k14rtta / tetocre / rosayfp mice induced at the onset of puberty ( 4 wk old ) and analyzed 1 wk ( c ) or 10 wk ( d ) later or during lactation ( e ) . immunostaining of k14 ( c and d ) or k5 ( e ) ( red ) and yfp ( green ) demonstrates the labeling of unipotent scs that ensure myoepithelial lineage expansion during puberty and pregnancy . ( f h ) lineage tracing of luminal cells ( green ) using k8creer / rosayfp mice induced at the onset of puberty ( 4 wk old ) and analyzed 1 wk ( f ) or 10 wk later ( g ) , or during lactation ( h ) shows the labeling of unipotent scs that ensure luminal lineage expansion during puberty and pregnancy . adapted from van keymeulen et al . the gut regulates the absorption of water , electrolytes , nutrients , and vitamins essential for the survival of the animal . the gut can be subdivided into the esophagus , stomach , intestine , and colon . the esophagus is a stratified epithelium resembling skin epidermis , whereas the stomach , intestine , and colon are simple epithelia composed of only one cell layer that contains the different cell lineages present in the different parts of the gut ( barker et al . , 2010a ) . the stomach can be subdivided into the corpus , the pylorus , the cardia , and the fundus . in the corpus , the parietal cells secrete the acid necessary to activate digestion , while the mucus cells secrete a protective barrier and the enteroendocrine cells regulate gut contractility and the secretion of the various digestive enzymes . although the mucus cells renew quite rapidly , the chief cells and the parietal cells responsible for acid secretion display a slow turnover . random marking of stomach scs after chemical mutagen administration demonstrated the coexistence of long - lived multipotent and unipotent stomach scs ( bjerknes and cheng , 2002 ) . in the pylorus , the vast majority of the proliferating cells are located in the middle zone called the isthmus . lgr5 lineage tracing , which marked cells located at the bottom of the gland , demonstrated that the progeny of lgr5-expressing cells migrate into the isthmus region , proliferate , and give rise to all lineages of the stomach ( barker et al . , 2010b ) . recently , sox2 lineage tracing , which appears to mark cells distinct from lgr5 + cells , showed that sox2 also marked long - lived multipotent stomach scs ( arnold et al . , further studies will be required to understand the relationship between sox2 + and lgr5 + stomach scs . the intestine is composed of proliferating crypts that produce the cells giving rise to the differentiated villi . villi are protrusions of the epithelium in the gut lumen that massively increase the surface area of the gut to allow for greater absorption . the intestine is one of the most rapidly proliferating tissues in the body , which completely self - renews in less than a week . several cell lineages are present in the intestine : the absorptive enterocytes are the most abundant cells , the goblet cells produce the mucus , and the enteroendocrine cells regulate the motility of the gut and the secretion of the digestive enzymes while the paneth cells residing at the base of the crypt produce microbicide proteins ( fig . 3 a ) . based on proliferation kinetics experiments , it has been suggested that intestinal scs reside in the crypt at position + 4 with respect to the base of the crypt ( potten et al . , 1978 ) . ( a ) schematic representation of the intestinal epithelium lineages ( enterocytes , enteroendocrine , goblet , and paneth cells ) and its scs . ( b and c ) lineage tracing of lgr5 + scs ( blue ) using lgr5-gfp - irescreer / rosalacz mice analyzed 12 h ( b ) or 60 d ( c ) after induction demonstrates the initial labeling of columnar basal cells ( b ) that give rise to the differentiated cells of a villus ( c ) . adapted from barker et al . ( d and e ) lineage tracing of bmi1 + scs ( green ) using bmi1creer / rosa yfp mice analyzed 5 d ( d ) or 2 mo ( e ) after induction . staining of yfp ( green ) and of chromogranin a ( chga ) labeling enteroendocrine cells ( red ) ; and lysozyme ( lys ) antibody , labeling paneth cells ( red ) , showing the initial labeling of cells above the paneth cells ( d ) that give rise to the differentiated cells of a villus ( e ) . ( f and g ) lineage tracing of crypts using ah - creer / rosaconfetti mice analyzed 1 wk ( f ) or 8 wk ( g ) after induction , showing the initial multicolor labeling of the crypt and villus unit that progressively become monoclonal ( one color per crypt ) over time . adapted from snippert et al . the existence of multipotent scs in the intestine has been suggested by the presence of long - lived clones of marked cells that contain several types of differentiated intestinal cells ( bjerknes and cheng , 1999 ) . ( 2007 ) identified lgr5 as a wnt target gene in colonic cancer lines , and demonstrated that lgr5 is expressed at the base of the crypt in intestinal crypt base columnar cells intercalated between paneth cells . in contrast to the vast majority of wnt target genes , which are expressed throughout the crypt , including in the transit amplifying cells , lgr5 expression is restricted to crypt base columnar cells . lgr5creer - ires - gfp lineage tracing demonstrated that indeed lgr5-expressing crypt base columnar cells are rapidly cycling multipotent scs of the intestine giving rise to all cell lineages of the intestine including enterocytes , goblet cells , and neuroendocrine cells , as well as paneth cells ( fig . bmi1 , a polycomb repressor , is expressed in the proximal intestine and preferentially marks the cells located in position + 4 . cells labeled in lineage - tracing experiments using bmi1creer , similarly to lgr5 + cells , give rise to all cell lineages of the intestine ( fig . 3 , d and e ; sangiorgi and capecchi , 2008 ) , suggesting the existence of two distinct classes of scs in the small intestine . two independent studies using creer show that preferentially but not exclusively labeled + 4 cells ( mtertcreer and hopxcreer ) give rise to all intestinal lineages ( montgomery et al . , 2011 ; takeda et al . , 2011 ) , confirming the presence of multipotent intestinal scs in the + 4 position . the faster expansion of lgr5 progeny in comparison to bmi1- or mtert - derived cells suggests that + 4 intestinal scs could be more quiescent than lgr5 + scs , but yet long - lived . interestingly , although the ablation of bmi1 + cells leads to the degeneration of the crypts ( sangiorgi and capecchi , 2008 ) , the ablation of lgr5 cells has no effect on intestinal homeostasis , as bmi1 + scs can compensate for the loss of lgr5 cells ( tian et al . , 2011 ) . under physiological conditions or after the destruction of lgr5 + cells similarly , mtert and hopx labeled cells also give rise to lgr5 + cells and conversely lgr5 cells can give rise to hopx cells ( takeda et al . , 2011 ) , suggesting that the lgr5 scs are in equilibrium with the + 4 cells scs . although there are around 16 lgr5 + cells per crypt , lgr5 lineage tracing using a multicolor reporter mouse demonstrated that the crypt rapidly evolves from polyclonal labeling ( multicolor ) to monoclonal labeling ( monocolor ) ( fig . 3 , f and g ) . analysis of lgr5 cell division revealed that they mostly divide symmetrically giving rise to 2 lgr5 + cells , incompatible with a model in which homeostasis is maintained by pure asymmetric cell division ( one sc gives one transit - amplifying cell ) . instead , this supports a model in which sc divisions are symmetric at the cellular level , but , at the level of the sc pool , the divisions seem asymmetric because for one sc that divides , one sc is lost , leading to neutral drift dynamics in which scs expand or are lost at random ( lopez - garcia et al . , 2010 ; snippert et al . , 2010b ) . how to reconcile the data supporting the existence of two populations of intestinal scs in equilibrium with each other , with the presence of neutral drift toward monoclonality of the intestinal crypt ? one possibility is that , despite their relatively distinct tissue localization , lgr5 and bmi1 are functionally equipotent scs competing with each other in the neutral drift proliferation dynamics . another possibility is that the long - term lineage tracing in both models results from the labeling of the same multipotent intestinal scs , as there is a small overlap between the cells traced with lgr5creer and the cells traced with bmi1 , hopx , mter creer . further studies analyzing the rate of the drift toward monoclonality using lgr5+creer and the other + 4 creer will help to address this open question . although lgr5 can also mark colonic scs ( barker et al . , 2007 ) , the low frequency of lgr5-marked crypts due to the mosaic expression lgr5creer in the colon renders it difficult to ascertain whether there is one or more colonic scs contributing to the homeostasis and the regenerative potential of the colonic epithelium . the airway system is compartmentalized along the proximal distal axis into three anatomically distinct regions : the trachea and bronchi , the bronchioles , and the alveoli . the cellular composition of the lung epithelium varies in the different regions , as well as the cellular hierarchy that regulates the maintenance and repair of these epithelia ( rock and hogan , 2011 ) . the trachea and bronchi are pseudostratified epithelia containing ciliated cells , secretory cells ( clara cells and goblet cells ) , and basal cells ( 30% of tracheal epithelial cells ; fig . 4 a ) . the function of this first part of the airway tract is to allow the circulation of the air but also to protect the lung epithelium by absorbing dust particles and microbes that are constantly inhaled . through movement of the cilia , rare neuroendocrine cells are also dispersed in the luminal layer of the trachea , which regulate the contraction of the respiratory tracts as well as the secretion of the mucus ( rock and hogan , 2011 ) . ( a and b ) schematic representation of the airway epithelium that can be divided into trachea and bronchi , bronchioli , and alveoli . ( c f ) lineage tracing of tracheal basal cells using k5creer / rosa - lacz and analyzed 6 d ( c and e ) , 3 wk ( f ) , or 12 wk ( d ) after tam administration . paraffin sections of x - gal stained ( blue ) ( k5-labeled cells and their progeny ) and anti - acetylated tubulin ( brown , cilia ) , showing the initial labeling of basal cells ( c ) that give rise to luminal cells during postnatal development ( d ) . ( e and f ) lineage tracing of tracheal basal cells using k5creer / rosa - yfp and analyzed 3 and 15 wk after tam administration showing the initial labeling of basal cells ( green ) that give rise to clara cells ( red ) . ( g i ) lineage tracing of the clara cells in the bronchioli using scgb1a1creer / rosayfp mice induced at e18.5 and analyzed 2 d ( g ) , 3 wk ( h ) , or 1 yr ( i ) after induction . immunostaining of gfp ( green ) , pro - spc ( aec2 ) ( red ) , and scgb1a1 ( clara cells ) ( blue ) shows the long - term renewal of clara cells and the expansion of ciliated cells over time . inset in h shows labeled ciliated cells ( arrowheads ) , but no neuroendocrine cells ( red , arrow ) . the stable frequency of lineage - labeled aec2 cells over time ( 13% ) suggests that basc cells do not contribute to alveoli expansion during postnatal growth . aec1 , alveolar type i ; aec2 , alveolar type ii ; badj , bronchioalveolar duct junction ; basc , bronchioalveolar stem cell . bars : ( c and d ) 20 m ; ( e and f ) 25 m ; ( g ) 50 m . in contrast to the constant and rapid turnover occurring in the intestine or the skin epidermis , the airway system presents a very low renewal under steady - state conditions . for example , lineage tracing using foxj1-creer , which labeled ciliated cells , supports the view that ciliated cells are postmitotic with a half time of several months under homeostatic conditions ( rawlins et al . , 2007 ) . there is increasing evidence that basal cells function as multipotent scs in the trachea and bronchi , able to self - renew and differentiate into both clara cells and ciliated cells under steady - state conditions and after injury . the first evidence that basal cells contain multipotent scs came from lineage - tracing experiments using the k14-creer to label basal cells in the trachea and bronchi during naphthalene - induced epithelium regeneration , demonstrating the massive contribution of basal cells during trachea and bronchi regeneration ( hong et al . however , under physiological conditions most mouse basal cells express k5 , whereas only a subset of basal cells expresses k14 , which is up - regulated in the basal cell population upon injury . these studies did not assess the contribution of the basal cells under steady - state conditions . ( 2009 ) used a k5-creer to label basal cells of the upper airway tract postnatally under physiological conditions and demonstrated that basal cells contain self - renewing multipotent scs , giving rise to clara and ciliated cells during postnatal growth , adult homeostasis , and epithelial repair ( fig . lineage tracing using scgb1a1 ( also called secretoglobin 1a1 or cc10 ) creer mice , which specifically marked clara cells in the trachea and the main bronchi , demonstrated that some of these cells can undergo several rounds of replication giving rise to clara and ciliated cells . however , most of them are progressively lost and replaced over time by cells that are not marked by scgb1a1creer ( rawlins et al . , 2009 ) , suggesting that clara cells in the trachea and the main bronchi do not contain scs and behave as a transit - amplifying cell population . bronchioles lack basal cells but are surrounded instead by myofibroblast cells ( fig . 4 b ) . they are mainly composed of ciliated and secretory cells , and also contain clusters of neuroendocrine cells . lineage tracing of clara cells with scgb1a1-creer showed that clara cells from bronchioles self - renew over a long period of time and give rise to ciliated cells , during postnatal growth , homeostasis , and repair of the bronchiolar epithelium . this is consistent with the presence of bipotent scgb1a1-expressing scs , which ensure the homeostasis of the bronchioles ( fig . alveoli are the sites of exchange between the inhaled air and the gas of the blood . to maximize the surface of exchange between the blood and the air , the bronchioles end in multiple sacs , called alveoli , which are encased by a dense capillary network . the alveoli contain two major cell types : the alveolar type 1 cells , which are squamous cells that comprise the major surface of the lung and express different ion transporters critical for fluidity of the mucus produced , whereas alveolar type 2 cells are cuboidal cells that secrete the surfactant protein c ( spc ) essential to maintain the alveoli open . proliferation kinetic experiments suggested that the alveolar type 1 cells are terminally differentiated cells that do not divide , neither under physiological conditions nor during tissue regeneration . on the other hand , type 2 cells divide during homeostasis and repair , and were assumed to contain scs of the alveoli ( adamson and bowden , 1975 ) , although no lineage - tracing experiment has formally demonstrated this hypothesis . cells expressing both scgb1a1 ( the clara marker ) and pro - spc ( a marker of cuboidal alveolar type 2 cells ) have been described at the bronchioalveolar junction . these cells do not die upon injury , become proliferatively active , and exhibit sc properties in culture , and therefore were called bronchioalveolar scs ( kim et al . , 2005 ) . labeling of bronchiolar cells , including the bronchioalveolar scs , with the scgb1a1-creer demonstrated that these cells do not contribute to the alveoli during postnatal growth and after hyperoxia injury but rather contribute to the bronchiolar regeneration ( rawlins et al . , . however , a new study showed that cells labeled with the scgb1a1-creer contribute extensively to the alveoli regeneration after bleomycin - induced lung injury , suggesting that the contribution of clara cells to lung regeneration can vary depending on the type of injury ( rock et al . , 2011 ) . consistent with the role of scgb1a1 + cells during lung repair after bleomycin - induced injury , lineage tracing of alveolar type 2 cells using spc - creer demonstrated that spc - derived cells are replaced by spc - negative progenitors during lung repair after bleomycin inhalation ( chapman et al . , 2011 ) . k14-creer lineage tracing suggested that k14/k5-positive cells appear in the bronchioles a few days post - infection of h1n1 virus and give rise to migrating k5-positive clusters of cells at the sites of interbronchiolar lung damage ( kumar et al . , 2011 ) . do they arise from sgbd1a1 cells that begin to express k5/k14 upon injury or do they come from already k5-positive cells from the main bronchia ? lineage - tracing experiments in different epithelia reveal the coexistence of several types of scs in each epithelium . epithelia such as the epidermis and the intestine contain rapidly cycling scs , dividing asymmetrically at the level of the population but symmetrically at the sc level , which balanced renewal and differentiation stochastically . in addition , these tissues present a slower cycling population of cells that represent a reserve pool of scs in case of sudden need . more studies will be required to precisely determine how the equilibrium between these two pools of scs is achieved and what the molecular mechanisms are that allow the stochastic choice between renewal and differentiation . another theme that emerges from these lineage - tracing studies is the existence of both multipotent and unipotent scs maintaining the diversity of cell lineages found in these epithelia during postnatal life . further studies will be necessary to better understand when the switch from multipotency to unipotency occurs during morphogenesis , how the differentiation of these epithelial scs is controlled , and what the relative importance of intrinsic versus extrinsic determinants is in regulating their fate . new lineage - tracing studies will be required to identify new types of scs in the different epithelial tissues . for example , how are the esophagus , pancreas , bladder , prostate , and ovary developed , maintained , and repaired ? the identification of the different scs , transit - amplifying cells , and differentiated cells in these different epithelia will be instrumental to uncover the cell lineages at the origin of the different epithelial cancers ( visvader , 2011 ) . what is the respective importance of oncogenic mutations versus the cellular origin in dictating the tumor phenotypes ? also , the identification of cellular origin of the different epithelial cancers will allow one to define more precisely the transcriptional and genetic changes accompanying tumor initiation and to understand the molecular mechanisms that shape the fate of tumor - initiating cells . cancer scs have been identified in several human and mouse cancers based on their ability to reform primary tumors after transplantation into immunodeficient mice ( lobo et al . , 2007 ) . further studies will be required to demonstrate the existence of cancer scs during in vivo tumor growth in intact tissues using lineage - tracing and clonal analysis .

epithelia ensure many critical functions of the body , including protection against the external environment , nutrition , respiration , and reproduction . stem cells ( scs ) located in the various epithelia ensure the homeostasis and repair of these tissues throughout the lifetime of the animal . genetic lineage tracing in mice has allowed the labeling of scs and their progeny . this technique has been instrumental in characterizing the origin and heterogeneity of epithelial scs , their tissue location , and their differentiation potential under physiological conditions and during tissue regeneration .

Introduction The skin epidermis The mammary gland The gut The airway system Perspectives

epithelia are sheets of cells that constitute the lining of most of the organs of the body such as the skin , the digestive and respiratory tracts , and the urogenital system and separate the inside of the body from the outside ( blanpain et al . an epithelium can be composed of one ( simple epithelium ) or multiple layers of cells ( stratified epithelium ) , and forms the majority of the glands . a variety of specialized differentiated cells present in these epithelia ensure the important diversity of physiological functions they control . the skin epidermis acts as a barrier that keeps fluids in and protects the animal from the aggressions of the external environment . other epithelia , such as those of the airway tracts , renew much more slowly under physiological conditions but nevertheless can rapidly and extensively proliferate to repair tissue upon damage or injuries ( rock and hogan , 2011 ) . stem cells ( scs ) located in these different adult tissues are essential to sustain tissue turnover and repair these different epithelia upon injuries . inspired by the field of hematopoietic scs , the most common assay to assess the renewal and differentiation of putative epithelial scs is their transplantation into immunodeficient animals ( blanpain et al . although transplantation assays are very informative about the differentiation potential of scs , they do not necessarily reflect physiological conditions because scs are dissociated from their normal environment and very often transplanted into a heterotopic site , such as into the renal capsule ( the fibrous layer surrounding the kidney ) or the dermis , with or without their normal underlying mesenchyme . lineage tracing has now become the method of choice to study the renewal and the differentiation potential of scs in intact tissue , as this approach avoids the many drawbacks of transplantation experiments . in this technique , a particular cell lineage is labeled and the fate of the labeled cells and their progeny is analyzed over time . these experiments are usually performed in mice coexpressing two different transgenes : a cre recombinase expressed under a lineage - specific promoter , and a reporter gene only expressed when the cre removes a stop cassette that precedes the reporter gene . administration of the synthetic ligand induces activation of the inducible cre recombinase , which in turn induces the expression of the reporter gene in the cells expressing the cre and all their subsequent progeny , allowing the fate of labeled cells to be followed over time . the limitation of the inducible lineage tracing experiments is the identification of a specific promoter that targets the sc of interest and the mosaic expression of the reporter gene within the sc population . because there is an important heterogeneity within epithelial scs , one should always leave open the possibility of the existence of another population of epithelial scs not labeled by this approach . lineage - tracing experiments provide important information about the cellular hierarchy that governs epithelium development and homeostasis , and uncover the diversity of epithelial scs , their origin , tissue location , renewal , migration , and differentiation potential , as well as their ability to contribute to tissue repair and tumor initiation . different methods to perform lineage tracing in vertebrates and invertebrates have been recently thoroughly reviewed elsewhere ( buckingham and meilhac , 2011 ; kretzschmar and watt , 2012 ) . in this review , we discuss the novel insights into epithelial scs and their lineages that have been gathered thanks to the use of this technique in mice . the skin epidermis is composed of the interfollicular epidermis , which forms the skin barrier , and its various appendages such as the hair follicle ( hf ) , the sebaceous gland , and the sweat gland ( fig . , 2008 ) , which are specifically expressed in the hair germ , demonstrated that all hf lineages including adult hf scs and the sebaceous gland arise from embryonic progenitors expressing these two markers during hf morphogenesis . similar long - term labeling of the hf lineages has been obtained using lgr6creer lineage tracing at e17.5 , which is also expressed in embryonic hair germ , although its expression is broader and included cells of the interfollicular epidermis ( snippert et al . ( a ) schematic representation of the skin epidermis and the different epidermal scs : the interfollicular epidermis ( ife ) scs , the isthmus scs , and the bulge scs . ( b and c ) lineage tracing of bulge stem cells ( green ) using k19creer - rosayfp mice induced with 10 mg tamoxifen between d 21 and 25 , and analyzed 1 wk ( b ) or 5 wk ( c ) after induction . immunostaining of cd34 ( b ) or integrin 4 ( c ) and yfp show the initial labeling of cd34 + bulge sc ( b ) and the presence of yfp cells in the newly formed hair follicle 5 wk after ( c ) . ( d ) lineage tracing of basal cells ( green ) from the interfollicular epidermis using k14creer - rosayfp mice induced with 1 mg tamoxifen and analyzed 5 wk later . ( g ) lineage tracing using lgr6-gfp - irescreer / rosa - lacz mice induced at d 20 and analyzed 1 yr later , showing the labeling of the sebaceous gland , and demonstrating the existence of long - lived sc of the sebaceous gland . transplantation experiments suggested that the permanent portion of the hf called the bulge contains multipotent scs able to differentiate into all epidermal lineages including hf , sebaceous gland , and interfollicular epidermis upon transplantation with embryonic skin mesenchyme into immunodeficient mice ( rochat et al . , 2010 ) markers confirmed that bulge scs are indeed multipotent scs responsible for the maintenance of all hf lineages under physiological conditions ( fig . these results suggest that the heterogeneity in the differentiation potential of bulge scs is either related to an intrinsic heterogeneity of bulge cells containing multipotent and unipotent scs or through a regulation of the lineage differentiation potential of multipotent scs . similarly to the embryonic hf lineage tracing , lineage tracing of adult bulge scs also revealed that the hf lineages do not contribute to the homeostasis the interfollicular epidermis under physiological conditions ( morris et al . however , upon wounding , bulge scs are rapidly activated and actively participate in the repair of the damaged interfollicular epidermis ( fig . lineage tracing of hf matrix cells demonstrated that these cells are transient amplifying cells that differentiate into one of the six lineages depending on their spatial position within the matrix ( legu and nicolas , 2005 ; youssef et al . the long - term labeling of the sebaceous gland observed after lgr6 lineage tracing in adult skin demonstrates the presence of resident lgr6-expressing sebaceous gland scs ( fig . similar to bulge scs , lgr6-expressing isthmus scs are also recruited toward the interfollicular epidermis upon wounding and contribute to the repair of the damaged epidermis ( snippert et al . further studies will be required to better understand the respective contribution of bulge versus resident lgr6 + scs in maintaining the homeostasis of the sebaceous gland and defining the mechanisms leading to the recruitment and the migration of bulge scs to this gland . as previously mentioned , the interfollicular epidermis is maintained by a source of scs that is distinct from the hf lineages during postnatal physiological conditions . many small units of proliferation called epidermal proliferative units ( epus ) scattered all along the interfollicular epidermis ensure the constant turnover of the epidermis and its proper differentiation . based on morphological and proliferation studies , it has been proposed that the interfollicular epidermis is maintained by hexagonal shaped epus containing one sc and 10 transit - amplifying cells ( potten , 1974 , 1981 ) . however , genetic lineage tracing showed that the epu was not hexagonal in shape as suggested by histological analysis and was sometimes bigger than the 10 basal cells expected from the model based on morphological observations ( ro and rannala , 2004 , 2005 ) . mathematical modeling of these data suggests that the epidermis could be maintained by one type of progenitor , without the presence of transit - amplifying cells , and these progenitors undergo population asymmetric renewal by balancing asymmetric and symmetric cell division in a stochastic manner ( clayton et al . in summary , lineage - tracing experiments in the skin epidermis indicate the existence of multiple populations of stem cells that ensure the maintenance of different compartments of the epidermis and play distinct roles during homeostasis and repair . the epithelial part of the mammary gland comprises two main cell types , the basal myoepithelial cells and the luminal cells , which can differentiate either into ductal cells or milk - producing alveolar cells ( fig . more recently , lineage tracing of the mammary gland using inducible cre expressed either in myoepithelial cells ( k14- or k5-expressing cells ) or in luminal cells ( k8- or k18-expressing cells ) demonstrated that the mammary gland initially develops from multipotent embryonic k14-expressing progenitors , which give rise to both myoepithelial cells and luminal cells . further studies will be necessary to define the mechanisms that restrict the differentiation potential of myoepithelial scs under physiological conditions , and whether pathophysiological conditions can expand the differentiation potential of scs in the intact mammary gland . immunostaining of k14 ( c and d ) or k5 ( e ) ( red ) and yfp ( green ) demonstrates the labeling of unipotent scs that ensure myoepithelial lineage expansion during puberty and pregnancy . ( f h ) lineage tracing of luminal cells ( green ) using k8creer / rosayfp mice induced at the onset of puberty ( 4 wk old ) and analyzed 1 wk ( f ) or 10 wk later ( g ) , or during lactation ( h ) shows the labeling of unipotent scs that ensure luminal lineage expansion during puberty and pregnancy . the gut regulates the absorption of water , electrolytes , nutrients , and vitamins essential for the survival of the animal . the esophagus is a stratified epithelium resembling skin epidermis , whereas the stomach , intestine , and colon are simple epithelia composed of only one cell layer that contains the different cell lineages present in the different parts of the gut ( barker et al . in the corpus , the parietal cells secrete the acid necessary to activate digestion , while the mucus cells secrete a protective barrier and the enteroendocrine cells regulate gut contractility and the secretion of the various digestive enzymes . in the pylorus , the vast majority of the proliferating cells are located in the middle zone called the isthmus . lgr5 lineage tracing , which marked cells located at the bottom of the gland , demonstrated that the progeny of lgr5-expressing cells migrate into the isthmus region , proliferate , and give rise to all lineages of the stomach ( barker et al . villi are protrusions of the epithelium in the gut lumen that massively increase the surface area of the gut to allow for greater absorption . the intestine is one of the most rapidly proliferating tissues in the body , which completely self - renews in less than a week . several cell lineages are present in the intestine : the absorptive enterocytes are the most abundant cells , the goblet cells produce the mucus , and the enteroendocrine cells regulate the motility of the gut and the secretion of the digestive enzymes while the paneth cells residing at the base of the crypt produce microbicide proteins ( fig . based on proliferation kinetics experiments , it has been suggested that intestinal scs reside in the crypt at position + 4 with respect to the base of the crypt ( potten et al . ( a ) schematic representation of the intestinal epithelium lineages ( enterocytes , enteroendocrine , goblet , and paneth cells ) and its scs . ( b and c ) lineage tracing of lgr5 + scs ( blue ) using lgr5-gfp - irescreer / rosalacz mice analyzed 12 h ( b ) or 60 d ( c ) after induction demonstrates the initial labeling of columnar basal cells ( b ) that give rise to the differentiated cells of a villus ( c ) . staining of yfp ( green ) and of chromogranin a ( chga ) labeling enteroendocrine cells ( red ) ; and lysozyme ( lys ) antibody , labeling paneth cells ( red ) , showing the initial labeling of cells above the paneth cells ( d ) that give rise to the differentiated cells of a villus ( e ) . ( f and g ) lineage tracing of crypts using ah - creer / rosaconfetti mice analyzed 1 wk ( f ) or 8 wk ( g ) after induction , showing the initial multicolor labeling of the crypt and villus unit that progressively become monoclonal ( one color per crypt ) over time . the existence of multipotent scs in the intestine has been suggested by the presence of long - lived clones of marked cells that contain several types of differentiated intestinal cells ( bjerknes and cheng , 1999 ) . in contrast to the vast majority of wnt target genes , which are expressed throughout the crypt , including in the transit amplifying cells , lgr5 expression is restricted to crypt base columnar cells . lgr5creer - ires - gfp lineage tracing demonstrated that indeed lgr5-expressing crypt base columnar cells are rapidly cycling multipotent scs of the intestine giving rise to all cell lineages of the intestine including enterocytes , goblet cells , and neuroendocrine cells , as well as paneth cells ( fig . bmi1 , a polycomb repressor , is expressed in the proximal intestine and preferentially marks the cells located in position + 4 . 3 , d and e ; sangiorgi and capecchi , 2008 ) , suggesting the existence of two distinct classes of scs in the small intestine . interestingly , although the ablation of bmi1 + cells leads to the degeneration of the crypts ( sangiorgi and capecchi , 2008 ) , the ablation of lgr5 cells has no effect on intestinal homeostasis , as bmi1 + scs can compensate for the loss of lgr5 cells ( tian et al . under physiological conditions or after the destruction of lgr5 + cells similarly , mtert and hopx labeled cells also give rise to lgr5 + cells and conversely lgr5 cells can give rise to hopx cells ( takeda et al . another possibility is that the long - term lineage tracing in both models results from the labeling of the same multipotent intestinal scs , as there is a small overlap between the cells traced with lgr5creer and the cells traced with bmi1 , hopx , mter creer . , 2007 ) , the low frequency of lgr5-marked crypts due to the mosaic expression lgr5creer in the colon renders it difficult to ascertain whether there is one or more colonic scs contributing to the homeostasis and the regenerative potential of the colonic epithelium . the cellular composition of the lung epithelium varies in the different regions , as well as the cellular hierarchy that regulates the maintenance and repair of these epithelia ( rock and hogan , 2011 ) . ( a and b ) schematic representation of the airway epithelium that can be divided into trachea and bronchi , bronchioli , and alveoli . paraffin sections of x - gal stained ( blue ) ( k5-labeled cells and their progeny ) and anti - acetylated tubulin ( brown , cilia ) , showing the initial labeling of basal cells ( c ) that give rise to luminal cells during postnatal development ( d ) . ( e and f ) lineage tracing of tracheal basal cells using k5creer / rosa - yfp and analyzed 3 and 15 wk after tam administration showing the initial labeling of basal cells ( green ) that give rise to clara cells ( red ) . ( g i ) lineage tracing of the clara cells in the bronchioli using scgb1a1creer / rosayfp mice induced at e18.5 and analyzed 2 d ( g ) , 3 wk ( h ) , or 1 yr ( i ) after induction . however , under physiological conditions most mouse basal cells express k5 , whereas only a subset of basal cells expresses k14 , which is up - regulated in the basal cell population upon injury . ( 2009 ) used a k5-creer to label basal cells of the upper airway tract postnatally under physiological conditions and demonstrated that basal cells contain self - renewing multipotent scs , giving rise to clara and ciliated cells during postnatal growth , adult homeostasis , and epithelial repair ( fig . lineage tracing using scgb1a1 ( also called secretoglobin 1a1 or cc10 ) creer mice , which specifically marked clara cells in the trachea and the main bronchi , demonstrated that some of these cells can undergo several rounds of replication giving rise to clara and ciliated cells . , 2009 ) , suggesting that clara cells in the trachea and the main bronchi do not contain scs and behave as a transit - amplifying cell population . lineage tracing of clara cells with scgb1a1-creer showed that clara cells from bronchioles self - renew over a long period of time and give rise to ciliated cells , during postnatal growth , homeostasis , and repair of the bronchiolar epithelium . this is consistent with the presence of bipotent scgb1a1-expressing scs , which ensure the homeostasis of the bronchioles ( fig . proliferation kinetic experiments suggested that the alveolar type 1 cells are terminally differentiated cells that do not divide , neither under physiological conditions nor during tissue regeneration . on the other hand , type 2 cells divide during homeostasis and repair , and were assumed to contain scs of the alveoli ( adamson and bowden , 1975 ) , although no lineage - tracing experiment has formally demonstrated this hypothesis . labeling of bronchiolar cells , including the bronchioalveolar scs , with the scgb1a1-creer demonstrated that these cells do not contribute to the alveoli during postnatal growth and after hyperoxia injury but rather contribute to the bronchiolar regeneration ( rawlins et al . in addition , these tissues present a slower cycling population of cells that represent a reserve pool of scs in case of sudden need . further studies will be necessary to better understand when the switch from multipotency to unipotency occurs during morphogenesis , how the differentiation of these epithelial scs is controlled , and what the relative importance of intrinsic versus extrinsic determinants is in regulating their fate . new lineage - tracing studies will be required to identify new types of scs in the different epithelial tissues . the identification of the different scs , transit - amplifying cells , and differentiated cells in these different epithelia will be instrumental to uncover the cell lineages at the origin of the different epithelial cancers ( visvader , 2011 ) .

mast cells are key agents in the direction of inflammatory processes throughout most tissues , especially at the mucosal interface . immediately identifiable consequences of mast cell activity are the symptoms of atopic disease : itching , sneezing , and allergic asthma . mast cells enact these processes through degranulation of stored histamine , various proteases , and the secretion of prostaglandins and leukotrienes . mast cells further affect inflammatory responses by the later ( hours ) de novo production and secretion of a plethora of cytokines , chemokines , and growth factors , including tnf- , gm - csf , il-4 , -6 , -13 , mip-1 , mcp-1 , and vegf . thus mast cells also act as effectors of other cellular responses , e.g. , maintenance of ige production , and the activities of regulatory t cells ( treg ) and myeloid derived suppressor cells ( mdsc ; gauchat et al . , 1993 ; frandji et al . , 1996 ; pawankar et al . , 1997 ; lu et al . , 2006 ; 2010b ) . notwithstanding their very important and deeply studied roles in atopy and parasite infections , mast cells are now implicated in the inflammatory responses associated with autoimmune disease , atherosclerosis , and resistance to bacterial infection ( levi - schaffer et al . , 2000 ; hara et al . , 2002 ; lee et al . , 2002 ; mclachlan et al . , this multifunctional role in the immune response makes mast cell biology a critical area of research that could greatly change the treatment of inflammatory diseases . investigation of signals proximal to the high affinity ige receptor , fcri , has been a topic of great interest due to the importance of ige in allergic pathologies . central points from these observations are that src family kinases lyn and fyn are critical to ige - mediated mast cell activation , and that the signal transducer and activator of transcription ( stat ) 5 transcription factor mediates mast cell vitality and function , particularly in the late phase of cytokine production . knowledge gained from fcri signaling could prove valuable in the setting of igg - mediated inflammation , which is the foundation for many autoimmune diseases , including lupus and rheumatoid arthritis . once deposited either as anti - host antibodies or as an immune complex these are expressed as pro - inflammatory ( fcri , fcriia / c , fcriiia / b , fcriv ) or anti - inflammatory ( fcriib ) receptors , some in a species - restricted manner , e.g. , fcriv in mice only , as discussed later . macrophages , basophils , neutrophils , and mast cells are activated by fcr signals , often via fcriii , with counter - regulation by fcriib ( malbec and daeron , 2007 ; nimmerjahn and ravetch , 2008 ) . understanding signals emanating from these receptors will offer new treatments for igg - mediated diseases . here . we will also describe interplay with regulatory cytokines il-4 , il-10 , and tgf1 , and the potential signaling overlap between ige and igg receptors in mast cells . mast cell activation and ige are almost synonymous topics that have been studied in - depth . an early phase of degranulation and eicosanoid secretion , and a late phase inflammatory cytokine production , are both potently stimulated by multivalent interactions between fcri - bound ige molecules and antigen at the mast cell surface . as a result , the crosslinking of these receptors into lipid raft domains brings into proximity protein tyrosine kinases ( ptk ) that may or may not be pre - associated with fcri . this makes for a ripe setting of immunoreceptor tyrosine - based activation motif ( itam ) trans - phosphorylation ( pribluda et al . , 1994 ) and further recruitment of other ptk , via src homology 2 ( sh2 ) domains , that propagate the signal . crosslinking of fcri activates a variety of src family kinases ( eiseman and bolen , 1992 ; gilfillan and rivera , 2009 ) , this review focuses on two wings of signaling that emerge from such an event . first , the stimulation of tyrosine kinase syk through activity of lyn ( a src family kinase ) ; syk phosphorylates linker of activated t cells ( lat ) which in turn discharges its duty as a sh2-based scaffold , eventually stimulating extracellular signal - regulated kinases ( erk ) and de novo transcription ( rivera and gilfillan , 2006 ; gilfillan and rivera , 2009 ) . the second pathway involves the activity of another src family kinase , fyn . through the grb2 associated binding protein ( gab ) 2 , fyn elicits signal transduction down the canonical pi3k - akt axis ( kim et al . , fyn is also essential for early phase degranulation of mast cells through the transient receptor potential channel 1 ( trpc1 ) , a non - selective cation channel that is required for ca influx , f - actin depolymerization , and resultant release of packed granules ( suzuki et al . , 2010 ) . both lyn and fyn are positive mediators of fcri activity ( parravicini et al . , 2002 ; however , a notable distinction exists in that lyn also contributes to the abatement of ige - mediated signaling ( hernandez - hansen et al . , 2004 ; odom et al . , 2004 ; kalesnikoff and galli , 2008 ) . beyond its role in syk recruitment , lyn influences several inhibitory proteins , including ship-1 , shp-2 , dok-1 , and cbp , leading to decreased signaling in the pi-3 kinase and the ras - mapk cascades ( satterthwaite et al . , 1998 ; takeshita et al . , 1998 ; lyn induces the activity of c - terminal src kinase ( csk ) by directly phosphorylating the membrane - anchored csk - binding protein ( cbp ; odom et al . , 2004 ; kovarova et al . , 2006 ) , also known as phosphoprotein associated with glycosphingolipid - enriched microdomains ( pag ) . this event places csk in close proximity to the fcri - associated lyn and fyn . two groups independently uncovered cbp / pag as the essential factor potentiating csk localization at the plasmalemma ( brdicka et al . cbp is ubiquitously expressed and constitutively tyrosine phosphorylated in some cell types ; with a number of sh2-mediated binding interactions occurring as a result of this ( brdicka et al . , 2000 ) . most notable is a positive interaction with csk upon phosphorylation of cbp y314 in rat ( takeuchi et al . , 2000 ) and y317 in human ( brdicka et al . , 2000 ) . the functional relationship between src family kinases and cbp was first defined in t cells and , raji and jurkat cell lines . csk and cbp are pre - associated in resting t - cells , however during t cell receptor ( tcr ) aggregation cbp is swiftly dephosphorylated , releasing csk , resulting in the delocalization of the latter from the membrane ( brdicka et al . , 2000 ) ; interestingly the same study demonstrated an association between fyn and cbp independent of an sh2 interaction ( brdicka et al . , 2000 ) . indeed in the setting of t cell activation , fyn eventually rephosphorylates cbp , reinstating the csk elicited brake on src family kinase ( lindquist et al . , 2003 ) . however , in the context of mast cells and fcri , csk recruitment / activity through cbp seems to be more a function of post - aggregation lyn activity ( honda et al . , 1997 ; ohtake et al . , 2002 ) , with lyn knockouts possessing un - phosphorylated cbp , therefore non - functional csk ( odom et al . , 2004 ) . moreover , unlike resting t cells , cbp is not constitutively active in mast cells ( ohtake et al . , 2002 ; importantly , genetic background has been shown to be key to the negative responses associated with lyn . it has been postulated that this could be due to a lyn : fyn balance , e.g. , c57bl/6 mice express significantly less fyn than the 129/sv strain . lyn null bone marrow - derived mast cells ( bmmc ) from the c57bl/6 strain have an impaired degranulation response to antigen , while a similar knock - out ( ko ) in 129/sv mast cells results in hyperdegranulation . in contrast , lyn deletion in either strain enhances ige - mediated cytokine production , revealing lyn as a negative regulator ( yamashita et al . , 2007 ) . as no similar negative roles for fyn have been observed in mast cells , one could hypothesize that increased fyn kinase , e.g. , on the 129/sv background , predisposes to fcri hyperactivity when the negative effects of lyn are absent . yet other work has demonstrated that the function of lyn changes with the intensity of ige - antigen stimulus on mast cells of similar background . low intensity receptor activation induces a positive signaling role for lyn , leading to eventual activation of akt and p38 , while high intensity activation of fcri elicits negative responses ( xiao et al . , 2005 ) . this bears resemblance to the dual roles of fyn in the context of tcr signaling . the stat family of transcription factors is best known as containing the intermediaries of cytokine and growth factor signals . typically , aggregating cytokine receptor subunits with their associated janus ( jak ) tyrosine kinases induce jak auto- or trans - phosphorylation and subsequent receptor phosphorylation , yielding sh2 binding sites to which stats dock . generally , stats are then targeted by the jak kinases , eliciting phosphotyrosine ( py)-induced stat dimers that shuttle into the nucleus and stimulate target gene transcription ( schindler et al . , 2007 ) . in mast cells one such process is the induction of stat5 phosphorylation by stem cell factor ( scf)/c - kit , or il-3 , via jak2 activation ( mui et al . , 1995 ; ryan et al . , 1997 ; one or both of these cytokines are essential for the development and survival of mast cells in vitro and in vivo . a series of work by our group and others has established the critical part fulfilled by stat5 in mast cell development and survival . in particular , stat5a appears to be more important for survival as demonstrated by dominant negative and gene ko approaches ( shelburne et al . bone marrow devoid of stat5a has markedly higher rates of apoptosis and lesser proliferation compared to wild type when cultured in il-3 or scf alone . it should be noted , however , in vitro mast cell development can be achieved by dual stimulation with il-3 + scf ( shelburne et al . , this could be due to the overlap of stat signaling , particularly from stat3 ( sonnenblick et al . , 2005 ) or stat6 ( malaviya and uckun , 2002 ) . in vivo , mast cells are present at birth , but can not be detected in total stat5 ko mice by 12 weeks of age ( shelburne et al . , stat5 ko bmmc have an attenuated yet functional early activation response , measured by histamine and leukotriene b4 release , but a near complete inability to induce cytokine production the hallmark of the late phase ( barnstein et al . , 2006 ) . instead , stat5 appears to inhibit mrna - destabilizing factors as one mechanism to regulate mast cell cytokines . the expression of one such protein , tristetrapolin ( ttp ) , is enhanced in stat5 ko bmmc during fcri crosslinkage ; inhibiting ttp partially restored cytokine production ( barnstein et al . , the potential effects of stat5 on other rna binding factors such as hur ( human antigen r ) , remains as yet unknown . until recently , the mechanism by which fcri stimulates phosphotyrosine ( py)-stat5 also remained unknown . through em co - localization studies ( similar to those in wilson et al . , 2007 ; xue et al . , 2007 ; andrews et al . , 2008 ) clustering of stat5 with fcri upon antigen exposure has been observed . our group has found that fyn is the indispensible kinase eliciting py - stat5 proximal to fcri in an ige - dependent , il-3/scf - independent manner ( pullen et al . , 2012 ) . stat5 and fyn were found to co - immunoprecipitate in resting mast cells , an association that dissipated with ige - antigen stimulation . surprisingly , knock out of gab2 the scaffolding protein linking fyn to pi3k signaling allowed a slight enhancement in ige - mediated py - stat5 . deficiency of sh2 tyrosine phosphatase ( shp ) -1 , a known gab2 associate , replicates the gab2 ko phenotype to a lesser extent ( pullen et al . , 2012 ) . these data with other accumulating evidence in non - mast cell studies ( yu et al . , 2002 ; kim et al . , 2009 ) , which can also include shp-2 , paint a more complicated picture of the role of gab2 . it is interesting to note that the latter work ( kim et al . , 2009 ) furthermore , a direct association between stat3 and gab2 has been reported ( ni et al . , 2007 ) , warranting its examination in mast cells in consideration of signal overlap with stat5 . in our hands , lyn suppression enhances py - stat5 in a manner similar to gab2 and shp-1 loss . this is in keeping with the known negative effects of lyn , where its deficiency on a c57bl/6 background enhances stat5 activation . while lyn and fyn offer a means of fine - tuning ige signaling , external regulation of the ige receptor can be achieved through cytokines , primarily tgf1 , il-10 , and il-4 . tgf1 is a multipotent cytokine regulating tissue repair , inflammation , proliferation , migration , and angiogenesis ( kehrl et al . , 1986 ; yang et al . it is linked to autoimmune diseases and cancer ( letterio and roberts , 1998 ; blobe et al . , 2000 ) , and recently shown to have a critical role in treg and th17 differentiation ( bettelli et al . , 2006 ; lee et al . , 2009 ; ziegler and buckner , 2009 ) . mast cells evoke tgf1-mediated paracrine signaling , by secreting tgf1 as well as proteases that activate its latent form ( pennington et al . il-10 belongs to a family of related cytokines including il-19 , il-20 , il-22 , il-24 , and il-26 ( kotenko , 2002 ) . cells involved in the allergic response , including th2 cells and mast cells , can produce il-10 ( moore et al . the well - documented ability of il-10 to inhibit macrophage activation and antigen presenting function suggests an important role in regulating the immune response . this is clearly demonstrated in il-10-deficient mice , which develop a severe autoimmune phenotype ( kuhn et al . , 1993 ) , as il-10-deficient dendritic cells are highly potent activators of th1 responses ( he et al . , 2005 ) . the presence of il-10 has been shown to regulate inflammatory responses to pathogen infection ( gazzinelli et al . , 1996 ; hunter et al . , 1997 ) , protect from endotoxic shock ( howard et al . , 1993 ; berg et al . , 1995 ) , and control both acute and chronic inflammatory responses ( roers et al . , 2004 ) clinically , il-10 inhibits progression of rheumatoid arthritis when delivered directly to the site of inflammation ( trachsel et al . , 2007 ) . as such , il-10 is being further studied as an inflammatory therapeutic ( murray , 2006 ) . our group and others have found that tgf1 and il-10 suppress mast cell function and survival , offering homeostatic feedback regulation ( bissonnette et al . , 1997 ; miller et al . , 1999 ; gomez et al . , 2005b ; norozian et al . , 2006 ; pemberton et al . , 2006 ; wiener et al . , 2007 ; woodman et al . , 2008 ) , 1989 ; toyota et al . , 1995 ) and blocked their rescue from apoptosis by scf ( mekori and metcalfe , 1994 ) . bissonnette et al . ( 1997 ) reported that antigen - induced histamine and tnf production from rat peritoneal mast cells were inhibited by tgf1 , whereas meade et al . ( 1992 ) showed that in vivo treatment with tgf1 blunted ige - mediated , mast cell - dependent , immediate hypersensitivity responses in mice . these effects could be partly due to tgf1-mediated suppression of fcri expression ( gomez et al . , 2005b ) . despite these studies supporting the theory that tgf1 inhibits mast cell function , contradicting evidence exists . this includes a report by kim and lee ( 1999 ) that tgf1 potentiates ige - dependent cutaneous anaphylaxis in rats . like tgf1 , il-10 can inhibit fcri expression and ige - mediated cytokine production in mast cells ( marshall et al . our recent work showed that il-10 is produced endogenously during mast cell development , and suppresses mast cell numbers and fcri expression ( speiran et al . , 2009 ) . exogenous il-10 can inhibit the differentiation and survival of immature mouse mast cells , eliciting apoptosis via reduction of c - kit signaling , caspase activation , and damage to the mitochondria ( bailey et al . we have investigated the means by which il-10 and tgf1 suppress mast cell activation . in recent work , we found that adding exogenous il-10 or tgf1 to mast cells leads to a gradual decline in fyn and stat5 protein levels , reaching its nadir in 34 days ( kennedy norton et al . , 2008 and data not yet published ) . this slow onset could be argued to allow protective mast cell functions prior to reinstating immune homeostasis . by targeting the fyn - stat5 pathway , these cytokines effectively disrupt mast cell cytokine production and survival . our most recent findings show that a set of micrornas ( mirs ) is regulated by il-10 and tgf1 ; mirs that could target fyn and stat5 , based on in silico analyses . we look forward to unraveling this area , in hopes of providing clues to molecular therapies targeting mast cells . il-4 is a pleiotropic cytokine that participates in driving the polarization of t cells toward a th2 phenotype by inhibiting ifn- production and inducing further production of il-4 by t cells ( foster et al . il-4 signals by binding to the il-4r chain , present as a heterodimer , paired with either the common gamma chain or the il-13r1 , creating the type i and type ii receptors , respectively . il-4 plays an important role in the development of asthma by supporting the early development of th2 cells and promoting b cell isotype switching to ige . along with il-13 , il-4 contributes to the pathophysiology of asthma by inducing bronchial hyperresponsiveness and goblet cell mucus production ( wills - karp and finkelman , 2008 ) . like il-10 , il-4 can be secreted by mast cells ( brown and hanifin , 1989 ) , and is produced constitutively in cultures of developing mast cells ( speiran et al . , 2009 ) . the combination of exogenous il-4 and il-10 can inhibit mast cell differentiation and survival ( yeatman et al . , 2000 ; bailey et al . , 2004 ; bouton et al . , 2004 ) through mechanisms moreover , deletion of il-4 [ or il-10 ] enhanced bmmc growth and fcri expression ( speiran et al . , 2009 ) . also similar to il-10 and tgf1 , il-4 acts on mature mouse mast cells to decrease fcri expression and signaling through a stat6 dependent pathway ( ryan et al . , 1998 ) however , il-4 has been repeatedly shown to have an enhancing effect on human fcri expression ( toru et al . , 1996 ; bischoff et al . , 1999 ; lorentz and bischoff , 2001 ; macey et al . , for this reason , il-4 and its receptor have been attractive candidates for the treatment of asthma . unfortunately , therapies targeting il-4 have not yielded promising results due to possible redundancy from il-13 through the type ii receptor . currently in clinical trials is a monoclonal antibody targeting il-4r , an antisense oligonucleotide that prevents translation of the il-4r chain , and an il-4 mutein that interferes with the binding of endogenous il-4 ( nguyen and casale , 2011 ) . tgf1 antagonizes il-4 signaling in mast cells , by down - regulating il-4 receptor expression and suppressing stat6 activation ( macey et al . , 2010 ) . tgf1 has also been shown to prevent il-4 expression , by inducing the adaptor protein nedd4 family - interacting protein 1 ( ndfip1 ) in treg . ndfip1 participates in the ubiquitination of the jun family transcription factors needed for il-4 transcription ( beal et al . , we found that il-4 suppresses tgf receptor expression and signaling in mast cells , showing these two cytokines are able to fine tune mast cell responses ( macey et al . as discussed earlier , mast cell activation can be achieved through a variety of stimuli , ranging from cytokines to allergen - specific immunoglobulins . in allergic and inflammatory pathologies , allergen - specific ige crosslinks mast cell fcri and triggers the activation of intracellular signaling molecules , ultimately resulting in mediator release and chemotaxis . however in addition to fcri , mast cells express the igg receptor family ( fcr ) . of the high - affinity igg receptors , both humans and mice possess fcri , while fcriv is restricted to mice . the low affinity fcrii and fcriii , are found in humans and mice . however , there are three fcrii ( a , b , and c ) and two fcriii ( a and b ) in humans , whereas mice have one receptor of each type ( fcriib and fcriiia ; nimmerjahn and ravetch , 2008 ) . further complicating receptor distribution among species is evidence that receptor type expression is also variable according to mast cell phenotype ( benhamou et al . , 1990 ) , e.g. , peritoneal vs. bmmc , along with the profile of mediator release ( katz et al . , 1992 ) , e.g. , leukotriene c4 vs. prostaglandin d2 . activating fcr on mouse cells consist of a ligand - binding alpha - chain ( ) and a signal - transducing gamma - chain ( ) bearing itams , which once phosphorylated upon receptor crosslinking , act as docking sites for signaling molecules such as syk kinase ; this results in the activation of signaling pathways . in contrast , ligand binding can also result in downregulation of antigen - dependent mediator release due to the existence of the immunoreceptor tyrosine - based inhibitory motifs ( itims ) on the cytosolic portion of the fcriib -chain . phosphorylation of itims upon fcriib ligation induces the activation of lyn kinase , which can recruit and activate phosphatases like ship in addition to directly acting upon itims , leading to decreased mediator release ( fong et al . , 1996 ; ono et al . , 1996 ) fcriib inhibits several aspects of mast cell and basophil functions such as ige - dependent degranulation and il-4 production ( daron et al . , 1993 , 1995 ) . in addition to mast cells , fcriib is expressed by other immune cells including dendritic cells , macrophages , activated neutrophils , basophils , and b cells . when expressed by these cells , fcriib inhibits the functions of activating fcrs , e.g. , phagocytosis and pro - inflammatory cytokine release ( daron et al . , 1993 , 1995 ; nimmerjahn and ravetch , 2008 ) . fc receptors share the common gamma subunit with fcri , and not surprisingly , similar signaling pathways ( kurosaki et al . , 1992 ; although the importance of syk kinase in igg signaling has been demonstrated ( crowley et al . little is known about src family kinase functions in fcr - mediated mast cell activation . our latest data demonstrates fcr - induced mast cell activation of fyn and lyn kinases , and downstream initiation of the pi3k - akt , erk , p38 , and jnk pathways ( falanga et al . , 2012 ) . in the same report , it was revealed that fyn and lyn kinases have opposing roles upon fcr crosslinking , since fyn - deficient ( ko ) mast cells displayed decreased degranulation and mediator release , whereas an exacerbated response was observed with lyn ko mast cells . this demonstrates that fcr - induced mast cell mediator release is regulated in a fyn- and lyn - dependent manner . in pathologic and extremely rare conditions , mast cell activation with antigen - specific ige historically , the onset and regulation of anaphylaxis was exclusively attributed to the generally known classical pathway of anaphylaxis involving ige - mediated fcri mast cell activation and subsequent histamine release ( miyajima et al . , 1997 ; however , elegantly conducted research on murine models demonstrated that an anaphylactic reaction can be induced through an igg - fcr pathway ( strait et al . , 2002 ; obata et al . , 2007 ; tsujimura et al . , 2008 ; in this alternative pathway , activation of basophils and macrophages ( fcri and fcriii ; strait et al . , 2002 ; obata et al . , 2007 ; tsujimura et al . , 2008 ) , , 2011a ) elicits anaphylactic symptoms , including a drop in core body temperature . although mast cell contribution in igg - mediated anaphylaxis was insignificant as mast cell - deficient mice still developed igg - induced anaphylaxis via platelet activating factor ( paf ) recent data demonstrate that mast cells account for the majority of circulating histamine during fcr - induced passive systemic anaphylaxis ( psa ) , which is regulated by fyn and lyn ( falanga et al . , 2012 ) . additionally , mast cells have recently been reported to be responsible for fcriia - dependent cutaneous anaphylaxis whereas monocytes / macrophages and neutrophils were found to be involved in fcriia - dependent systemic anaphylaxis ( jonsson et al . , 2011b ) . taken together , these findings bring new insights to the function of fyn and lyn kinases downstream of fcr stimulation and the role of mast cells in igg - mediated pathologies . exploration of the biological chemistry behind igg signaling will be an important activity contributing to efforts in understanding igg - mediated hypersensitivity disorders . lessons from fcri studies serve as a valuable model offering clues in this pursuit . we posit that the fyn / stat5 pathway will be an important regulator in igg - mediated mast cell pathology , and here we have reviewed key areas of ige - mediated signaling where igg cues are likely to intersect , summarized by figure 1 . the similarities also offer attractive lines of study with respect to the interplay of igg and other exogenous signals such as il-4 , il-10 , and tgf1 , which can have their own effects on fyn and stat5 . notwithstanding this potential , there are still many avenues of investigation that need attention . overlap in fcri , fcriii , and fcriib signaling . ige - bound multivalent antigen or igg immune complexes both positively influence downstream signaling through syk and lat , but lyn also produces negative feedback by phosphorylating itims and by inducing the csk recruitment via sh2 interactions with phosphorylated cbp / pag . fyn activates stat5 , transcription factors that are required for mast cell survival ( stat5a ) and cytokine production ( stat5b ) . regulatory cytokines such as il-10 and tgf1 can inhibit fyn and stat5 production , possibly through the induction of specific mirs . first is the need for a more precise dissection of stat5 activation and mobilization as an effect of ige and igg receptor crosslinking . stat5 is actually represented by two similar but distinct proteins : stat5a and stat5b ( grimley et al . , 1999 ) . as mentioned earlier stat5a is important for the development and proliferation of mast cells ( shelburne et al . , 2003 ) , whereas stat5b has been identified as the regulator of cytokine production ( pullen et al . , 2012 ) . thus far these roles do not appear interchangeable , e.g. , stat5a depletion does not significantly affect ige - mediated cytokine production . while the role of stat5b in mast cell development has not been directly addressed , its impact is likely redundant since total stat5ko bone marrow can be rescued by reconstituting stat5a ( shelburne et al . , one question needing pursuit asks if there is a specificity for which stat5 isoform associates with fyn and fcri . furthermore , which stat5 mobilizes to the nucleus when , and what are their targets ? complicating this matter is the fact that in addition to tyrosine at least two serines are phosphorylated , and the function of each phosphorylated residue could possibly diverge between each stat5 . for example , phosphorylation of stat5a serines 725 and 779 has been identified as a requirement for leukemogenic transformation ( friedbichler et al . , 2010 ) , and there are slight primary sequence variations in the location of the known target serines and tyrosine between stat5a and stat5b . second is the need to further dissect the roles of other fc receptor signal mediators . for example , double ko of the tec kinases itk and btk was found to inhibit degranulation / histamine release and impaired mast cell granularity ( iyer et al . , 2011 ) . interestingly , in the same work c - kit expression and mast cell development were not affected , however the double kos showed enhanced proliferation . furthermore , fcri and the fcr share common -chains containing itams that , when phosphorylated , can recruit syk ( crowley et al . , 1997 ; lach - trifilieff et al . , 2000 ; nimmerjahn and ravetch , 2008 ) . in fcri signaling , the activation of syk through lyn is a well - known effector of mast cell function ; if this happens in the context of fcr crosslinking is open for investigation . the association of lyn [ and fyn ] with fcri is an enigmatic matter , as it is known to be dynamic and possibly compartmentalized ( gilfillan and rivera , 2009 ) . this in combination with the diversity of igg receptors on mast cells suggests that , while the high affinity ige receptor serves as a good model , there is much more to these signaling stories than is currently understood . for example , additional exploration of fcr expression profiles in mast cells of different sources , phenotypes , and species will be important for interpreting data . finally , unraveling these discrete biochemical questions should be matched with in vivo studies of stat5 functions in allergic and autoimmune diseases , therapies targeting mast cell activation and secreted mediators have proven effective in allergic disease . if the fyn - stat5 pathway operates as a key signaling pathway as we hypothesize , selective targeting of this axis could prove therapeutic to many disorders . these clinical goals are critical , and should come as a natural product of efforts to understand the fundamentals of immune homeostasis . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .

mast cells are central players in immune surveillance and activation , positioned at the host environment interface . understanding the signaling events controlling mast cell function , especially those that maintain host homeostasis , is an important and still less understood area of mast cell - mediated disease . with respect to allergic disease , it is well established that ige and its high affinity receptor fcri are major mediators of mast cell activation . however , igg - mediated signals can also modulate mast cell activities . signals elicited by igg binding to its cognate receptors ( fcr ) are the basis for autoimmune disorders such as lupus and rheumatoid arthritis . using knowledge of ige - mediated mast cell signaling , recent work has begun to illuminate potential overlap between fcri and fcr signal transduction . herein we review the importance of src family kinases in fcri and fcr signaling , the role of the transcription factor stat5 , and impingement of the regulatory cytokines il-4 , il-10 , and tgf1 upon this network .

Introduction Src Family Kinases are Integral to IgE Signaling STAT5 Activity is Modulated by FcRI in a Fyn-Dependent Manner Cytokine Regulation of FcRI Signaling IgG-Mediated Mast Cell Activity Closing Remarks Conflict of Interest Statement

mast cells are key agents in the direction of inflammatory processes throughout most tissues , especially at the mucosal interface . immediately identifiable consequences of mast cell activity are the symptoms of atopic disease : itching , sneezing , and allergic asthma . mast cells enact these processes through degranulation of stored histamine , various proteases , and the secretion of prostaglandins and leukotrienes . mast cells further affect inflammatory responses by the later ( hours ) de novo production and secretion of a plethora of cytokines , chemokines , and growth factors , including tnf- , gm - csf , il-4 , -6 , -13 , mip-1 , mcp-1 , and vegf . thus mast cells also act as effectors of other cellular responses , e.g. , maintenance of ige production , and the activities of regulatory t cells ( treg ) and myeloid derived suppressor cells ( mdsc ; gauchat et al . notwithstanding their very important and deeply studied roles in atopy and parasite infections , mast cells are now implicated in the inflammatory responses associated with autoimmune disease , atherosclerosis , and resistance to bacterial infection ( levi - schaffer et al . , this multifunctional role in the immune response makes mast cell biology a critical area of research that could greatly change the treatment of inflammatory diseases . investigation of signals proximal to the high affinity ige receptor , fcri , has been a topic of great interest due to the importance of ige in allergic pathologies . central points from these observations are that src family kinases lyn and fyn are critical to ige - mediated mast cell activation , and that the signal transducer and activator of transcription ( stat ) 5 transcription factor mediates mast cell vitality and function , particularly in the late phase of cytokine production . knowledge gained from fcri signaling could prove valuable in the setting of igg - mediated inflammation , which is the foundation for many autoimmune diseases , including lupus and rheumatoid arthritis . macrophages , basophils , neutrophils , and mast cells are activated by fcr signals , often via fcriii , with counter - regulation by fcriib ( malbec and daeron , 2007 ; nimmerjahn and ravetch , 2008 ) . understanding signals emanating from these receptors will offer new treatments for igg - mediated diseases . we will also describe interplay with regulatory cytokines il-4 , il-10 , and tgf1 , and the potential signaling overlap between ige and igg receptors in mast cells . mast cell activation and ige are almost synonymous topics that have been studied in - depth . an early phase of degranulation and eicosanoid secretion , and a late phase inflammatory cytokine production , are both potently stimulated by multivalent interactions between fcri - bound ige molecules and antigen at the mast cell surface . as a result , the crosslinking of these receptors into lipid raft domains brings into proximity protein tyrosine kinases ( ptk ) that may or may not be pre - associated with fcri . crosslinking of fcri activates a variety of src family kinases ( eiseman and bolen , 1992 ; gilfillan and rivera , 2009 ) , this review focuses on two wings of signaling that emerge from such an event . first , the stimulation of tyrosine kinase syk through activity of lyn ( a src family kinase ) ; syk phosphorylates linker of activated t cells ( lat ) which in turn discharges its duty as a sh2-based scaffold , eventually stimulating extracellular signal - regulated kinases ( erk ) and de novo transcription ( rivera and gilfillan , 2006 ; gilfillan and rivera , 2009 ) . the second pathway involves the activity of another src family kinase , fyn . through the grb2 associated binding protein ( gab ) 2 , fyn elicits signal transduction down the canonical pi3k - akt axis ( kim et al . , fyn is also essential for early phase degranulation of mast cells through the transient receptor potential channel 1 ( trpc1 ) , a non - selective cation channel that is required for ca influx , f - actin depolymerization , and resultant release of packed granules ( suzuki et al . , 2002 ; however , a notable distinction exists in that lyn also contributes to the abatement of ige - mediated signaling ( hernandez - hansen et al . beyond its role in syk recruitment , lyn influences several inhibitory proteins , including ship-1 , shp-2 , dok-1 , and cbp , leading to decreased signaling in the pi-3 kinase and the ras - mapk cascades ( satterthwaite et al . the functional relationship between src family kinases and cbp was first defined in t cells and , raji and jurkat cell lines . indeed in the setting of t cell activation , fyn eventually rephosphorylates cbp , reinstating the csk elicited brake on src family kinase ( lindquist et al . however , in the context of mast cells and fcri , csk recruitment / activity through cbp seems to be more a function of post - aggregation lyn activity ( honda et al . moreover , unlike resting t cells , cbp is not constitutively active in mast cells ( ohtake et al . lyn null bone marrow - derived mast cells ( bmmc ) from the c57bl/6 strain have an impaired degranulation response to antigen , while a similar knock - out ( ko ) in 129/sv mast cells results in hyperdegranulation . in contrast , lyn deletion in either strain enhances ige - mediated cytokine production , revealing lyn as a negative regulator ( yamashita et al . as no similar negative roles for fyn have been observed in mast cells , one could hypothesize that increased fyn kinase , e.g. yet other work has demonstrated that the function of lyn changes with the intensity of ige - antigen stimulus on mast cells of similar background . in mast cells one such process is the induction of stat5 phosphorylation by stem cell factor ( scf)/c - kit , or il-3 , via jak2 activation ( mui et al . , 1997 ; one or both of these cytokines are essential for the development and survival of mast cells in vitro and in vivo . it should be noted , however , in vitro mast cell development can be achieved by dual stimulation with il-3 + scf ( shelburne et al . , this could be due to the overlap of stat signaling , particularly from stat3 ( sonnenblick et al . in vivo , mast cells are present at birth , but can not be detected in total stat5 ko mice by 12 weeks of age ( shelburne et al . the expression of one such protein , tristetrapolin ( ttp ) , is enhanced in stat5 ko bmmc during fcri crosslinkage ; inhibiting ttp partially restored cytokine production ( barnstein et al . , the potential effects of stat5 on other rna binding factors such as hur ( human antigen r ) , remains as yet unknown . until recently , the mechanism by which fcri stimulates phosphotyrosine ( py)-stat5 also remained unknown . stat5 and fyn were found to co - immunoprecipitate in resting mast cells , an association that dissipated with ige - antigen stimulation . surprisingly , knock out of gab2 the scaffolding protein linking fyn to pi3k signaling allowed a slight enhancement in ige - mediated py - stat5 . these data with other accumulating evidence in non - mast cell studies ( yu et al . , 2009 ) , which can also include shp-2 , paint a more complicated picture of the role of gab2 . , 2007 ) , warranting its examination in mast cells in consideration of signal overlap with stat5 . while lyn and fyn offer a means of fine - tuning ige signaling , external regulation of the ige receptor can be achieved through cytokines , primarily tgf1 , il-10 , and il-4 . tgf1 is a multipotent cytokine regulating tissue repair , inflammation , proliferation , migration , and angiogenesis ( kehrl et al . it is linked to autoimmune diseases and cancer ( letterio and roberts , 1998 ; blobe et al . , 2000 ) , and recently shown to have a critical role in treg and th17 differentiation ( bettelli et al . mast cells evoke tgf1-mediated paracrine signaling , by secreting tgf1 as well as proteases that activate its latent form ( pennington et al . , 1993 ) , as il-10-deficient dendritic cells are highly potent activators of th1 responses ( he et al . , 2004 ) clinically , il-10 inhibits progression of rheumatoid arthritis when delivered directly to the site of inflammation ( trachsel et al . as such , il-10 is being further studied as an inflammatory therapeutic ( murray , 2006 ) . our group and others have found that tgf1 and il-10 suppress mast cell function and survival , offering homeostatic feedback regulation ( bissonnette et al . ( 1997 ) reported that antigen - induced histamine and tnf production from rat peritoneal mast cells were inhibited by tgf1 , whereas meade et al . ( 1992 ) showed that in vivo treatment with tgf1 blunted ige - mediated , mast cell - dependent , immediate hypersensitivity responses in mice . despite these studies supporting the theory that tgf1 inhibits mast cell function , contradicting evidence exists . this includes a report by kim and lee ( 1999 ) that tgf1 potentiates ige - dependent cutaneous anaphylaxis in rats . like tgf1 , il-10 can inhibit fcri expression and ige - mediated cytokine production in mast cells ( marshall et al . our recent work showed that il-10 is produced endogenously during mast cell development , and suppresses mast cell numbers and fcri expression ( speiran et al . exogenous il-10 can inhibit the differentiation and survival of immature mouse mast cells , eliciting apoptosis via reduction of c - kit signaling , caspase activation , and damage to the mitochondria ( bailey et al . we have investigated the means by which il-10 and tgf1 suppress mast cell activation . in recent work , we found that adding exogenous il-10 or tgf1 to mast cells leads to a gradual decline in fyn and stat5 protein levels , reaching its nadir in 34 days ( kennedy norton et al . this slow onset could be argued to allow protective mast cell functions prior to reinstating immune homeostasis . by targeting the fyn - stat5 pathway , these cytokines effectively disrupt mast cell cytokine production and survival . our most recent findings show that a set of micrornas ( mirs ) is regulated by il-10 and tgf1 ; mirs that could target fyn and stat5 , based on in silico analyses . il-4 signals by binding to the il-4r chain , present as a heterodimer , paired with either the common gamma chain or the il-13r1 , creating the type i and type ii receptors , respectively . like il-10 , il-4 can be secreted by mast cells ( brown and hanifin , 1989 ) , and is produced constitutively in cultures of developing mast cells ( speiran et al . also similar to il-10 and tgf1 , il-4 acts on mature mouse mast cells to decrease fcri expression and signaling through a stat6 dependent pathway ( ryan et al . , for this reason , il-4 and its receptor have been attractive candidates for the treatment of asthma . currently in clinical trials is a monoclonal antibody targeting il-4r , an antisense oligonucleotide that prevents translation of the il-4r chain , and an il-4 mutein that interferes with the binding of endogenous il-4 ( nguyen and casale , 2011 ) . , we found that il-4 suppresses tgf receptor expression and signaling in mast cells , showing these two cytokines are able to fine tune mast cell responses ( macey et al . as discussed earlier , mast cell activation can be achieved through a variety of stimuli , ranging from cytokines to allergen - specific immunoglobulins . in allergic and inflammatory pathologies , allergen - specific ige crosslinks mast cell fcri and triggers the activation of intracellular signaling molecules , ultimately resulting in mediator release and chemotaxis . however in addition to fcri , mast cells express the igg receptor family ( fcr ) . however , there are three fcrii ( a , b , and c ) and two fcriii ( a and b ) in humans , whereas mice have one receptor of each type ( fcriib and fcriiia ; nimmerjahn and ravetch , 2008 ) . further complicating receptor distribution among species is evidence that receptor type expression is also variable according to mast cell phenotype ( benhamou et al . in contrast , ligand binding can also result in downregulation of antigen - dependent mediator release due to the existence of the immunoreceptor tyrosine - based inhibitory motifs ( itims ) on the cytosolic portion of the fcriib -chain . , 1996 ) fcriib inhibits several aspects of mast cell and basophil functions such as ige - dependent degranulation and il-4 production ( daron et al . in addition to mast cells , fcriib is expressed by other immune cells including dendritic cells , macrophages , activated neutrophils , basophils , and b cells . fc receptors share the common gamma subunit with fcri , and not surprisingly , similar signaling pathways ( kurosaki et al . , 1992 ; although the importance of syk kinase in igg signaling has been demonstrated ( crowley et al . little is known about src family kinase functions in fcr - mediated mast cell activation . our latest data demonstrates fcr - induced mast cell activation of fyn and lyn kinases , and downstream initiation of the pi3k - akt , erk , p38 , and jnk pathways ( falanga et al . in the same report , it was revealed that fyn and lyn kinases have opposing roles upon fcr crosslinking , since fyn - deficient ( ko ) mast cells displayed decreased degranulation and mediator release , whereas an exacerbated response was observed with lyn ko mast cells . in pathologic and extremely rare conditions , mast cell activation with antigen - specific ige historically , the onset and regulation of anaphylaxis was exclusively attributed to the generally known classical pathway of anaphylaxis involving ige - mediated fcri mast cell activation and subsequent histamine release ( miyajima et al . , 1997 ; however , elegantly conducted research on murine models demonstrated that an anaphylactic reaction can be induced through an igg - fcr pathway ( strait et al . , 2008 ; in this alternative pathway , activation of basophils and macrophages ( fcri and fcriii ; strait et al . although mast cell contribution in igg - mediated anaphylaxis was insignificant as mast cell - deficient mice still developed igg - induced anaphylaxis via platelet activating factor ( paf ) recent data demonstrate that mast cells account for the majority of circulating histamine during fcr - induced passive systemic anaphylaxis ( psa ) , which is regulated by fyn and lyn ( falanga et al . taken together , these findings bring new insights to the function of fyn and lyn kinases downstream of fcr stimulation and the role of mast cells in igg - mediated pathologies . exploration of the biological chemistry behind igg signaling will be an important activity contributing to efforts in understanding igg - mediated hypersensitivity disorders . we posit that the fyn / stat5 pathway will be an important regulator in igg - mediated mast cell pathology , and here we have reviewed key areas of ige - mediated signaling where igg cues are likely to intersect , summarized by figure 1 . the similarities also offer attractive lines of study with respect to the interplay of igg and other exogenous signals such as il-4 , il-10 , and tgf1 , which can have their own effects on fyn and stat5 . overlap in fcri , fcriii , and fcriib signaling . ige - bound multivalent antigen or igg immune complexes both positively influence downstream signaling through syk and lat , but lyn also produces negative feedback by phosphorylating itims and by inducing the csk recruitment via sh2 interactions with phosphorylated cbp / pag . fyn activates stat5 , transcription factors that are required for mast cell survival ( stat5a ) and cytokine production ( stat5b ) . regulatory cytokines such as il-10 and tgf1 can inhibit fyn and stat5 production , possibly through the induction of specific mirs . first is the need for a more precise dissection of stat5 activation and mobilization as an effect of ige and igg receptor crosslinking . as mentioned earlier stat5a is important for the development and proliferation of mast cells ( shelburne et al . , stat5a depletion does not significantly affect ige - mediated cytokine production . while the role of stat5b in mast cell development has not been directly addressed , its impact is likely redundant since total stat5ko bone marrow can be rescued by reconstituting stat5a ( shelburne et al . , 2010 ) , and there are slight primary sequence variations in the location of the known target serines and tyrosine between stat5a and stat5b . for example , double ko of the tec kinases itk and btk was found to inhibit degranulation / histamine release and impaired mast cell granularity ( iyer et al . interestingly , in the same work c - kit expression and mast cell development were not affected , however the double kos showed enhanced proliferation . in fcri signaling , the activation of syk through lyn is a well - known effector of mast cell function ; if this happens in the context of fcr crosslinking is open for investigation . the association of lyn [ and fyn ] with fcri is an enigmatic matter , as it is known to be dynamic and possibly compartmentalized ( gilfillan and rivera , 2009 ) . this in combination with the diversity of igg receptors on mast cells suggests that , while the high affinity ige receptor serves as a good model , there is much more to these signaling stories than is currently understood . for example , additional exploration of fcr expression profiles in mast cells of different sources , phenotypes , and species will be important for interpreting data . finally , unraveling these discrete biochemical questions should be matched with in vivo studies of stat5 functions in allergic and autoimmune diseases , therapies targeting mast cell activation and secreted mediators have proven effective in allergic disease . these clinical goals are critical , and should come as a natural product of efforts to understand the fundamentals of immune homeostasis .

the desire for the discovery of renewable liquid fuels , as well as commodity chemicals , has escalated due to the environmental impact , supply security , and decreasing total reserve of petroleum - based fuels and chemicals . petroleum consumption reached 37.1 quadrillion btu in the united states in 2008 , of which a large majority ( 71% ) was used as a liquid fuel in the transportation sector . this has lead to an increased focus to find sustainable replacements or supplements to petroleum derived diesel fuel , jet fuel , and motor gasoline [ 35 ] . the largest effort thus far has been the production of ethanol , which is often used as a supplement to gasoline but is also available in high percentage blends such as e85 . ethanol production via fermentation reached 9.2 billion gallons in the united states in 2008 , an increase of over 40% from 2007 . according to the newest renewable fuels standard ( rfs2 ) set aside in 2010 , the mandate for renewable fuels production is 36 billion gallons by 2022 . these renewable fuels are classified into 4 categories : cellulosic biofuels , which must be derived from renewable lignocellulosics and achieve a 60% lifecycle greenhouse gas ( ghg ) emission reduction over their petroleum - derived counterparts ; biomass - based diesel ( 50% ghg emission reduction ) ; advanced biofuels , which consist of any renewable fuel other than corn ethanol that reduces ghg emissions by 50% ; total renewable fuel , of which any fuel that achieves a 20% reduction of ghg emissions is counted . of the biomass - derived diesel fuels , biodiesel has gained momentum as a supplement or replacement to traditional petrodiesel , with production reaching just under 700 million gallons in 2008 . biodiesel can be made by several methods , but is most commonly synthesized by the transesterification of oils and fat triglycerides with methanol to make fatty acid methyl esters ( fame ) . although each of these fuel alternatives provides initial platforms for biofuel development , their increased commercialization to replace petroleum is not without its limitations . ethanol is incompatible with the current fuel infrastructure , and the supply of raw materials for biodiesel production from plant oils and waste animal fats may become a concern . these opportunities for refinement have lead researchers to look for alternative fuels and production processes to replace petroleum derived fuels , including fermentative alcohols [ 712 ] , nonfermentative higher chain alcohols , isoprenoid and lipid fuels [ 1518 ] , and fuels synthesized directly from co2 via photosynthesis [ 19 , 20 ] . these microbial - based processes are critical first steps in designing processes to provide renewable drop - in liquid fuels . aiding in the design and continued development of these processes , among a host of others , has been synthetic biology . synthetic biology aims to design , synthesize , and characterize new biological elements , or redesign natural systems , that can be lumped together in a toolbox . these elements can include promoters [ 2123 ] , regulatory proteins and rnas [ 2428 ] , and scaffolds [ 29 , 30 ] . with this synthetic biologists assemble these individually characterized parts into hierarchal structures to perform new , novel , or nonnative tasks , such as synthetic oscillators and toggle switches . this toolbox also allows for the investigation of several designs to achieve the same function , often with varying levels of success , as in the case of heterologous 1-butanol production [ 7 , 9 , 10 ] . this differs from traditional engineering approaches in that the design focal point is on the core components , which can be fine - tuned to meet strict guidelines for specific tasks . much of the work accomplished in biofuel research until now has relied on the identification of target pathways and the design of synthetic expression systems for enzymes responsible for fuel production . as these technologies progress and mature , the design , implementation , and optimization of new functions , as well as the upgrading and rewiring of existing components , will be essential for the successful discovery and production of new biofuels , as many challenges still limit their productivity . this review will investigate recent progress made in the microbial production of biofuels to supplant petrodiesel and motor gasoline and will discuss how existing and newly developed synthetic biology tools may aid in the advancement of these processes . isopropanol can be used directly as a fuel supplement to gasoline or as a feedstock for the transesterification of fats into biodiesel . both isopropanol and 1-butanol are produced in a mixed product fermentation in various strains of clostridium , with maximum production levels reaching 2 g / l , respectively [ 37 , 38 ] . with a renewed interest in alternative fuels , the production of isopropanol and 1-butanol has been recently investigated in genetically tractable heterologous organisms . these organisms , such as escherichia coli and saccharomyces cerevisiae , facilitate the design and optimization of new biofuels processes by combining an increasing synthetic biology toolbox with a well - studied metabolism . isopropanol production in e. coli has surpassed that of clostridium by assembling the pathway for acetone production and a secondary alcohol dehydrogenase [ 8 , 12 ] . construction of a new strain harboring a single construct resulted in an increase in production to 1.2 in addition to e. coli , 1-butanol production has been investigated in pseudomonas putida , bacillus subtilis , and s. cerevisiae [ 10 , 11 ] , although production in e. coli has thus far shown the most promise . each of these processes , however , is far from industrial feasibility , as yields ( ~0.05 g / g ) and productivities ( ~0.01 g / l / h ) must increase significantly to match the same figures for corn ethanol ( ~0.5 the advancement of these processes is thought to be limited by the low activity of pathway enzymes due to poor expression , solubility , or oxygen sensitivity , as well as the metabolic imbalance introduced by these heterologous pathways . while productivity in each of these platforms is low in comparison with clostridial fermentation , the ability to engineer and manipulate these user - friendly hosts will facilitate the development of these processes . the production of biofuels from native organisms can present unique challenges to synthetic biologists as oftentimes the availability of genetic tools and physiological knowledge of the hosts is limited . additionally , the engineering of heterologous hosts for biofuel production may decrease the overall fitness of the cell and require delicate pathway balancing that is oftentimes difficult . it is therefore advantageous to use native pathways to generate immediate precursors for biofuel production . this was accomplished in e. coli by using the hosts ' amino acid biosynthesis pathways to generate 2-keto acid precursors , which can be converted to alcohols through a single heterologous reaction ( figure 1 ) . these alcohols can serve as direct replacements to gasoline , or can be polymerized to form a variety of potential fuel molecules . expression of keto acid decarboxylase ( kdc ) from lactococcus lactis enabled e. coli to convert these 2-keto acids to aldehydes , which can be reduced to alcohols using alcohol dehydrogenase ( adh ) . a total of 6 of these higher chain alcohols were detected after expression of kdc . g / l after host optimization and amplification of genes responsible for the synthesis of 2-ketoisovalerate , the precursor to both isobutanol and valine . subsequent efforts have also been made to engineer the production of 1-propanol and 1-butanol , 2-methyl-1-butanol , and 3-methyl-1-butanol . an alternative and more direct route to 2-ketobutyrate , the citramalate pathway from methanococcus jannaschii , was utilized to produce 1-butanol and 1-propanol independently of threonine . by leveraging a growth selection based on a requirement for 2-keto acids , the directed evolution of citramalate synthase was able to enhance the activity of this heterologous pathway and increase the production of 1-propanol and 1-butanol by 9-fold and 22-fold , respectively . these pathways were also expanded to produce longer chain alcohols from nonstandard 2-keto acids . these longer chain alcohols can be used as commodity chemicals and also possess advantageous fuel properties similar to other high chain alcohols . the leucine biosynthesis pathway was engineered to catalyze the elongation of larger substrates by mutation of leua . similarly , kdc was also designed to fit larger substrates , after which e. coli was able to produce several longer chain alcohols from c5c8 from these nonstandard 2-keto acids . one distinct advantage in the production of these alcohols is the ability to apply existing amino acid production technology . amino acids are produced microbially from several microorganisms , yet corynebacterium glutamicum has been the most successful host for the production of many amino acids , including valine . in addition to industrial success in amino acid production , c. glutamicum shows an increased tolerance to isobutanol relative to e. coli , making the production of isobutanol from c. glutamicum promising . optimization of the host by gene deletion and overexpression of isobutanol synthesis genes resulted in the production of 4.9 g / l of isobutanol from glucose . similarly , an amino acid strain development technique was adapted for the production of 3-methyl-1-butanol ( 3 mb ) , which shares a common precursor with leucine . whole cell mutagenesis and selection with a leucine analogue resulted in a strain able to produce 2.8 g / l 3 mb , greater than a 5-fold improvement from wild - type ( wt ) . addition of an in situ extraction technique to remove 3 mb from the aqueous culture media resulted in the production of 9.5 isoprenoids represent a diverse group of hydrocarbons synthesized from the c5 isomers isopentyl diphosphate ( ipp ) and dimethylallyl diphosphate ( dmapp ) . these precursors can be produced from acetyl coenzyme a ( coa ) via the mevalonate pathway or from glyceraldehyde-3-phosphate and pyruvate through the methylerythritol pathway ( mep ) ( figure 1 ) . isoprenoids are found naturally as hormones , photosynthetic pigments , and a variety of other specialized secondary metabolites , and have been previously investigated as nutraceuticals and pharmaceuticals . because isoprenoids possess vast structural diversity , including saturated , unsaturated , branched , or cyclic alkenes or alkanes , their potential as fuel candidates , such as isopentenol ( c5 ) for motor gasoline , or farnesene ( c15 ) for diesel fuel , is promising . recently , the production of isopentenol ( 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol ) , a c5 unsaturated alcohol , has been investigated in e. coli . isopentenol can be produced by the dephosphorylation of the isoprenoid building blocks ipp and dmapp ( figure 1 ) . by screening a bacillus subtilis genomic library for relief of prenyl diphosphate ( ipp and dmapp ) accumulation , two isopentenol biosynthetic genes , nudf and yhfr , were identified . by overexpression of nudf in a previously optimized strain , production of isopentenol reached 110 mg / l . although the production of isoprenoid fuels is still limited , synthetic biology can provide the framework for improving isopentenol production as well as the development of processes for other potential isoprenoid fuels . the fatty acid biosynthesis pathways are of great importance to the production of renewable fuels . fatty acids are commonly used now in the synthesis of biodiesel , and the production of long chain alkanes , alkenes , aldehydes , and alcohols offer promise in the development of alternative diesel and jet fuels . the fatty acid elongation cycle begins with the carboxylation of acetyl - coa to malonyl - coa . after transacylation of acetyl - coa and malonyl - coa to acyl carrier protein ( acp ) , acetyl - acp and malonyl - acp are condensed into acetoacetyl - acp . after a reduction , dehydration , and second reduction reaction , a saturated fatty acyl - acp ( butyryl - acp ) is formed . in each subsequent elongation cycle , malonyl - acp is condensed with the saturated fatty acyl - acp to add 2 carbons to the growing hydrocarbon . after elongation , these hydrocarbons can be released as free fatty acids by a thioesterase . additionally , fatty acyl - coa can be reduced to a fatty aldehyde , which itself can be reduced to a fatty alcohol , or decarbonylated to an alkane or alkene ( figure 1 ) . the microbial production of biodiesel has been approached from two angles first , by producing short chain alcohols and performing the transesterification in vivo with exogenously added fatty acids , and second , by producing free fatty acids that can be harvested for transesterification in vitro . the in vivo production of biodiesel using endogeneously produced ethanol was recently demonstrated in e. coli . expression of the ethanol production pathway from zymomonas mobilis , along with a broad substrate range acyltransferase ( atfa ) from acinetobacter baylyi , lead to the production of 1.3 g / l of fatty acid ethyl esters ( faee ) after addition of exogenous oleic acid . research on the production of fatty acids have centered around the discovery of oligeanous algae , yeast , and even bacteria , in which the lipid content , mainly composed of triacylglycerols ( tag ) , can reach 60%80% of the total biomass produced [ 51 , 52 ] . the identification of the genetic elements involved in fatty acid synthesis and the implementation and development of synthetic biology tools to facilitate strain development will become critical for process refinement . recently , efforts have also been made to produce fatty acids from user - friendly organisms such as e. coli . the overexpression of an endogenous and exogenous thioesterase along with acetyl - coa carboxylase in a fadd strain resulted in the production of 2.5 g / l of fatty acids from glycerol . by redesigning the expression of these genes the production of fatty acids was increased to 4.5 g / l at a 6% yield with a specific productivity of 0.04 g / h / g dry cell weight . although each of these approaches has been successful in producing precursors for biodiesel synthesis , the supply of raw materials ( lipid ) or downstream processing ( transesterification ) can be cost - intensive . it would be advantageous , therefore , to create a consolidated process to reduce costs . consolidated bioprocessing ( cbp ) , which involves the simultaneous production of saccharolytic enzymes with the hydrolysis of pretreated biomass and the fermentation of hexose and pentose sugars , significantly reduces processing costs by converting abundant , inexpensive biomass into useful fuels or chemicals in a single step . unfortunately , no organisms possess both the ability to digest lignocellulosic biomass and ferment sugars to fuels at high yields . the solution to this challenge is being approached in two directions : the introduction of high yield fuel production pathways into cellulolytic organisms , and the engineering of substrate ( lignocellulosic ) utilizing pathways into organisms with superior product formation . much of the work until this point has focused around the production of ethanol [ 53 , 54 ] , until recently when this strategy was applied by engineering e. coli for the production of biodiesel ( faee ) , fatty alcohols , and wax esters . e. coli was chosen as a host due to its high fatty acid synthesis rate ( 0.2 g / l / h / g dry cell mass ) and straightforwardness in genetic manipulation . as in previous studies , the ethanol production pathway from z. mobilis ( pdc , adhb ) was overexpressed to produce ethanol for faee production . by combining this pathway with a cytosolic version of an endogenous thioesterase ( tesa ) and an ester synthase from a. baylyi ( atfa ) , a fatty acid oxidation deficient strain of e. coli ( fade ) was able to produce 37 mg / l of faee directly from glucose . to increase the production of faee , two coa ligases , fadd from e. coli and faa2 from s. cerevisiae , were overexpressed along with another copy of atfa to bring production of faee up to 674 mg / l . in order to mitigate the cost of processing of raw cellulosic biomass into refined sugars , this process was engineered to use xylan , a pentose polysaccharide component of hemicellulose . expression of the endoxylanase xyn10b from clostridium stercorarium and the xylanase xsa from bacteroides ovatus as chimeras with osmy allowed e. coli to grow on xylan as a sole carbon source . assembly of the xylan degradation pathway with the previously described faee production strain resulted in a strain able to produce 12 mg / future work may focus on the development of secreted cellulases to increase the substrate utilization capacity of e. coli , in addition to optimizing the fatty acid pathway by prospecting for enzymes or expression systems with increased activity or stability . this work demonstrates the first consolidated process for the production of fatty acid based fuels and chemicals from complex polysaccharides , and while the process yields and productivity remained to be improved to merit commercialization , this work gives engineers and synthetic biologists the foundation to advance this process . many current technologies , such as biomass derived biofuels and algal lipids , have received attention as viable fuel replacement technologies [ 55 , 56 ] , yet rely on intermediate stages to incorporate co2 or recover biomass to process precursors into useable fuels , which can increase costs . photosynthetic organisms such as cyanobacteria , algae , and plants use light energy to generate reducing power to directly incorporate co2 into organic metabolites . the use of these organisms to directly produce fuels can limit production costs and co2 emissions during intermediate processing , and may also help reduce net co2 emissions by scrubbing co2 enriched flue gases from traditional power plants and producing useful fuels or chemicals , although their potential is not limited to this scenario . initial efforts have focused on the production of ethanol in rhodobacter and synechococcus . the production of advanced biofuels such as isobutyraldehyde , isobutanol , and isoprene has recently been investigated in cyanobacteria . this was first demonstrated by transferring the 2-keto acid pathways to higher chain alcohols into the cyanobacterium synechococcus elongatus ( figure 2 ) . isobutyraldehyde was chosen as an initial target since its boiling point ( 63c ) allows it to be easily stripped from the culture medium , thus avoiding any toxicity effects . chromosomal integration of 2-ketoisovalerate biosynthesis genes ( alss , ilvcd ) and 2-ketoisovalerate decarboxylase ( kivd ) resulted in the production of 723 mg / l of isobutyraldehyde from dissolved co2 ( nahco3 ) . to improve the low activity of ribulose-1,5-bisphophate carboxylase / oxygenase ( rubisco ) , the rbcls genes from a similar cyanobacterium were integrated downstream of the endogenous rbcls genes . g / l , with a productivity of 6.2 mg / l / h , an encouraging figure considering microalgal biodiesel has been estimated to be near 4 mg / l / h . the production of isobutanol was also investigated by expressing the nadp dependent alcohol dehydrogenase yqhd from e. coli . the production of isobutanol reached 450 mg / l , and although encouraging , is currently thought to be limited by end product toxicity . a similar study was also recently conducted in which the isoprenoid biosynthesis pathways were exploited for biofuel production in the cyanobacterium synechocystis sp . the volatile hydrocarbon isoprene , most notably produced in plants , is a potential feedstock for biofuel or chemical production . the isps gene from kudzu vine ( pueraria montana ) , encoding for an isoprene synthase , which catalyzes the conversion of dmapp to isoprene , was codon optimized and cloned into synechocystis under the control of a light dependent promoter . expression of isps under high intensity light resulted in a small accumulation of isoprene ( 50 g / day / g dry cell mass ) relative to healthy oak leaves ( ~1,650 g / day / g dry cell mass ) , demonstrating that while successful , this process remains to be improved . a significant obstacle to biofuel production in photosynthetic organisms is the design of scale up processes . photosynthetic organisms require more complex reactor designs and their growth and productivity in suboptimal conditions ( temperature , salt concentration , etc . ) is not well understood . however , two reactor designs in use today , the raceway pond and the photobioreactor , have shown promise in their ability to accumulate photosynthetic biomass using sunlight . the raceway pond is inexpensive and simple to construct , but it is subject to contamination and is less photosynthetically efficient than a photobioreactor . photobioreactors , having several designs , have increased capital costs compared to raceway ponds , but have superior productivities due to their increased biomass concentration , and therefore , greater photosynthetic efficiency . hybrid systems comprised of both raceway ponds and photobioreactors have also been investigated to maximize the advantages of each design . synthetic biology is an increasingly expanding discipline focusing on the design and construction of artificial systems to achieve a desired goal . these systems are derived from the assembly of standardized components in a hierarchal manner to create a population of programmed cells carrying out a desired function . for biofuels , this is of particular interest as the production of these chemicals requires efficient integration of foreign genes and pathways into central metabolism . delicate optimization and fine - tuning of these processes to maximize productivity and yield is of equal concern as the viability of any biofuel processes is extremely sensitive to production costs , such as raw material supply , total production , and downstream processing . synthetic biology can provide tools and design principles to guide the development of such processes ( figure 3 ) . a goal of synthetic biology is to create a library of biological parts that can be used independently or as part of a larger assembly for a higher function . these biological parts can have simple or complex behaviors relayed through a variety of outputs such as gene expression . a simple but powerful example is the design of synthetically regulated promoters [ 2123 ] to accurately control gene expression . more complex examples include the modulation of the expression of multiple genes through tunable intergenic regions ( tigr ) , the activation and silencing of gene expression by riboregulators and ribozymes [ 24 , 26 , 28 ] , or successive increases in gene expression through chromosomal amplification , which will become particularly important in the design of stable strains for industrial biofuel production . additionally , thermodynamic models have been developed to rationally design ribosome binding sites to achieve robust expression levels varying by as much as 100,000 fold . preliminary designs for these biofuel gene expression systems will need to evolve to regulate and fine - tune the gene expression of these pathways , as was previously discovered for isoprenoids , a pathway discussed earlier for biofuel production . synthetic biology has also achieved the adaptation of posttranslation systems to regulate enzyme activity such as allosteric protein gates and synthetic scaffolds . the balance between the metabolic capacity of biofuel production pathways and host fitness will also play a key role in the productivity and yield of the process . this balancing of biofuel pathways , which aims to maximize the flow of carbon toward product formation without drastically altering the metabolic load or intracellular cofactors ( nad(p)h , atp , acp , etc . ) can be achieved through the use of these synthetic biology tools . this was again demonstrated in the mevalonate pathway to isoprenoids by employing a synthetic scaffold to increase the metabolic capacity while limiting protein expression . in addition to the balancing of biofuel production pathways , the expression of multienzyme complexes , which often require delicate balancing of catalytic subunits , is of great importance . the most direct example is the production of complexed cellulases , which will be crucial to increase the substrate utilization capacity of biomass - based biofuel processes . the regulation of flux through divergent or branched pathways will also be critical , as the balance of carbon and electronic cofactors such as nadh must be considered to achieve an efficient process . these divergent pathways are especially common in the nonfermentative higher chain alcohol and isoprenoid pathways . to direct carbon flow in the desired manner , scaffolds can be engineered to connect the preferred branches of the pathway together . regulatory rna molecules can also be employed to minimize the expression of competing but essential pathways . product yields may also be dramatically affected by the availability of nadh or nadph . one possible solution is to engineer pathways with alternative cofactor specificity to increase their availability or interconversion . the design of new regulatory pathways from synthetic genetic elements will be important in sensing the extracellular or intracellular environment and producing a programmed cellular response . for biofuel production from lignocellulosic biomass , this is of great importance as the efficient uptake of a mixture of hexose and pentose sugars simultaneously is desirable . synthetic biology can provide tools to construct new circuits devoid of unwanted regulation from the bottom up to sense the extracellular environment and produce the necessary response to digest the sugars . ultimately , the bottom - up construction of biological circuits may extend beyond individual pathways toward all of metabolism , as the synthesis of entirely synthetic , replicable , and functional genomes has recently been accomplished . in the future , this will allow synthetic biologists to build heterologous pathways into organisms devoid of unwanted pathways or properties , therefore , increasing the selectivity and yield of the process . the development and optimization of many aspects of biofuel production technology can benefit from the work already accomplished through synthetic biology . as the number of available tools increases one of the current challenges facing synthetic biology is the reproducibility of the developed tools in different systems , as the variability in cellular regulation and physiology can vary greatly from host to host . proposals for standardization have been made , although as the complexity of these systems increase so will their variability . however , current technology developed by synthetic biology has already allowed for the successful design of many heterologously expressed biofuel production and substrate utilization pathways extending beyond the most user - friendly organisms . the advancement of synthetic biology toward new diagnostic tools and high throughput screening systems will aid in the further development of these biofuel processes for pathway optimization and enzyme discovery or improvement . the success that synthetic biology has already afforded biofuel production technology lends confidence to future synergistic developments and breakthroughs . the desire for renewable liquid fuel replacements to petroleum has steadily increased with concerns about the current fuel economy 's stability and environmental impact . the development of new biofuel production processes has sought to mitigate some of these issues . these fuels , whether designed for motor gasoline , diesel fuel , or jet fuel , will face challenges in strain development and productivity in a cost - sensitive market . the integration of synthetic biology with the development of these processes will be significant in bringing the biofuels industry from its infancy to a commercially viable alternative to petroleum .

the advancement of microbial processes for the production of renewable liquid fuels has increased with concerns about the current fuel economy . the development of advanced biofuels in particular has risen to address some of the shortcomings of ethanol . these advanced fuels have chemical properties similar to petroleum - based liquid fuels , thus removing the need for engine modification or infrastructure redesign . while the productivity and titers of each of these processes remains to be improved , progress in synthetic biology has provided tools to guide the engineering of these processes through present and future challenges .

1. Introduction 2. Current Biofuels Research 3. Synthetic Biology for Biofuels 4. Conclusions

the desire for the discovery of renewable liquid fuels , as well as commodity chemicals , has escalated due to the environmental impact , supply security , and decreasing total reserve of petroleum - based fuels and chemicals . this has lead to an increased focus to find sustainable replacements or supplements to petroleum derived diesel fuel , jet fuel , and motor gasoline [ 35 ] . the largest effort thus far has been the production of ethanol , which is often used as a supplement to gasoline but is also available in high percentage blends such as e85 . these renewable fuels are classified into 4 categories : cellulosic biofuels , which must be derived from renewable lignocellulosics and achieve a 60% lifecycle greenhouse gas ( ghg ) emission reduction over their petroleum - derived counterparts ; biomass - based diesel ( 50% ghg emission reduction ) ; advanced biofuels , which consist of any renewable fuel other than corn ethanol that reduces ghg emissions by 50% ; total renewable fuel , of which any fuel that achieves a 20% reduction of ghg emissions is counted . of the biomass - derived diesel fuels , biodiesel has gained momentum as a supplement or replacement to traditional petrodiesel , with production reaching just under 700 million gallons in 2008 . although each of these fuel alternatives provides initial platforms for biofuel development , their increased commercialization to replace petroleum is not without its limitations . ethanol is incompatible with the current fuel infrastructure , and the supply of raw materials for biodiesel production from plant oils and waste animal fats may become a concern . these opportunities for refinement have lead researchers to look for alternative fuels and production processes to replace petroleum derived fuels , including fermentative alcohols [ 712 ] , nonfermentative higher chain alcohols , isoprenoid and lipid fuels [ 1518 ] , and fuels synthesized directly from co2 via photosynthesis [ 19 , 20 ] . these microbial - based processes are critical first steps in designing processes to provide renewable drop - in liquid fuels . aiding in the design and continued development of these processes , among a host of others , has been synthetic biology . synthetic biology aims to design , synthesize , and characterize new biological elements , or redesign natural systems , that can be lumped together in a toolbox . this toolbox also allows for the investigation of several designs to achieve the same function , often with varying levels of success , as in the case of heterologous 1-butanol production [ 7 , 9 , 10 ] . much of the work accomplished in biofuel research until now has relied on the identification of target pathways and the design of synthetic expression systems for enzymes responsible for fuel production . as these technologies progress and mature , the design , implementation , and optimization of new functions , as well as the upgrading and rewiring of existing components , will be essential for the successful discovery and production of new biofuels , as many challenges still limit their productivity . this review will investigate recent progress made in the microbial production of biofuels to supplant petrodiesel and motor gasoline and will discuss how existing and newly developed synthetic biology tools may aid in the advancement of these processes . isopropanol can be used directly as a fuel supplement to gasoline or as a feedstock for the transesterification of fats into biodiesel . with a renewed interest in alternative fuels , the production of isopropanol and 1-butanol has been recently investigated in genetically tractable heterologous organisms . these organisms , such as escherichia coli and saccharomyces cerevisiae , facilitate the design and optimization of new biofuels processes by combining an increasing synthetic biology toolbox with a well - studied metabolism . each of these processes , however , is far from industrial feasibility , as yields ( ~0.05 g / g ) and productivities ( ~0.01 g / l / h ) must increase significantly to match the same figures for corn ethanol ( ~0.5 the advancement of these processes is thought to be limited by the low activity of pathway enzymes due to poor expression , solubility , or oxygen sensitivity , as well as the metabolic imbalance introduced by these heterologous pathways . while productivity in each of these platforms is low in comparison with clostridial fermentation , the ability to engineer and manipulate these user - friendly hosts will facilitate the development of these processes . the production of biofuels from native organisms can present unique challenges to synthetic biologists as oftentimes the availability of genetic tools and physiological knowledge of the hosts is limited . additionally , the engineering of heterologous hosts for biofuel production may decrease the overall fitness of the cell and require delicate pathway balancing that is oftentimes difficult . it is therefore advantageous to use native pathways to generate immediate precursors for biofuel production . a total of 6 of these higher chain alcohols were detected after expression of kdc . g / l after host optimization and amplification of genes responsible for the synthesis of 2-ketoisovalerate , the precursor to both isobutanol and valine . subsequent efforts have also been made to engineer the production of 1-propanol and 1-butanol , 2-methyl-1-butanol , and 3-methyl-1-butanol . an alternative and more direct route to 2-ketobutyrate , the citramalate pathway from methanococcus jannaschii , was utilized to produce 1-butanol and 1-propanol independently of threonine . by leveraging a growth selection based on a requirement for 2-keto acids , the directed evolution of citramalate synthase was able to enhance the activity of this heterologous pathway and increase the production of 1-propanol and 1-butanol by 9-fold and 22-fold , respectively . these longer chain alcohols can be used as commodity chemicals and also possess advantageous fuel properties similar to other high chain alcohols . one distinct advantage in the production of these alcohols is the ability to apply existing amino acid production technology . amino acids are produced microbially from several microorganisms , yet corynebacterium glutamicum has been the most successful host for the production of many amino acids , including valine . in addition to industrial success in amino acid production , c. glutamicum shows an increased tolerance to isobutanol relative to e. coli , making the production of isobutanol from c. glutamicum promising . optimization of the host by gene deletion and overexpression of isobutanol synthesis genes resulted in the production of 4.9 g / l of isobutanol from glucose . similarly , an amino acid strain development technique was adapted for the production of 3-methyl-1-butanol ( 3 mb ) , which shares a common precursor with leucine . whole cell mutagenesis and selection with a leucine analogue resulted in a strain able to produce 2.8 g / l 3 mb , greater than a 5-fold improvement from wild - type ( wt ) . addition of an in situ extraction technique to remove 3 mb from the aqueous culture media resulted in the production of 9.5 isoprenoids represent a diverse group of hydrocarbons synthesized from the c5 isomers isopentyl diphosphate ( ipp ) and dimethylallyl diphosphate ( dmapp ) . isoprenoids are found naturally as hormones , photosynthetic pigments , and a variety of other specialized secondary metabolites , and have been previously investigated as nutraceuticals and pharmaceuticals . recently , the production of isopentenol ( 3-methyl-3-buten-1-ol or 3-methyl-2-buten-1-ol ) , a c5 unsaturated alcohol , has been investigated in e. coli . isopentenol can be produced by the dephosphorylation of the isoprenoid building blocks ipp and dmapp ( figure 1 ) . by screening a bacillus subtilis genomic library for relief of prenyl diphosphate ( ipp and dmapp ) accumulation , two isopentenol biosynthetic genes , nudf and yhfr , were identified . by overexpression of nudf in a previously optimized strain , production of isopentenol reached 110 mg / l . although the production of isoprenoid fuels is still limited , synthetic biology can provide the framework for improving isopentenol production as well as the development of processes for other potential isoprenoid fuels . the fatty acid biosynthesis pathways are of great importance to the production of renewable fuels . fatty acids are commonly used now in the synthesis of biodiesel , and the production of long chain alkanes , alkenes , aldehydes , and alcohols offer promise in the development of alternative diesel and jet fuels . in each subsequent elongation cycle , malonyl - acp is condensed with the saturated fatty acyl - acp to add 2 carbons to the growing hydrocarbon . after elongation , these hydrocarbons can be released as free fatty acids by a thioesterase . the microbial production of biodiesel has been approached from two angles first , by producing short chain alcohols and performing the transesterification in vivo with exogenously added fatty acids , and second , by producing free fatty acids that can be harvested for transesterification in vitro . the in vivo production of biodiesel using endogeneously produced ethanol was recently demonstrated in e. coli . expression of the ethanol production pathway from zymomonas mobilis , along with a broad substrate range acyltransferase ( atfa ) from acinetobacter baylyi , lead to the production of 1.3 g / l of fatty acid ethyl esters ( faee ) after addition of exogenous oleic acid . research on the production of fatty acids have centered around the discovery of oligeanous algae , yeast , and even bacteria , in which the lipid content , mainly composed of triacylglycerols ( tag ) , can reach 60%80% of the total biomass produced [ 51 , 52 ] . the identification of the genetic elements involved in fatty acid synthesis and the implementation and development of synthetic biology tools to facilitate strain development will become critical for process refinement . the overexpression of an endogenous and exogenous thioesterase along with acetyl - coa carboxylase in a fadd strain resulted in the production of 2.5 g / l of fatty acids from glycerol . by redesigning the expression of these genes the production of fatty acids was increased to 4.5 g / l at a 6% yield with a specific productivity of 0.04 g / h / g dry cell weight . although each of these approaches has been successful in producing precursors for biodiesel synthesis , the supply of raw materials ( lipid ) or downstream processing ( transesterification ) can be cost - intensive . consolidated bioprocessing ( cbp ) , which involves the simultaneous production of saccharolytic enzymes with the hydrolysis of pretreated biomass and the fermentation of hexose and pentose sugars , significantly reduces processing costs by converting abundant , inexpensive biomass into useful fuels or chemicals in a single step . the solution to this challenge is being approached in two directions : the introduction of high yield fuel production pathways into cellulolytic organisms , and the engineering of substrate ( lignocellulosic ) utilizing pathways into organisms with superior product formation . much of the work until this point has focused around the production of ethanol [ 53 , 54 ] , until recently when this strategy was applied by engineering e. coli for the production of biodiesel ( faee ) , fatty alcohols , and wax esters . to increase the production of faee , two coa ligases , fadd from e. coli and faa2 from s. cerevisiae , were overexpressed along with another copy of atfa to bring production of faee up to 674 mg / l . expression of the endoxylanase xyn10b from clostridium stercorarium and the xylanase xsa from bacteroides ovatus as chimeras with osmy allowed e. coli to grow on xylan as a sole carbon source . assembly of the xylan degradation pathway with the previously described faee production strain resulted in a strain able to produce 12 mg / future work may focus on the development of secreted cellulases to increase the substrate utilization capacity of e. coli , in addition to optimizing the fatty acid pathway by prospecting for enzymes or expression systems with increased activity or stability . this work demonstrates the first consolidated process for the production of fatty acid based fuels and chemicals from complex polysaccharides , and while the process yields and productivity remained to be improved to merit commercialization , this work gives engineers and synthetic biologists the foundation to advance this process . many current technologies , such as biomass derived biofuels and algal lipids , have received attention as viable fuel replacement technologies [ 55 , 56 ] , yet rely on intermediate stages to incorporate co2 or recover biomass to process precursors into useable fuels , which can increase costs . the use of these organisms to directly produce fuels can limit production costs and co2 emissions during intermediate processing , and may also help reduce net co2 emissions by scrubbing co2 enriched flue gases from traditional power plants and producing useful fuels or chemicals , although their potential is not limited to this scenario . initial efforts have focused on the production of ethanol in rhodobacter and synechococcus . the production of advanced biofuels such as isobutyraldehyde , isobutanol , and isoprene has recently been investigated in cyanobacteria . isobutyraldehyde was chosen as an initial target since its boiling point ( 63c ) allows it to be easily stripped from the culture medium , thus avoiding any toxicity effects . chromosomal integration of 2-ketoisovalerate biosynthesis genes ( alss , ilvcd ) and 2-ketoisovalerate decarboxylase ( kivd ) resulted in the production of 723 mg / l of isobutyraldehyde from dissolved co2 ( nahco3 ) . to improve the low activity of ribulose-1,5-bisphophate carboxylase / oxygenase ( rubisco ) , the rbcls genes from a similar cyanobacterium were integrated downstream of the endogenous rbcls genes . g / l , with a productivity of 6.2 mg / l / h , an encouraging figure considering microalgal biodiesel has been estimated to be near 4 mg / l / h . the production of isobutanol was also investigated by expressing the nadp dependent alcohol dehydrogenase yqhd from e. coli . the production of isobutanol reached 450 mg / l , and although encouraging , is currently thought to be limited by end product toxicity . expression of isps under high intensity light resulted in a small accumulation of isoprene ( 50 g / day / g dry cell mass ) relative to healthy oak leaves ( ~1,650 g / day / g dry cell mass ) , demonstrating that while successful , this process remains to be improved . a significant obstacle to biofuel production in photosynthetic organisms is the design of scale up processes . hybrid systems comprised of both raceway ponds and photobioreactors have also been investigated to maximize the advantages of each design . synthetic biology is an increasingly expanding discipline focusing on the design and construction of artificial systems to achieve a desired goal . for biofuels , this is of particular interest as the production of these chemicals requires efficient integration of foreign genes and pathways into central metabolism . delicate optimization and fine - tuning of these processes to maximize productivity and yield is of equal concern as the viability of any biofuel processes is extremely sensitive to production costs , such as raw material supply , total production , and downstream processing . synthetic biology can provide tools and design principles to guide the development of such processes ( figure 3 ) . a goal of synthetic biology is to create a library of biological parts that can be used independently or as part of a larger assembly for a higher function . a simple but powerful example is the design of synthetically regulated promoters [ 2123 ] to accurately control gene expression . more complex examples include the modulation of the expression of multiple genes through tunable intergenic regions ( tigr ) , the activation and silencing of gene expression by riboregulators and ribozymes [ 24 , 26 , 28 ] , or successive increases in gene expression through chromosomal amplification , which will become particularly important in the design of stable strains for industrial biofuel production . preliminary designs for these biofuel gene expression systems will need to evolve to regulate and fine - tune the gene expression of these pathways , as was previously discovered for isoprenoids , a pathway discussed earlier for biofuel production . synthetic biology has also achieved the adaptation of posttranslation systems to regulate enzyme activity such as allosteric protein gates and synthetic scaffolds . the balance between the metabolic capacity of biofuel production pathways and host fitness will also play a key role in the productivity and yield of the process . this balancing of biofuel pathways , which aims to maximize the flow of carbon toward product formation without drastically altering the metabolic load or intracellular cofactors ( nad(p)h , atp , acp , etc . ) can be achieved through the use of these synthetic biology tools . the most direct example is the production of complexed cellulases , which will be crucial to increase the substrate utilization capacity of biomass - based biofuel processes . to direct carbon flow in the desired manner , scaffolds can be engineered to connect the preferred branches of the pathway together . synthetic biology can provide tools to construct new circuits devoid of unwanted regulation from the bottom up to sense the extracellular environment and produce the necessary response to digest the sugars . in the future , this will allow synthetic biologists to build heterologous pathways into organisms devoid of unwanted pathways or properties , therefore , increasing the selectivity and yield of the process . the development and optimization of many aspects of biofuel production technology can benefit from the work already accomplished through synthetic biology . as the number of available tools increases one of the current challenges facing synthetic biology is the reproducibility of the developed tools in different systems , as the variability in cellular regulation and physiology can vary greatly from host to host . proposals for standardization have been made , although as the complexity of these systems increase so will their variability . however , current technology developed by synthetic biology has already allowed for the successful design of many heterologously expressed biofuel production and substrate utilization pathways extending beyond the most user - friendly organisms . the advancement of synthetic biology toward new diagnostic tools and high throughput screening systems will aid in the further development of these biofuel processes for pathway optimization and enzyme discovery or improvement . the success that synthetic biology has already afforded biofuel production technology lends confidence to future synergistic developments and breakthroughs . the desire for renewable liquid fuel replacements to petroleum has steadily increased with concerns about the current fuel economy 's stability and environmental impact . the development of new biofuel production processes has sought to mitigate some of these issues . these fuels , whether designed for motor gasoline , diesel fuel , or jet fuel , will face challenges in strain development and productivity in a cost - sensitive market . the integration of synthetic biology with the development of these processes will be significant in bringing the biofuels industry from its infancy to a commercially viable alternative to petroleum .

( irvingiaceae ) has become known as african mango and is unrelated botanically to the common , or indian , mango [ mangifera indica ( anacardiaceae ) ] . the fruits of african mango are edible , and their use in traditional medicine has been documented for the treatment of gastrointestinal or hepatic disorders , diarrhea , infections , and as a purgative . market as a dietary supplement and have shown high in vitro antioxidant capacity , significant effects on body weight loss , blood lipid decreases , and a lowering of plasma glucose in experimental animal or human subject studies . phytochemical investigations of the stem bark of i. gabonensis have led to the isolation of antioxidant and hepatoprotective triterpenes and phenols . nevertheless , very limited studies on the chemical constituents of the seeds have been reported . in 2011 , atawodi and co - workers published the first report on the phenolic compound profile of i. gabonensis seeds , in which methyl gallate , ellagic acid , and other ellagic acid derivatives were detected by lc - uv - ms in the methanol extract of the seeds , which also demonstrated strong in vitro antioxidant activity . a recent study of dietary supplements wholly or partially containing african mango seed extracts using uhplc - hrms found evidence of contamination , adulteration , and/or mislabeling among multiple commercial samples when compared to authentic samples . it is noteworthy that no alkaloids have been previously isolated or detected spectroscopically from african mango , according to the accessible and surveyed literature . scavenging reactive oxygen species by antioxidants and enhancing carcinogen detoxification via induction of phase ii enzymes such as quinone reductase ( qr ) are two important cancer chemopreventive strategies . in a continuing search for natural inhibitors of carcinogenesis , the chloroform - soluble extract of a commercially sourced african mango seed extract showed in vitro hydroxyl radical - scavenging and qr - inducing activities , and thus was selected for further study . bioactivity - guided fractionation of this sample , using both hydroxyl radical - scavenging and qr - inducing assays , led to the isolation of a new pyrrole alkaloid , 1 , along with seven known compounds , 28 . compounds 14 represent structural analogues of substituted pyrrole alkaloids reported recently from lycium chinense mill . the fruits of l. chinense and of the closely related species lycium barbarum l. are commonly known as goji berry or wolfberry and are used almost interchangeably . goji has a long history of usage in asian countries as a culinary ingredient , traditional tonic medicine , and functional food for its perceived benefits in antiaging , as well as enhancements in vision and liver and kidney functions . in recent years , goji has become increasingly popular in europe and north america as a superfruit and botanical dietary supplement . the in vivo inhibitory activity of powdered l. barbarum ( wolfberry ) fruits when evaluated in an n - nitrosomethylbenzylamine - induced esophageal cancer model was reported recently in rats . other recent studies have indicated that goji berry extracts and l. barbarum polysaccharides possess a range of biological effects , including antiaging , antioxidant , antitumor , immunomodulatory , and cytoprotective activities . the published chemical constituents of goji have been thoroughly reviewed , with the occurrence of pyrrole alkaloids well established . due to the unexpected presence of the pyrrole alkaloids isolated , 14 , and their structural similarity to constituents found in goji berries , as well as the recent report of contamination in commercial african mango samples , the identity of the source material investigated came into question . the present study includes the bioactivity - guided isolation , identification , and biological evaluation of compounds 18 in addition to comparison of the commercial product with authentic african mango seeds and l. barbarum samples by chemotaxonomy using lc - ms and h nmr fingerprinting as well as microscopic analysis . optical rotations were measured using a perkinelmer 343 automatic polarimeter ( perkinelmer , waltham , ma , usa ) . uv spectra were collected on a hitachi u-2910 spectrophotometer ( hitachi , tokyo , japan ) . ir spectra were obtained with a nicolet 6700 ft - ir spectrometer ( thermo scientific , waltham , ma , usa ) . nmr spectroscopic data were recorded at room temperature on a bruker avance drx-400 mhz spectrometer ( bruker , billerica , ma , usa ) using standard bruker pulse sequences . high - resolution electrospray ionization mass spectra ( hresims ) were obtained on a micromass q - tof ii ( micromass , wythenshawe , uk ) mass spectrometer operated in the positive - ion mode , with sodium iodide being used for mass calibration . column chromatography was performed with sephadex lh-20 ( supelco , bellefonte , pa , usa ) and 65 250 or 230 400 mesh silica gel ( sorbent technologies , atlanta , ga , usa ) . analytical thin - layer chromatography ( tlc ) was conducted on precoated 250 m thickness partisil si gel 60f254 glass plates . a 150 mm 19 mm i.d . , 5 m , xbridge prepc18 column with a 10 mm 19 mm i.d . guard column of the same material ( waters , milford , ma , usa ) was used for semipreparative hplc , along with a hitachi system composed of an l-2130 prep pump , an l-2200 autosampler , and an l-2450 diode array detector ( hitachi , tokyo , japan ) . all solvents used for chromatographic separations were purchased from fisher scientific ( fair lawn , nj , usa ) . 2,7-dichlorodihydrofluorescein diacetate ( h2dcf - da ) , esterase , ferrous sulfate ( feso4 ) , hydrogen peroxide ( h2o2 ) , quercetin , dimethyl sulfoxide ( dmso ) , digitonin , edta , trizma base , tween 20 , flavin adenine dinucleotide phosphate ( fad ) , glucose-6-phosphate ( g-6-p ) , nicotinamide adenine dinucleotide phosphate ( nadp ) , glucose-6-phosphate dehydrogenase ( g-6-p - d ) , menadione , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) , bovine serum albumin ( bsa ) , l - sulforaphane , and deuterated nmr solvents were purchased from sigma - aldrich ( st . gallic acid , methyl gallate , and ellagic acid that served as standards for hplc - pda analysis were authentic samples isolated in previous studies and stored in a compound library at the ohio state university . the commercial sample labeled as powdered seeds of african mango ( i. gabonensis ) , used for isolation , was obtained by nature s sunshine products , inc . , from a manufacturer in the people s republic of china . a representative sample ( code osuadk - ccp0024 ) was deposited in the division of medicinal chemistry and pharmacognosy , college of pharmacy , the ohio state university . an authentic sample of african mango seed powder , collected in nigeria and imported by nutralliance ( yorba linda , ca , usa ) , was purchased by nature s sunshine products , inc . three samples of authentic goji berries were obtained , two from the ningxia province of people s republic of china , by nature s sunshine , inc . , and another directly from a drug store in chengdu , sichuan , people s republic of china ( codes osuadk - ccp0011 , osuadk - ccp0015 , and osuadk - ccp0026 , respectively ; same repository ) . the seeds of the last - mentioned product were separated manually from the fruit pulp . these five samples ( osuadk - ccp0024 , osuadk - ccp0026 , osuadk - ccp0015 , osuadk - ccp0011 , and osuadk - ccp0025 ) are referred to as products a e , respectively , henceforth in this paper . the commercial sample labeled as the powdered seeds of african mango ( product a , 5 kg ) was extracted by maceration with meoh ( 3 10 l ) at room temperature , for 3 days each . after filtration and evaporation of the solvent under reduced pressure , the combined crude methanol extract ( ca . 1000 g ) was suspended in h2o ( 1 l ) and then partitioned in turn with hexanes ( 3 1 l ) , chcl3 ( 3 1 l ) , etoac ( 3 1 l ) , and n - buoh ( 3 1 l ) to afford dried hexanes ( 40.1 g ) , chcl3 ( 5.0 g ) , etoac ( 6.5 g ) , n - buoh ( 166.0 g ) , and h2o - soluble ( ca . the chcl3-soluble extract showed activities in both hydroxyl radical - scavenging ( ed50 6.3 g / ml ) and qr induction ( cd 12.4 g / ml ) assays and hence was selected for further fractionation . the chcl3-soluble extract was subjected initially to chromatography over a silica gel column , eluted with chcl3/meoh ( 50:1 to pure meoh , stepwise ) , to afford 12 fractions ( fractions f01f12 ) . fractions f01f03 showed activity in either the hydroxyl radical - scavenging assay or the qr induction assay and were chosen for further purification . 890 mg ) was chromatographed over an open c18 reversed - phase column , eluted with meoh / h2o ( 5:95 to pure meoh , stepwise ) to afford eight subfractions . 400 mg , eluted by meoh / h2o 20:80 ) was subjected to passage over a sephadex lh-20 column with meoh / h2o ( 20:80 to pure meoh , stepwise ) as the eluting solvent system to yield compound 8 ( eluted by meoh / h2o 50:50 ; 3.0 mg ) and a further subfraction f010211 ( ca . subfraction f010211 was separated by hplc using a 150 mm 19 mm i.d . , 5 m , xbridge prepc18 column with a 10 mm 19 mm i.d . guard column of the same material ( waters ) and with ch3cn / h2o by isocratic elution ( 3:97 ; 8 ml / min ) to afford compounds 5 ( tr 68.6 min ; 5.7 mg ) and 6 ( tr 55.0 min ; 0.8 mg ) and a further subfraction , f01021103 ( ca . f01021103 was purified further by hplc on the same column with ch3cn / h2o ( 0.025% nh4oh ) isocratic elution ( 3:97 ; 8 ml / min ) to obtain compound 2 ( tr 12.3 min ; 4.5 mg ) . fractions f02 ( ca . 120 mg ) were subjected separately to sephadex lh-20 column chromatography , eluted by meoh / h2o ( 50:50 , 75:25 , and pure meoh ) to remove pigments and afforded several subfractions . 100 mg ) was purified further by hplc on the same xbridge prepc18 column as above , by a gradient elution ( a , ch3cn ; b , h2o , 315% a over 70 min , 15100% a from 70 to 85 min ; 8 ml / min ) , to render the purification of compounds 1 ( tr 49.5 min ; 4.8 mg ) , 3 ( tr 46.7 min ; 1.3 mg ) , and 7 ( tr 16.5 min ; 20.0 mg ) . 80 mg ) was also purified by hplc on the same xbridge prepc18 column , eluted by a ch3cn / h2o gradient ( a , ch3cn ; b , h2o , 315% a over 70 min , 15100% a from 70 to 85 min ; 8 ml / min ) , to yield compound 4 ( tr 22.7 min ; 6.8 mg ) . 0.0001% of dry weight ) as a colorless resin : [ ]d + 28.0 ( c 0.1 , meoh ) ; uv ( meoh ) max ( log ) 291 ( 4.09 ) nm ; ir ( film ) max 3424 , 3120 , 2946 , 2850 , 2803 , 2727 , 1749 , 1655 cm ; h nmr ( 400 mhz , cd3od ) 9.31 ( 1h , s , cho ) , 7.07 ( 1h , d , j = 4.0 hz , h-3 ) , 6.28 ( 1h , d , j = 4.0 hz , h-4 ) , 5.43 ( 1h , q , j = 7.0 hz , h-1 ) , 4.63 ( 2h , abq , = 8.1 hz , j = 13.9 hz , h-6 ) , 3.67 ( 3h , s , och3 ) , 1.67 ( 3h , d , j = 7.0 hz , ch3 ) ; c nmr ( 100 mhz , cd3od ) 180.5 ( cho ) , 172.5 ( coo ) , 144.6 ( c-5 ) , 133.4 ( c-2 ) , 127.4 ( c-3 ) , 111.4 ( c-4 ) , 56.6 ( c-6 ) , 55.8 ( c-1 ) , 52.8 ( och3 ) , 18.0 ( ch3 ) ; hresims m / z 234.0733 [ m + na ] ( calcd for c10h13no4na , 234.0742 ) . hydroxyl radical - scavenging activity was tested according to the method previously reported . the in vitro qr - inducing activity of the extracts , fractions , and pure isolates was assayed according to a previously published method . the clean samples of products b d were pulverized and passed through a no . products a and e were obtained as powders and thus passed through the sieve directly . the powdered materials of products a e were each placed on a slide and stirred with a fine - pointed needle to distribute the powders evenly , followed by the application of 2 drops of clearing agent , visikol ( phytosys llc , new brunswick , nj , usa ) , and gentle heat until air bubbles moved to the edge of the slide . two drops of diluted glycerin and a coverslip were added to the slide , followed by immediate observation under a nikon eclipse te300 optical microscope ( nikon , tokyo , japan ) , along with an a01f881036 camera module ( roper scientific , trenton , nj , usa ) for digital imaging of the microscopic structures observed . each sample ( 3 g ) was extracted with 10 ml of methanol by sonication at room temperature for 30 min . the methanol extracts were dried in vacuo ( 40 c ) , followed by another ultrasonic extraction with 10 ml of chloroform at room temperature for 30 min , to obtain a chloroform - soluble extract of each sample , which was then dried in vacuo at 40 c and stored at 20 c before analysis . the hplc - pda analysis was performed using a 150 mm 4.6 mm i.d . , 5 m , xbridge c18 analytical column ( waters ) , along with a hitachi system composed of an l-2130 pump and an l-2450 diode array detector ( hitachi ) . the mobile phase consisted of 0.05% trifluoroacetic acid in water ( a ) and acetonitrile ( b ) using a gradient program of 320% b from 0 to 60 min and 20100% b from 60 to 70 min . the mobile phase flow rate and the injection volume were 1 ml / min and 10 l , respectively . the dried chloroform - soluble extract of each sample was dissolved in 1 ml of hplc grade methanol , and filtered through a fisher scientific 13 mm syringe filter ( 0.2 m ) prior to injection . after comparison of the chromatograms of the chloroform - soluble extract solution recorded at wavelengths within 200550 nm , it was found that 254 nm could best represent the profile of the analytes . the lc - it - ms analysis employed the same separation conditions and xbridge c18 analytical column as used for the hplc - pda analysis mentioned above , on a waters alliance 2690 separation module . the mobile phase flow rate was maintained at 1 ml / min and was split approximately 50:1 postcolumn using a microsplitter valve ( upchurch scientific , oak harbor , wa , usa ) for the introduction to the esi source . the electrospray ionization ion trap mass spectrometry ( esi - it - ms ) was performed on a bruker dual funnel amazon etd ion trap mass spectrometer ( bremen , germany ) equipped with an orthogonal electrospray source operated in positive - ion mode . sodium iodide was used for mass calibration for a calibration range of m / z 1001000 . optimal esi conditions were as follows : capillary voltage , 4500 v ; source temperature , 250 c ; esi drying gas ( nitrogen ) , 4.0 l / min ; and nebulizer , 10 psi . the ion trap was set to ultrascan mode with a target mass of m / z 500 pass ions from m / z 100 to 1000 . the h nmr spectroscopic fingerprinting was conducted using the previously described chloroform - soluble extracts of products a e . the samples were dissolved in 600 l of cdcl3 , and spectra were measured at 300 k using a bruker avance - iii hd400 spectrometer . the chcl3-soluble extract of product a ( a commercial sample labeled as powdered seeds of african mango ) was found to be the most potent among the hexanes- , chcl3- , etoac- , n - buoh- , and h2o - soluble extracts in the in vitro hydroxyl radical - scavenging ( ed50 , concentration scavenging hydroxyl radical by 50% , 6.3 g / ml ) and qr induction ( cd , concentration required to double qr activity , 12.4 g / ml ) assays . fractions f01f03 of the chcl3-soluble extract showed hydroxyl radical - scavenging activity , with ed50 values of 3.3 , 5.6 , and 6.8 g / ml , respectively . in addition , fractions f01 and f02 exhibited qr - inducing activity , with cd values of 6.9 and 10.2 g / ml , respectively . accordingly , fractions f01f03 were used for further detailed purification . in this way , bioactivity - guided fractionation of product a led to the isolation of a new pyrrole alkaloid , 1 , and seven known compounds , 28 , as shown in figure 1 . structures of compounds isolated from a goji berry - contaminated commercial sample labeled african mango ( irvingia gabonensis ) . the molecular formula was determined as c10h13no4 on the basis of the sodiated molecular ion peak in the hresims . the ir spectrum showed absorptions of hydroxy ( 3424 cm ) , ester carbonyl ( 1749 cm ) , and conjugated aldehyde ( 2803 , 2727 , and 1655 cm ) groups . the uv spectrum of 1 exhibited a maximum absorption at 291 nm , which is characteristic of pyrrole 2-carbaldehyde . the proton signal at h 9.31 and the carbon signal at c 180.5 correlated in the hsqc spectrum and indicated the presence of an aldehyde group . two proton signals at h 4.63 ( 2h , abq , = 8.1 hz , j = 13.9 hz , h-6 ) resulted from a magnetically inequivalent oxygenated methylene group with geminal coupling . the hmbc correlation ( figure 2 ) of the methoxy group ( h 3.67 , 3h , s , och3 ) to the carbonyl at c 172.5 revealed that this methoxy group forms an ester with this carbonyl . the quartet of h-1 ( h 5.43 , j = 7.0 hz ) and doublet of the methyl group ( h 1.67 , j = 7.0 hz ) , as well as their correlation in the cosy spectrum , clearly indicated the connection of the methyl group ( c 18.1 ) to c-1 ( c 55.8 ) . furthermore , hmbc correlation of this methyl group ( h 1.67 ) to the ester carbonyl c-2 ( c 172.5 ) suggested the connection of c-1 ( c 55.8 ) to c-2. the chemical shifts and coupling constants of the two protons at h 7.07 ( 1h , d , j = 4.0 hz , h-3 ) and h 6.28 ( 1h , d , j = 4.0 hz , h-4 ) implied the presence of a pyrrole ring . moreover , the substitution pattern of the pyrrole ring should be 2,5- because a 2,3- or a 2,4-disubstituted pyrrole would have coupling constants of 1.32.9 or 2.33.2 hz , respectively . also , the two carbons of the pyrrole ring at the lower field region ( c 144.6 and 133.4 ) were not present in the c dept 135 spectrum , suggesting that c-2 and c-5 of this ring system are substituted ( quaternary carbons ) . this was confirmed by the hmbc correlation of h-6 ( h 4.63 ) to c-4 ( c 111.4 ) and c-5 ( c 144.6 ) , h-4 ( h 6.28 ) to c-6 ( c 56.6 ) , h-3 ( h 7.07 ) to the aldehyde carbon ( c 180.5 ) , and the aldehyde proton ( h 9.31 ) to c-2 ( c 133.4 ) . in addition , the connection between c-1 and n-1 was clearly indicated by the hmbc correlation of h-1 ( h 5.43 ) to c-2 ( c 133.4 ) . therefore , the structure of 1 was established unambiguously as methyl 2-[2-formyl-5-(hydroxymethyl)-1h - pyrrol-1-yl]propanoate . h h cosy ( bold lines ) and key hmbc ( arrows ) correlations of the new compound 1 . on the basis of the spectroscopic data measurements and the comparison of the data obtained with published values , the structures of the known compounds 28 were identified as 4-[formyl-5-(methoxymethyl)-1h - pyrrol-1-yl]butanoic acid , 2 , 5-(methoxymethyl)-1h - pyrrole-2-carbaldehyde , 3 , 5-(hydroxymethyl)-1h - pyrrole-2-carbaldehyde , 4 , methyl-5-hydroxy-2-pyridinecarboxylate , 5 , 5-hydroxy-2-pyridyl methyl ketone , 6 , 5-hydroxymethyl-2-furancarbaldehyde , 7 , and 4-hydroxybenzoic acid , 8 . in the present study , all of the isolates , 18 , were evaluated in vitro using the hydroxyl radical - scavenging and qr induction assays . the new compound , 1 , and the known pyrrole alkaloid , 2 , showed activities in both assays , as shown in table 1 . when the qr - inducing activities were compared among the structurally related pyrrole alkaloids , 14 , it was observed that 2 , containing a butanoic acid side chain at position n-1 , exhibited the greatest potency ( cd 2.4 m ) ; 1 , with a methyl propanoate substitution attached to n-1 , showed much less potency ( cd 43.1 m ) , whereas 3 and 4 , each bearing no substituent to n-1 , displayed no discernible activity ( both cd > 100 m ) , suggesting that the length of the side chain at n-1 may significantly affect the qr - inducing activity of this type of compound . compounds 35 , 7 , and 8 did not show hydroxyl radical - scavenging or qr - inducing activity at the concentrations tested . compounds with cd values of < 20 , 20100 , and > 100 m are considered significantly active , weakly active , and inactive , respectively . ci , chemoprevention index (= ic50/cd ) . positive control for hydroxyl radical - scavenging assay compounds 1 , 3 , and 4 are analogues of 2 and are also pyrrole alkaloids . in contrast , no alkaloid has been previously isolated or detected spectroscopically from african mango . in addition , both goji berry and african mango reportedly can be used as antiobesity medicinal plants . due to the unexpected presence of these pyrrole alkaloids and their structural similarity to and occurrence as constituents found in goji berries , as well as the public concern and a recent report about frequent contamination in commercial african mango samples , a comprehensive comparison of the commercial sample labeled as african mango seeds ( product a ) with authentic goji berry samples ( products b d ) and african mango seeds ( product e ) was conducted via microscopic analysis as well as chemotaxonomy using lc - ms and h nmr fingerprinting , as shown in the following sections . in the powders of products a d , irregular polygonal testa sclereids ( stone cells ) were observed with thickened and curved walls as well as distinct striations in surface view ( figure 3 ) , characteristic of the seeds of goji berry . this microscopic feature was not found in the authentic sample of african mango seed powder ( product e ) . microscopic structures of testa sclereids ( stone cells ) observed in products a d . products a d shared very similar hplc chromatograms , which differed significantly from that of product e ( figure 4 ) . compound 3 had been all consumed for bioassay and thus could not be used as a standard . however , compounds 1 , 2 , and 48 isolated from product a were present in the authentic goji berry samples ( products b d ) , as identified by comparison of the retention times , uv profiles , and mass spectra of the peaks with standard compounds that had been identified by nmr ( table 2 ) . in contrast , the hplc chromatogram of the authentic sample of african mango seeds ( product e ) was shown to be very different , containing gallic acid , methyl gallate , ellagic acid , and their derivatives as major constituents , as identified by the same analytical methods mentioned above . hplc chromatograms ( 254 nm ) of products a e and standard compounds isolated from product a. visual inspection of the h nmr spectra revealed that all samples tested contained triacylglycerols as major constituents . by comparison , however , products a d each contained pyrrole alkaloids as minor constituents with characteristic signals at h 6.06.4 ppm that were not observed in product e. the presence of signals in the region of h 9.49.8 ppm can be attributed to the aldehyde functionality of the isolated pyrrole-2-carbaldehyde compounds and further distinguished the botanical identity of product a from product e. additionally , the signals that were detected only in product e may be ascribed to the previously reported constituents of african mango , namely , methyl gallate , ellagic acid , or their derivatives . the region of the nmr spectra pertinent to these comparisons is shown in figure 5 . the relative cleanness of the proton nmr spectrum of the authentic african mango seed extract suggests that h nmr fingerprinting may be a useful tool for the future authentication and/or quality control of such samples . h nmr fingerprinting ( h 5.510.1 ppm ) of products a e ( cdcl3 , 400 mhz ) . accordingly , chemotaxonomic examination by lc - it - ms and h nmr fingerprinting using authentic samples of goji berries and african mango suggested that there was evidence of contamination , adulteration , and/or mislabeling of the commercial african mango sample evaluated ( product a ) , in agreement with a concerning trend previously reported . it is worth noting that goji berry from the same batch as product c was previously reported to exhibit in vivo inhibitory activity in a rat n - nitrosomethylbenzylamine ( nmba)-induced esophageal cancer model , and the detection of the in vitro active compounds 1 , 2 , and 6 in product c suggests that these compounds may possess potential in vivo cancer chemopreventive activity and hence merit further investigation .

bioassay - guided fractionation of a commercial sample of african mango ( irvingia gabonensis ) that was later shown to be contaminated with goji berry ( lycium sp . ) led to the isolation of a new pyrrole alkaloid , methyl 2-[2-formyl-5-(hydroxymethyl)-1h - pyrrol-1-yl]propanoate , 1 , along with seven known compounds , 28 . the structures of the isolated compounds were established by analysis of their spectroscopic data . the new compound 1 g showed hydroxyl radical - scavenging activity with an ed50 value of 16.7 m , whereas 4-[formyl-5-(methoxymethyl)-1h - pyrrol-1-yl]butanoic acid ( 2 ) was active in both the hydroxyl radical - scavenging ( ed50 11.9 m ) and quinone reductase - induction [ cd ( concentration required to double qr activity ) 2.4 m ) ] assays used . the isolated compounds were shown to be absent in a taxonomically authenticated african mango sample but present in three separate authentic samples of goji berry ( lycium barbarum ) using lc - ms and 1h nmr fingerprinting analysis , including one sample that previously showed inhibitory activity in vivo in a rat esophageal cancer model induced with n - nitrosomethylbenzylamine . additionally , microscopic features characteristic of goji berry were observed in the commercial african mango sample .

Introduction Materials and Methods Results and Discussion

( irvingiaceae ) has become known as african mango and is unrelated botanically to the common , or indian , mango [ mangifera indica ( anacardiaceae ) ] . phytochemical investigations of the stem bark of i. gabonensis have led to the isolation of antioxidant and hepatoprotective triterpenes and phenols . in 2011 , atawodi and co - workers published the first report on the phenolic compound profile of i. gabonensis seeds , in which methyl gallate , ellagic acid , and other ellagic acid derivatives were detected by lc - uv - ms in the methanol extract of the seeds , which also demonstrated strong in vitro antioxidant activity . a recent study of dietary supplements wholly or partially containing african mango seed extracts using uhplc - hrms found evidence of contamination , adulteration , and/or mislabeling among multiple commercial samples when compared to authentic samples . it is noteworthy that no alkaloids have been previously isolated or detected spectroscopically from african mango , according to the accessible and surveyed literature . in a continuing search for natural inhibitors of carcinogenesis , the chloroform - soluble extract of a commercially sourced african mango seed extract showed in vitro hydroxyl radical - scavenging and qr - inducing activities , and thus was selected for further study . bioactivity - guided fractionation of this sample , using both hydroxyl radical - scavenging and qr - inducing assays , led to the isolation of a new pyrrole alkaloid , 1 , along with seven known compounds , 28 . the fruits of l. chinense and of the closely related species lycium barbarum l. are commonly known as goji berry or wolfberry and are used almost interchangeably . the in vivo inhibitory activity of powdered l. barbarum ( wolfberry ) fruits when evaluated in an n - nitrosomethylbenzylamine - induced esophageal cancer model was reported recently in rats . other recent studies have indicated that goji berry extracts and l. barbarum polysaccharides possess a range of biological effects , including antiaging , antioxidant , antitumor , immunomodulatory , and cytoprotective activities . due to the unexpected presence of the pyrrole alkaloids isolated , 14 , and their structural similarity to constituents found in goji berries , as well as the recent report of contamination in commercial african mango samples , the identity of the source material investigated came into question . the present study includes the bioactivity - guided isolation , identification , and biological evaluation of compounds 18 in addition to comparison of the commercial product with authentic african mango seeds and l. barbarum samples by chemotaxonomy using lc - ms and h nmr fingerprinting as well as microscopic analysis . nmr spectroscopic data were recorded at room temperature on a bruker avance drx-400 mhz spectrometer ( bruker , billerica , ma , usa ) using standard bruker pulse sequences . guard column of the same material ( waters , milford , ma , usa ) was used for semipreparative hplc , along with a hitachi system composed of an l-2130 prep pump , an l-2200 autosampler , and an l-2450 diode array detector ( hitachi , tokyo , japan ) . gallic acid , methyl gallate , and ellagic acid that served as standards for hplc - pda analysis were authentic samples isolated in previous studies and stored in a compound library at the ohio state university . the commercial sample labeled as powdered seeds of african mango ( i. gabonensis ) , used for isolation , was obtained by nature s sunshine products , inc . an authentic sample of african mango seed powder , collected in nigeria and imported by nutralliance ( yorba linda , ca , usa ) , was purchased by nature s sunshine products , inc . the commercial sample labeled as the powdered seeds of african mango ( product a , 5 kg ) was extracted by maceration with meoh ( 3 10 l ) at room temperature , for 3 days each . 1000 g ) was suspended in h2o ( 1 l ) and then partitioned in turn with hexanes ( 3 1 l ) , chcl3 ( 3 1 l ) , etoac ( 3 1 l ) , and n - buoh ( 3 1 l ) to afford dried hexanes ( 40.1 g ) , chcl3 ( 5.0 g ) , etoac ( 6.5 g ) , n - buoh ( 166.0 g ) , and h2o - soluble ( ca . the chcl3-soluble extract showed activities in both hydroxyl radical - scavenging ( ed50 6.3 g / ml ) and qr induction ( cd 12.4 g / ml ) assays and hence was selected for further fractionation . fractions f01f03 showed activity in either the hydroxyl radical - scavenging assay or the qr induction assay and were chosen for further purification . 400 mg , eluted by meoh / h2o 20:80 ) was subjected to passage over a sephadex lh-20 column with meoh / h2o ( 20:80 to pure meoh , stepwise ) as the eluting solvent system to yield compound 8 ( eluted by meoh / h2o 50:50 ; 3.0 mg ) and a further subfraction f010211 ( ca . guard column of the same material ( waters ) and with ch3cn / h2o by isocratic elution ( 3:97 ; 8 ml / min ) to afford compounds 5 ( tr 68.6 min ; 5.7 mg ) and 6 ( tr 55.0 min ; 0.8 mg ) and a further subfraction , f01021103 ( ca . 0.0001% of dry weight ) as a colorless resin : [ ]d + 28.0 ( c 0.1 , meoh ) ; uv ( meoh ) max ( log ) 291 ( 4.09 ) nm ; ir ( film ) max 3424 , 3120 , 2946 , 2850 , 2803 , 2727 , 1749 , 1655 cm ; h nmr ( 400 mhz , cd3od ) 9.31 ( 1h , s , cho ) , 7.07 ( 1h , d , j = 4.0 hz , h-3 ) , 6.28 ( 1h , d , j = 4.0 hz , h-4 ) , 5.43 ( 1h , q , j = 7.0 hz , h-1 ) , 4.63 ( 2h , abq , = 8.1 hz , j = 13.9 hz , h-6 ) , 3.67 ( 3h , s , och3 ) , 1.67 ( 3h , d , j = 7.0 hz , ch3 ) ; c nmr ( 100 mhz , cd3od ) 180.5 ( cho ) , 172.5 ( coo ) , 144.6 ( c-5 ) , 133.4 ( c-2 ) , 127.4 ( c-3 ) , 111.4 ( c-4 ) , 56.6 ( c-6 ) , 55.8 ( c-1 ) , 52.8 ( och3 ) , 18.0 ( ch3 ) ; hresims m / z 234.0733 [ m + na ] ( calcd for c10h13no4na , 234.0742 ) . hydroxyl radical - scavenging activity was tested according to the method previously reported . two drops of diluted glycerin and a coverslip were added to the slide , followed by immediate observation under a nikon eclipse te300 optical microscope ( nikon , tokyo , japan ) , along with an a01f881036 camera module ( roper scientific , trenton , nj , usa ) for digital imaging of the microscopic structures observed . , 5 m , xbridge c18 analytical column ( waters ) , along with a hitachi system composed of an l-2130 pump and an l-2450 diode array detector ( hitachi ) . the mobile phase consisted of 0.05% trifluoroacetic acid in water ( a ) and acetonitrile ( b ) using a gradient program of 320% b from 0 to 60 min and 20100% b from 60 to 70 min . the lc - it - ms analysis employed the same separation conditions and xbridge c18 analytical column as used for the hplc - pda analysis mentioned above , on a waters alliance 2690 separation module . the electrospray ionization ion trap mass spectrometry ( esi - it - ms ) was performed on a bruker dual funnel amazon etd ion trap mass spectrometer ( bremen , germany ) equipped with an orthogonal electrospray source operated in positive - ion mode . the chcl3-soluble extract of product a ( a commercial sample labeled as powdered seeds of african mango ) was found to be the most potent among the hexanes- , chcl3- , etoac- , n - buoh- , and h2o - soluble extracts in the in vitro hydroxyl radical - scavenging ( ed50 , concentration scavenging hydroxyl radical by 50% , 6.3 g / ml ) and qr induction ( cd , concentration required to double qr activity , 12.4 g / ml ) assays . fractions f01f03 of the chcl3-soluble extract showed hydroxyl radical - scavenging activity , with ed50 values of 3.3 , 5.6 , and 6.8 g / ml , respectively . in this way , bioactivity - guided fractionation of product a led to the isolation of a new pyrrole alkaloid , 1 , and seven known compounds , 28 , as shown in figure 1 . structures of compounds isolated from a goji berry - contaminated commercial sample labeled african mango ( irvingia gabonensis ) . the molecular formula was determined as c10h13no4 on the basis of the sodiated molecular ion peak in the hresims . the hmbc correlation ( figure 2 ) of the methoxy group ( h 3.67 , 3h , s , och3 ) to the carbonyl at c 172.5 revealed that this methoxy group forms an ester with this carbonyl . the quartet of h-1 ( h 5.43 , j = 7.0 hz ) and doublet of the methyl group ( h 1.67 , j = 7.0 hz ) , as well as their correlation in the cosy spectrum , clearly indicated the connection of the methyl group ( c 18.1 ) to c-1 ( c 55.8 ) . the chemical shifts and coupling constants of the two protons at h 7.07 ( 1h , d , j = 4.0 hz , h-3 ) and h 6.28 ( 1h , d , j = 4.0 hz , h-4 ) implied the presence of a pyrrole ring . also , the two carbons of the pyrrole ring at the lower field region ( c 144.6 and 133.4 ) were not present in the c dept 135 spectrum , suggesting that c-2 and c-5 of this ring system are substituted ( quaternary carbons ) . this was confirmed by the hmbc correlation of h-6 ( h 4.63 ) to c-4 ( c 111.4 ) and c-5 ( c 144.6 ) , h-4 ( h 6.28 ) to c-6 ( c 56.6 ) , h-3 ( h 7.07 ) to the aldehyde carbon ( c 180.5 ) , and the aldehyde proton ( h 9.31 ) to c-2 ( c 133.4 ) . therefore , the structure of 1 was established unambiguously as methyl 2-[2-formyl-5-(hydroxymethyl)-1h - pyrrol-1-yl]propanoate . h h cosy ( bold lines ) and key hmbc ( arrows ) correlations of the new compound 1 . on the basis of the spectroscopic data measurements and the comparison of the data obtained with published values , the structures of the known compounds 28 were identified as 4-[formyl-5-(methoxymethyl)-1h - pyrrol-1-yl]butanoic acid , 2 , 5-(methoxymethyl)-1h - pyrrole-2-carbaldehyde , 3 , 5-(hydroxymethyl)-1h - pyrrole-2-carbaldehyde , 4 , methyl-5-hydroxy-2-pyridinecarboxylate , 5 , 5-hydroxy-2-pyridyl methyl ketone , 6 , 5-hydroxymethyl-2-furancarbaldehyde , 7 , and 4-hydroxybenzoic acid , 8 . in the present study , all of the isolates , 18 , were evaluated in vitro using the hydroxyl radical - scavenging and qr induction assays . the new compound , 1 , and the known pyrrole alkaloid , 2 , showed activities in both assays , as shown in table 1 . when the qr - inducing activities were compared among the structurally related pyrrole alkaloids , 14 , it was observed that 2 , containing a butanoic acid side chain at position n-1 , exhibited the greatest potency ( cd 2.4 m ) ; 1 , with a methyl propanoate substitution attached to n-1 , showed much less potency ( cd 43.1 m ) , whereas 3 and 4 , each bearing no substituent to n-1 , displayed no discernible activity ( both cd > 100 m ) , suggesting that the length of the side chain at n-1 may significantly affect the qr - inducing activity of this type of compound . compounds 35 , 7 , and 8 did not show hydroxyl radical - scavenging or qr - inducing activity at the concentrations tested . positive control for hydroxyl radical - scavenging assay compounds 1 , 3 , and 4 are analogues of 2 and are also pyrrole alkaloids . due to the unexpected presence of these pyrrole alkaloids and their structural similarity to and occurrence as constituents found in goji berries , as well as the public concern and a recent report about frequent contamination in commercial african mango samples , a comprehensive comparison of the commercial sample labeled as african mango seeds ( product a ) with authentic goji berry samples ( products b d ) and african mango seeds ( product e ) was conducted via microscopic analysis as well as chemotaxonomy using lc - ms and h nmr fingerprinting , as shown in the following sections . in the powders of products a d , irregular polygonal testa sclereids ( stone cells ) were observed with thickened and curved walls as well as distinct striations in surface view ( figure 3 ) , characteristic of the seeds of goji berry . this microscopic feature was not found in the authentic sample of african mango seed powder ( product e ) . microscopic structures of testa sclereids ( stone cells ) observed in products a d . however , compounds 1 , 2 , and 48 isolated from product a were present in the authentic goji berry samples ( products b d ) , as identified by comparison of the retention times , uv profiles , and mass spectra of the peaks with standard compounds that had been identified by nmr ( table 2 ) . in contrast , the hplc chromatogram of the authentic sample of african mango seeds ( product e ) was shown to be very different , containing gallic acid , methyl gallate , ellagic acid , and their derivatives as major constituents , as identified by the same analytical methods mentioned above . by comparison , however , products a d each contained pyrrole alkaloids as minor constituents with characteristic signals at h 6.06.4 ppm that were not observed in product e. the presence of signals in the region of h 9.49.8 ppm can be attributed to the aldehyde functionality of the isolated pyrrole-2-carbaldehyde compounds and further distinguished the botanical identity of product a from product e. additionally , the signals that were detected only in product e may be ascribed to the previously reported constituents of african mango , namely , methyl gallate , ellagic acid , or their derivatives . the relative cleanness of the proton nmr spectrum of the authentic african mango seed extract suggests that h nmr fingerprinting may be a useful tool for the future authentication and/or quality control of such samples . accordingly , chemotaxonomic examination by lc - it - ms and h nmr fingerprinting using authentic samples of goji berries and african mango suggested that there was evidence of contamination , adulteration , and/or mislabeling of the commercial african mango sample evaluated ( product a ) , in agreement with a concerning trend previously reported . it is worth noting that goji berry from the same batch as product c was previously reported to exhibit in vivo inhibitory activity in a rat n - nitrosomethylbenzylamine ( nmba)-induced esophageal cancer model , and the detection of the in vitro active compounds 1 , 2 , and 6 in product c suggests that these compounds may possess potential in vivo cancer chemopreventive activity and hence merit further investigation .

the thermochromism of simple dye molecules is a good indicator of changes in the dye environment polarity following changes in temperature . in many cases thermochromic shifts in absorption and emission reflect the temperature induced changes in refractive index , n , and electric permittivity , , of the solvent as observed by our group in for 4-aminophthalimide ( 4-ap ) and coumarin 153 ( c153 , scheme 1 ) dissolved in several polar non - protic 1-chloroalkanes , or by suppan et al . in [ 36 ] for several probes including 4-ap.scheme 1coumarin 153 ( c153 ) structure coumarin 153 ( c153 ) structure however , for a dye interacting specifically with the solvent molecules , temperature influence on the energy of this interaction can affect much more significantly the scale of thermochromic shifts than for a dye showing nonspecific interaction , related to n(t ) and (t ) dependencies . this additional shift can be of the sign the same as or the opposite to that resulting from nonspecific interactions . in we have found both 4-ap and c153 to form hydrogen bonds ( h - bond ) acting as hydrogen acceptors and donors . both types of h - bonding interactions result in an additional stabilisation of the first excited singlet state s1 of both probes . however , in the case of the probes acting as hydrogen acceptors , this additional stabilization has been found to weaken with decreasing temperature . in the case of the dyes acting as hydrogen donors , a slightly rise in the stabilization was observed with decreasing temperature . to identify the origin of such temperature changes in h - bond energies , a preliminary study on the influence of temperature on absorption and emission , including fluorescence time - resolved measurements , was performed for c153 dissolved in 1-chloropropane ( clp ) . the purpose of this study was to determine the influence of temperature on the kinetics of c153 deactivation from s1 in nonspecifically interacting polar solvents . surprisingly , c153 fluorescence lifetime ( f ) and fluorescence quantum yield ( f ) have been found to follow in this solvent a complex temperature dependence , indicating the intramolecular deactivation is not only controlled by the energy gap law of radiationless deactivation rate . in this report we would like to present results of our further studies of temperature influence on c153 excitation and deactivation . in order to compare new results with the previous ones obtained for clp , the absorption and emission spectra as well as quantum yield and fluorescence lifetime were measured for c153 dissolved in 1-chlorohexane ( clh ) in the temperature range of 183 k323 k. additionally , the same spectra and quantities were determined in propionitrile ( ppn)a hydrogen acceptor ( kamlet - taft polarity scale = 0 , = 0.4 [ 810 ] ) , in trifluoroethanol ( tfetoh)a hydrogen donor ( = 1.51 , = 0 ) and in ethanol ( etoh)a hydrogen donor and acceptor ( = 0.86 , = 0.75 ) . the choice of these solvents was dictated by their h - bonding character and by their melting points , the lower the better . as previously , emission spectra were accumulated using a modified aminco spf-500 spectrofluorimeter with single photon counting detection . time - resolved fluorescence measurements were made using a tcspc system with an instrument response function ( irf ) of 30 ps full - width at half of the maximum ( fwhm ) . the time per channel was set to 12.2 ps and fluorescence decays were collected into 4,096 channels . a home - made analytical software was used to fit the decays with the simplex approximation algorithm . the signal scattered at excitation light wavelength from a ludox in water solution c153 ( fluka ) was used as received , 1-chlorohexane ( aldrich ) , propionitrile ( sigma - aldrich ) , ethanol ( sigma - aldrich ) and tri - fluoroethanol ( sigma - aldrich ) were dehydrated using 3 ( merck ) and 4 ( fluka ) molecular sieves and the sample were prepared under argon atmosphere after solvent dehydration . c153 concentration was kept at ~10 m. it is worth underlining the necessity of careful dehydration , whose failure can lead to incorrect results as shown for 4-ap in . in this report steady - state absorption and emission spectra were measured in selected solvents at different temperature ranges with a 10 k step . figure 1 present normalized absorption spectra of c153 in all solvents used , obtained at room temperature and at 233 k , for the sake of comparison also the ones obtained in 1-chloropropane are shown.fig . 1c153 absorption spectra at room temperature ( a ) and at 233 k ( b ) in : clp ( solid ) , clh ( long dash ) , ppn ( short dash ) , etoh ( dot dash ) and tfetoh ( dot ) c153 absorption spectra at room temperature ( a ) and at 233 k ( b ) in : clp ( solid ) , clh ( long dash ) , ppn ( short dash ) , etoh ( dot dash ) and tfetoh ( dot ) a subtle difference in the shape of the spectra recorded at the two temperatures can be noted for clp and clh . to better resolve this change it is convenient to determine the differences in absorbance at consecutive temperatures at which measurements were made , separated by 10 k. figure 2 presents such derivatives for c153 in clh.fig . 2changes in absorbance of c153 in clh induced by decrease in temperature : 323 k313 k ( solid line ) , 263 k253 k ( dot ) and 193 k183 k ( dash ) changes in absorbance of c153 in clh induced by decrease in temperature : 323 k313 k ( solid line ) , 263 k253 k ( dot ) and 193 k183 k ( dash ) note that the curves shown in fig . 2 include the effects resulting from solvatochromic shifts of absorption , thus they can not be directly interpreted as a result of temperature induced intramolecular changes . nevertheless , in clh ( and clp , not shown ) a vibronic structure of the changes in absorbance can be seen . in fig . 2 the distance in energy of 1,380 cm this value is close to the 1,150 cm frequency of the deactivation accepting mode found in the emission in clp and in gas - phase . in other solvents no structure can be observed which would well correspond to the lack of any clear change in absorption spectra shape when decreasing temperature . note that the ratio of the amplitude of two maxima visible in each curve in fig . 2 changes with decreasing t in favour of the highest most red shifted peak . figure 3 shows the temperature dependence of the peak positions , p , of absorption spectrum s0s1 band in all five solvents . lines represent p(t ) slopes predicted theoretically in the way described later in the text.fig . 3c153 steady - state absorption spectra maximum positions ( p ) vs temperature in : clh ( filled squares ) , clp ( filled triangles ) , ppn ( empty diamonds ) , etoh ( empty triangles ) and tfetoh ( filled circles ) . lines correspond to theoretically determined positions c153 steady - state absorption spectra maximum positions ( p ) vs temperature in : clh ( filled squares ) , clp ( filled triangles ) , ppn ( empty diamonds ) , etoh ( empty triangles ) and tfetoh ( filled circles ) . lines correspond to theoretically determined positions in ppn , etoh and tfetoh the peak positions correspond to p of lognormal curves fitted to the absorption spectra . for clp and clh , due to the vibronic structure of the spectra , no correct fit could be obtained by means of lognormal functions . thus , in this case p corresponds to frequencies at which the maxima of the consecutive absorption spectra were observed . the difference in approaches results in a smoother t dependence in the three specifically interacting solvents . using quinine sulphate in 0.05 m h2so4 ( f = 0.52 ) as a standard , c153 fluorescence quantum yield , f , in all solvents was determined at room temperature . then , by assuming the room temperature emission spectrum in selected solvent as a standard , temperature dependencies of f were determined , shown in fig . 4.fig . 4temperature dependencies of the fluorescence quantum yield , f , of c153 in clh ( a ) , ppn ( b ) , etoh ( c ) and tfetoh ( d ) temperature dependencies of the fluorescence quantum yield , f , of c153 in clh ( a ) , ppn ( b ) , etoh ( c ) and tfetoh ( d ) values of f(t ) follow similar dependencies in clh and ppn , while in etoh and tfetoh they are significantly different . note , that f(t ) in tfetoh is quite similar to the one found in clp . only in etoh analysis of time - resolved emission spectra ( tres ) , discussed in the next section , have shown that this rise is a consequence of a significant retardation of solvation dynamics in etoh at low temperatures . in all solvents f(t ) slope changes from negative into positive at distinct temperatures : 243 k in clh , 273 k in ppn , 273 k in etoh and 283 k in tfetoh . in clp it was 273 k. the changes observed in f are twice as high in clh and ppn as those in etoh and tfetoh . first of all , fluorescence decays were measured for c153 in all solvents in the same temperature ranges as in the case of steady - state absorption and emission spectra . due to some limitation of our experimental setup , during the experiment the excitation wavelength was constant and set to the maximum of the room temperature absorption spectrum in a selected solvent , while the emission wavelength was set to the maximum of the emission spectrum at a selected temperature . in clh and ppn the decay was found to be properly described by a single exponential decay function in the full temperature range . in etoh below 293 k and in tfetoh in the full t range a decrease in temperature induced a change in the second component decay time from 100 ps ( contribution f = 0.2 % ) to 2,200 ps ( f = 25 % ) in etoh , and from 100 ps ( f = 0.6 % ) to 900 ps ( f = 4 % ) in tfetoh . the long component decay time had been found in both protic solvents to follow a similar temperature dependence as the single exponential decay time , also fitted to the data . therefore , fig . 5 shows f of the single exponential fit for clh , ppn and the long component of the double exponential fit for both protic solvents.fig . 5temperature dependencies of the fluorescence decay time for c153 in clh ( a ) , ppn ( b ) , etoh ( c ) and tfetoh ( d ) . in the alcohols the dominant long component time ( filled circles ) of the double - exponential decay function fitted to the experimental decays is presented along with the single exponential decay time for tfetoh ( empty circles ) temperature dependencies of the fluorescence decay time for c153 in clh ( a ) , ppn ( b ) , etoh ( c ) and tfetoh ( d ) . in the alcohols the dominant long component time ( filled circles ) of the double - exponential decay function fitted to the experimental decays is presented along with the single exponential decay time for tfetoh ( empty circles ) there is a similarity in f(t ) dependencies in clh and ppn , as well as in etoh and tfetoh . similarly , as for f(t ) there are characteristic temperatures at which changes in f(t ) slope are observed in clh ( 263 k ) and ppn ( 273 k ) . in alcohols a modulation of f(t ) can be observed in t ranges between 300 k and 273 k , the same in which changes in f(t ) slope occur . the scale of the changes in f seems to be linked to the polarity of the solvent , the higher the polarity the larger the range of the f values . for c153 in clp and clh we also noticed that incorrect dehydration of these solvents led to the f(t ) dependencies significantly different from that shown in fig . 5 and in ref . . we had preliminarily dehydrated 50 ml of clh and next we added to it 2 l of distilled water , which corresponded to a 2.210 m water concentration . fluorescence decays in such a clh+water mixture were measured at the wavelengths corresponding to the maxima of steady - state spectra collected at subsequent temperatures . they were found to be described by double exponential decay functions with a dominant 56 ns component and a minor ~200 ps second component whose contribution did not exceed 1 % . figure 6a presents the temperature dependence of the long component for c153 dissolved in clh+water mixture along with the dependence found in dehydrated clh shown already in fig . the properly dehydrated sample ( filled circles ) was prepared with ppn dried with molecular sieves twice as long as for the improperly dehydrated one ( empty circles ) , after changing the molecular sieves once . it was also prepared under argon atmosphere in contrast to the sample still contaminated by water.fig . 6temperature dependence of fluorescence decay time of c153 in dehydrated clh ( a)filled circles , in clh+water mixture at 2.210 m water concentration ( a)empty circles , in dehydrated ppn ( b)filled circles and in improperly dehydrated ppn ( b)empty circles temperature dependence of fluorescence decay time of c153 in dehydrated clh ( a)filled circles , in clh+water mixture at 2.210 m water concentration ( a)empty circles , in dehydrated ppn ( b)filled circles and in improperly dehydrated ppn ( b)empty circles water presence makes fluorescence decays much shorter at high temperatures . lowering temperature induces f rise , which asymptotically approaches the values found in dehydrated solvents at the lowest temperatures . these results , combined with f values obtained for c153 in etoh and tfetoh , show that the interaction of c153 with protic solvents leads to a shortening of f . to check if c153+water complexes are formed only in the ground state or also c153+water exciplexes are formed after excitation , fluorescence decays were measured in clh+water solution at three different wavelengths and at 323 k , 293 k and 263 k. the wavelengths were selected from the blue and red ranges of the steady - state spectrum and at its maximum . at the fourth temperature of 213 k fluorescence decays this indicates that water forms complexes with c153 only in the ground state of the probe , in contrast to 4-ap dissolved in the same solvent mixture . such a conclusion is justified as c153 lifetime is three times shorter than that of 4-ap , while water association is much more effective in the latter dye . however , in contrast to 4-ap for which two decay components were found in clh+water mixture ; one corresponding to free 4-ap and the other to 4-ap - water complex / exciplex , c153 decay is described mainly by a single exponential component which must correspond to the c153+water complex . table 1 gives the fluorescence decay components times and amplitudes found at selected wavelengths.table 1times and amplitudes of fluorescence decay components found for c153 in clh+water mixture at 2.2 10 m water concentration and at selected temperatures and wavelengthst [ k] [ nm]1 [ ps]a12 [ ps]a232344544940.821300.1848350080.931900.0755650110.982810.0229345051520.75870.2549252040.942070.0656052211.07160.0726345053970.8820450.1249053900.901310.1056354620.981700.0221344554660.361310.6449056030.902170.1056056091.38500.38 times and amplitudes of fluorescence decay components found for c153 in clh+water mixture at 2.2 10 m water concentration and at selected temperatures and wavelengths increase in 1 with fluorescence wavelength reflects solvation of clh and/or preferential solvation of water . lack of rise in 2 with decreasing t indicates this component is not related to free c153 , but is also a manifestation of solvation dynamics . the multi - exponential character of the fluorescence decays observed for c153 in etoh and tfetoh is similarly as the (t ) dependence in etoh , connected to significantly slower solvation dynamics of the probe in alcohols than in the two other solvents . such a conclusion follows from analysis of tres determined for c153 in etoh at 293 k , 233 k and 193 k and in tfetoh at 293 k and 233 k. tres were determined in a usual way . first , fluorescence decays at 12 selected wavelengths with a 15 nm step were determined in etoh , starting from 480 nm , and in tfetoh starting from 490 nm . next , the decay functions resulting from the fit were normalized in such a way as to equalize the area under the decay to the steady - state fluorescence spectrum intensity at selected wavelength and temperature . steady - state spectra were corrected for the detector sensitivity and scaled by the factor . from the normalized decays tres were reconstructed . next , each spectrum of the tres corresponding to subsequent time delay was fitted by a lognormal function , with the amplitude , asymmetry , peak frequency p and half - width as the parameters of the fit . from p values the correlation function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c(t ) = \left ( { { v_p}(t ) - { v_p}\left ( \infty \right ) } \right)/\left ( { { v_p}(0 ) - { v_p}\left ( \infty \right ) } \right ) $ $ \end{document } was determined . here , p( ) corresponds to the peak frequency of the lognormal curve fitted to the tres spectrum at the longest time delay . finally , c(t ) dependencies were fitted by single- and double - exponential decays , depending on which number of exponential components was found necessary to reproduce c(t ) properly . table 2 presents the obtained c(t ) decay time values , si , along with the component amplitudes , bi , and the average decay time defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left\langle { { { \tau } _ { s } } } \right\rangle = \left ( { \sum\limits_{i } { { { b}_{i}}{{\tau } _ { { si } } } } } \right)/\sum\limits_{i } { { { b}_{i } } } $ $ \end{document}.table 2decay time values , si , and amplitudes , bi , of the exponential decay components fitted to the solvation correlation function , c(t ) , determined for c153 in etoh and tfetoh at selected temperatures . s average decay timesolventt [ k]s1 [ ps]b1s2 [ ps]b2s [ ps]etoh29335135233700.452280.551571932080.359740.65704tfetoh293770.662570.34138233500.233780.77302 decay time values , si , and amplitudes , bi , of the exponential decay components fitted to the solvation correlation function , c(t ) , determined for c153 in etoh and tfetoh at selected temperatures . the values reported in table 2 describe only the slowest part of c(t ) decays , because of a small time - resolution used here . there is a good agreement between the value obtained for c153 in etoh at 293 k and the longest c(t ) component reported in . the solvation dynamics is slower in tfetoh at room t. however , if we analyze the relative change in s in etoh and tfetoh with decreasing t , we see that the solvation is slowed by decreasing t much more efficiently in etoh . this is the reason why f in fig . 4 and f in fig . 5 increase in etoh at the lowest t. strongly retarded solvation at this t range prevents the steady - state spectrum from shifting fully to the red . as a result , fluorescence deactivation rate ( kf ) proportional to as well as to f , increases additionally , f which is calculated by integration of the fluorescence steady - state spectrum , rises because the integration range is shifted to the blue , thus to higher energies . finally , as mentioned earlier , the retarded solvation is responsible for the bi - exponential character of the fluorescence decays in the alcohols measured at the maximum of the steady - state fluorescence spectra . we assume in the next section that the dominant longest component of the fluorescence decays in etoh and tfetoh bring reliable information about c153 deactivation rate , at least in the range of high temperatures . however , for the lowest t such assumption is clearly invalid , thus the interpretation of the kinetic results presented below is correct for c153 in alcohols only in the high temperature range . steady - state absorption and emission spectra were measured in selected solvents at different temperature ranges with a 10 k step . figure 1 present normalized absorption spectra of c153 in all solvents used , obtained at room temperature and at 233 k , for the sake of comparison also the ones obtained in 1-chloropropane are shown.fig . 1c153 absorption spectra at room temperature ( a ) and at 233 k ( b ) in : clp ( solid ) , clh ( long dash ) , ppn ( short dash ) , etoh ( dot dash ) and tfetoh ( dot ) c153 absorption spectra at room temperature ( a ) and at 233 k ( b ) in : clp ( solid ) , clh ( long dash ) , ppn ( short dash ) , etoh ( dot dash ) and tfetoh ( dot ) a subtle difference in the shape of the spectra recorded at the two temperatures can be noted for clp and clh . to better resolve this change it is convenient to determine the differences in absorbance at consecutive temperatures at which measurements were made , separated by 10 k. figure 2 presents such derivatives for c153 in clh.fig . 2changes in absorbance of c153 in clh induced by decrease in temperature : 323 k313 k ( solid line ) , 263 k253 k ( dot ) and 193 k183 k ( dash ) changes in absorbance of c153 in clh induced by decrease in temperature : 323 k313 k ( solid line ) , 263 k253 k ( dot ) and 193 k183 k ( dash ) note that the curves shown in fig . 2 include the effects resulting from solvatochromic shifts of absorption , thus they can not be directly interpreted as a result of temperature induced intramolecular changes . nevertheless , in clh ( and clp , not shown ) a vibronic structure of the changes in absorbance can be seen . in fig . 2 the distance in energy of 1,380 cm this value is close to the 1,150 cm frequency of the deactivation accepting mode found in the emission in clp and in gas - phase . in other solvents no structure can be observed which would well correspond to the lack of any clear change in absorption spectra shape when decreasing temperature . note that the ratio of the amplitude of two maxima visible in each curve in fig . 2 changes with decreasing t in favour of the highest most red shifted peak . figure 3 shows the temperature dependence of the peak positions , p , of absorption spectrum s0s1 band in all five solvents . lines represent p(t ) slopes predicted theoretically in the way described later in the text.fig . 3c153 steady - state absorption spectra maximum positions ( p ) vs temperature in : clh ( filled squares ) , clp ( filled triangles ) , ppn ( empty diamonds ) , etoh ( empty triangles ) and tfetoh ( filled circles ) . lines correspond to theoretically determined positions c153 steady - state absorption spectra maximum positions ( p ) vs temperature in : clh ( filled squares ) , clp ( filled triangles ) , ppn ( empty diamonds ) , etoh ( empty triangles ) and tfetoh ( filled circles ) . lines correspond to theoretically determined positions in ppn , etoh and tfetoh the peak positions correspond to p of lognormal curves fitted to the absorption spectra . for clp and clh , due to the vibronic structure of the spectra , no correct fit could be obtained by means of lognormal functions . thus , in this case p corresponds to frequencies at which the maxima of the consecutive absorption spectra were observed . the difference in approaches results in a smoother t dependence in the three specifically interacting solvents . using quinine sulphate in 0.05 m h2so4 ( f = 0.52 ) as a standard , c153 fluorescence quantum yield , f , in all solvents was determined at room temperature . then , by assuming the room temperature emission spectrum in selected solvent as a standard , temperature dependencies of f were determined , shown in fig . 4.fig . 4temperature dependencies of the fluorescence quantum yield , f , of c153 in clh ( a ) , ppn ( b ) , etoh ( c ) and tfetoh ( d ) temperature dependencies of the fluorescence quantum yield , f , of c153 in clh ( a ) , ppn ( b ) , etoh ( c ) and tfetoh ( d ) values of f(t ) follow similar dependencies in clh and ppn , while in etoh and tfetoh they are significantly different . note , that f(t ) in tfetoh is quite similar to the one found in clp . only in etoh analysis of time - resolved emission spectra ( tres ) , discussed in the next section , have shown that this rise is a consequence of a significant retardation of solvation dynamics in etoh at low temperatures . in all solvents f(t ) slope changes from negative into positive at distinct temperatures : 243 k in clh , 273 k in ppn , 273 k in etoh and 283 k in tfetoh . in clp it was 273 k. the changes observed in f are twice as high in clh and ppn as those in etoh and tfetoh . first of all , fluorescence decays were measured for c153 in all solvents in the same temperature ranges as in the case of steady - state absorption and emission spectra . due to some limitation of our experimental setup , during the experiment the excitation wavelength was constant and set to the maximum of the room temperature absorption spectrum in a selected solvent , while the emission wavelength was set to the maximum of the emission spectrum at a selected temperature . in clh and ppn the decay was found to be properly described by a single exponential decay function in the full temperature range . in etoh below 293 k and in tfetoh in the full t range a decrease in temperature induced a change in the second component decay time from 100 ps ( contribution f = 0.2 % ) to 2,200 ps ( f = 25 % ) in etoh , and from 100 ps ( f = 0.6 % ) to 900 ps ( f = 4 % ) in tfetoh . the long component decay time had been found in both protic solvents to follow a similar temperature dependence as the single exponential decay time , also fitted to the data . 5 shows f of the single exponential fit for clh , ppn and the long component of the double exponential fit for both protic solvents.fig . 5temperature dependencies of the fluorescence decay time for c153 in clh ( a ) , ppn ( b ) , etoh ( c ) and tfetoh ( d ) . in the alcohols the dominant long component time ( filled circles ) of the double - exponential decay function fitted to the experimental decays is presented along with the single exponential decay time for tfetoh ( empty circles ) temperature dependencies of the fluorescence decay time for c153 in clh ( a ) , ppn ( b ) , etoh ( c ) and tfetoh ( d ) . in the alcohols the dominant long component time ( filled circles ) of the double - exponential decay function fitted to the experimental decays is presented along with the single exponential decay time for tfetoh ( empty circles ) there is a similarity in f(t ) dependencies in clh and ppn , as well as in etoh and tfetoh . similarly , as for f(t ) there are characteristic temperatures at which changes in f(t ) slope are observed in clh ( 263 k ) and ppn ( 273 k ) . in alcohols a modulation of f(t ) can be observed in t ranges between 300 k and 273 k , the same in which changes in f(t ) slope occur . the scale of the changes in f seems to be linked to the polarity of the solvent , the higher the polarity the larger the range of the f values . for c153 in clp and clh we also noticed that incorrect dehydration of these solvents led to the f(t ) dependencies significantly different from that shown in fig . 5 and in ref . . we had preliminarily dehydrated 50 ml of clh and next we added to it 2 l of distilled water , which corresponded to a 2.210 m water concentration . fluorescence decays in such a clh+water mixture were measured at the wavelengths corresponding to the maxima of steady - state spectra collected at subsequent temperatures . they were found to be described by double exponential decay functions with a dominant 56 ns component and a minor ~200 ps second component whose contribution did not exceed 1 % . figure 6a presents the temperature dependence of the long component for c153 dissolved in clh+water mixture along with the dependence found in dehydrated clh shown already in fig . the properly dehydrated sample ( filled circles ) was prepared with ppn dried with molecular sieves twice as long as for the improperly dehydrated one ( empty circles ) , after changing the molecular sieves once . it was also prepared under argon atmosphere in contrast to the sample still contaminated by water.fig . 6temperature dependence of fluorescence decay time of c153 in dehydrated clh ( a)filled circles , in clh+water mixture at 2.210 m water concentration ( a)empty circles , in dehydrated ppn ( b)filled circles and in improperly dehydrated ppn ( b)empty circles temperature dependence of fluorescence decay time of c153 in dehydrated clh ( a)filled circles , in clh+water mixture at 2.210 m water concentration ( a)empty circles , in dehydrated ppn ( b)filled circles and in improperly dehydrated ppn ( b)empty circles water presence makes fluorescence decays much shorter at high temperatures . lowering temperature induces f rise , which asymptotically approaches the values found in dehydrated solvents at the lowest temperatures . these results , combined with f values obtained for c153 in etoh and tfetoh , show that the interaction of c153 with protic solvents leads to a shortening of f . to check if c153+water complexes are formed only in the ground state or also c153+water exciplexes are formed after excitation , fluorescence decays were measured in clh+water solution at three different wavelengths and at 323 k , 293 k and 263 k. the wavelengths were selected from the blue and red ranges of the steady - state spectrum and at its maximum . at the fourth temperature of 213 k fluorescence decays this indicates that water forms complexes with c153 only in the ground state of the probe , in contrast to 4-ap dissolved in the same solvent mixture . such a conclusion is justified as c153 lifetime is three times shorter than that of 4-ap , while water association is much more effective in the latter dye . however , in contrast to 4-ap for which two decay components were found in clh+water mixture ; one corresponding to free 4-ap and the other to 4-ap - water complex / exciplex , c153 decay is described mainly by a single exponential component which must correspond to the c153+water complex . table 1 gives the fluorescence decay components times and amplitudes found at selected wavelengths.table 1times and amplitudes of fluorescence decay components found for c153 in clh+water mixture at 2.2 10 m water concentration and at selected temperatures and wavelengthst [ k] [ nm]1 [ ps]a12 [ ps]a232344544940.821300.1848350080.931900.0755650110.982810.0229345051520.75870.2549252040.942070.0656052211.07160.0726345053970.8820450.1249053900.901310.1056354620.981700.0221344554660.361310.6449056030.902170.1056056091.38500.38 times and amplitudes of fluorescence decay components found for c153 in clh+water mixture at 2.2 10 m water concentration and at selected temperatures and wavelengths increase in 1 with fluorescence wavelength reflects solvation of clh and/or preferential solvation of water . lack of rise in 2 with decreasing t indicates this component is not related to free c153 , but is also a manifestation of solvation dynamics . the multi - exponential character of the fluorescence decays observed for c153 in etoh and tfetoh is similarly as the (t ) dependence in etoh , connected to significantly slower solvation dynamics of the probe in alcohols than in the two other solvents . such a conclusion follows from analysis of tres determined for c153 in etoh at 293 k , 233 k and 193 k and in tfetoh at 293 k and 233 k. tres were determined in a usual way . first , fluorescence decays at 12 selected wavelengths with a 15 nm step were determined in etoh , starting from 480 nm , and in tfetoh starting from 490 nm . next , the decay functions resulting from the fit were normalized in such a way as to equalize the area under the decay to the steady - state fluorescence spectrum intensity at selected wavelength and temperature . steady - state spectra were corrected for the detector sensitivity and scaled by the factor . from the normalized decays tres were reconstructed . next , each spectrum of the tres corresponding to subsequent time delay was fitted by a lognormal function , with the amplitude , asymmetry , peak frequency p and half - width as the parameters of the fit . from p values the correlation function \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ c(t ) = \left ( { { v_p}(t ) - { v_p}\left ( \infty \right ) } \right)/\left ( { { v_p}(0 ) - { v_p}\left ( \infty \right ) } \right ) $ $ \end{document } was determined . here , p( ) corresponds to the peak frequency of the lognormal curve fitted to the tres spectrum at the longest time delay . finally , c(t ) dependencies were fitted by single- and double - exponential decays , depending on which number of exponential components was found necessary to reproduce c(t ) properly . table 2 presents the obtained c(t ) decay time values , si , along with the component amplitudes , bi , and the average decay time defined as \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left\langle { { { \tau } _ { s } } } \right\rangle = \left ( { \sum\limits_{i } { { { b}_{i}}{{\tau } _ { { si } } } } } \right)/\sum\limits_{i } { { { b}_{i } } } $ $ \end{document}.table 2decay time values , si , and amplitudes , bi , of the exponential decay components fitted to the solvation correlation function , c(t ) , determined for c153 in etoh and tfetoh at selected temperatures . s average decay timesolventt [ k]s1 [ ps]b1s2 [ ps]b2s [ ps]etoh29335135233700.452280.551571932080.359740.65704tfetoh293770.662570.34138233500.233780.77302 decay time values , si , and amplitudes , bi , of the exponential decay components fitted to the solvation correlation function , c(t ) , determined for c153 in etoh and tfetoh at selected temperatures . s average decay time the values reported in table 2 describe only the slowest part of c(t ) decays , because of a small time - resolution used here . there is a good agreement between the value obtained for c153 in etoh at 293 k and the longest c(t ) component reported in . the solvation dynamics is slower in tfetoh at room t. however , if we analyze the relative change in s in etoh and tfetoh with decreasing t , we see that the solvation is slowed by decreasing t much more efficiently in etoh . this is the reason why f in fig . 4 and f in fig . 5 increase in etoh at the lowest t. strongly retarded solvation at this t range prevents the steady - state spectrum from shifting fully to the red . as a result , fluorescence deactivation rate ( kf ) proportional to as well as to f , increases . additionally , f which is calculated by integration of the fluorescence steady - state spectrum , rises because the integration range is shifted to the blue , thus to higher energies . finally , as mentioned earlier , the retarded solvation is responsible for the bi - exponential character of the fluorescence decays in the alcohols measured at the maximum of the steady - state fluorescence spectra . we assume in the next section that the dominant longest component of the fluorescence decays in etoh and tfetoh bring reliable information about c153 deactivation rate , at least in the range of high temperatures . however , for the lowest t such assumption is clearly invalid , thus the interpretation of the kinetic results presented below is correct for c153 in alcohols only in the high temperature range . thermochromic shifts of c153 emission spectra reported in indicated that the probe forms h - bond with hydrogen accepting solvents , and that the change in energy of this bond , stimulated by probe excitation , increases with decreasing temperature , at least in the vibrationally and solvent relaxed first singlet excited state , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } $ $ \end{document}. these results were obtained as a difference between the emission spectrum positions predicted by the onsager model and the experimental position values . to evaluate in a similar way the possible influence of specific interactions on absorption peak positions 1 .1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \matrix { { \delta { v_{abs } } = 2\frac{{{\mu_g}\left ( { { \mu_g } - { \mu_e } } \right)}}{{{a^3 } } } \cdot \left ( { \frac{{\varepsilon - 1}}{{\varepsilon + 2 } } - \frac{{{n^2 } - 1}}{{{n^2 } + 2 } } } \right ) } \\ { + \frac{{\mu_g^2 - \mu_e^2}}{{{a^3 } } } \cdot \left ( { \frac{{{n^2 } - 1}}{{2{n^2 } + 2 } } } \right ) } \\ { + \frac{{6\mu_g^2\left ( { { a_g } - { a_e } } \right)}}{{{a^6 } } } \cdot { { \left ( { \frac{{\varepsilon - 1}}{{\varepsilon + 2 } } - \frac{{{n^2 } - 1}}{{{n^2 } + 2 } } } \right)}^2 } } \\ { + \frac{{3\left ( { { a_g } - { a_e } } \right)j{j_s}}}{{2hc{a^3}\left ( { j + { j_s } } \right ) } } \cdot \frac{{{n^2 } - 1}}{{{n^2 } + 2 } } } \\ } $ $ \end{document } above , g and e are c153 dipole moments in the ground and excited states , a is the onsager radius of the probe molecule , g and e are the ground and excited state probe polarizabilities , j and js are the probe and solvent ionization potentials and h and c have the usual meanings . these calculations were made assuming the same parameter values for c153 in the franck - condon \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left ( { s_0^{fc},\,s_1^{fc } } \right ) $ $ \end{document } and in the relaxed \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left ( { s_0^{rel},\,s_1^{rel } } \right ) $ $ \end{document } states , thus assuming the same dipole moment g = 6.6 d in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_0^{fc } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_0^{rel } $ $ \end{document } , e = 9.7 d in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{fc } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } $ $ \end{document } , and the same g in both s0 states and e in both s1 states . etoh ionization potential js = 11.05 ev was determined using am1 hamiltonian with the mopac suite , etoh n(t ) and (t ) were determined from the data given in . for the other c153 and solvents the values of vabs were compared with experimental p(t ) in the following way : for a selected solvent the absorption spectrum peak value p(293 k ) was added to the absolute value of the shift vabs ( 293 k ) . in this way a pseudo gas - phase \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \nu_p^{g\prime \prime } $ $ \end{document } position of the spectrum was obtained . next , from \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \nu_p^{g\prime \prime } $ $ \end{document } the absolute values of vabs(t ) were subtracted at subsequent temperatures different from 293 k. the results of such a procedure are shown in fig . 3 as solid lines . it led to the following \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \nu_p^{g\prime \prime } $ $ \end{document } values : 26,060 cm ( clh ) , 26170 ( clp ) , 26120 ( ppn ) , 25840 ( etoh ) and 24920 ( tfetoh ) . as expected , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \nu_p^{g\prime \prime } $ $ \end{document } values are not the same as they were determined assuming that all solvents interacted exclusively nonspecifically which is obviously incorrect . but , they indicate which solvents most probably interact specifically with c153 in the ground state and these solvents are etoh and tfetoh . it is especially important that \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \nu_p^{g\prime \prime } $ $ \end{document } in ppn fall into the same range as found for 1-chloroalkanes , and that the vp ( t ) dependence slope in ppn corresponds exactly to the vabs ( t ) dependence slope , see fig . both these observations indicate that ppn interacts only nonspecifically with c153 in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_0^{rel } $ $ \end{document } , or that the h - bond formed by ppn with c153 do not change in energy after c153 excitation to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{fc } $ $ \end{document } , contrary to what was deduced from the emissive results for c153 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } $ $ \end{document } in the same solvent . on the other hand , a comparison of \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \nu_p^{g\prime \prime } $ $ \end{document } values obtained in 1-chloroalkanes and ppn with those found in alcohols indicates that in etoh a slight additional specific stabilisation take place already in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{fc } $ $ \end{document } , while in tfetoh the specific interaction with c153 in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{fc } $ $ \end{document } is significantly higher in energy than in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_0^{rel } $ $ \end{document}. no temperature dependence of this additional specific stabilisation is observed in etoh , while in tfetoh its energy increases with decreasing temperature . this last result is in contrast to that found for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } $ $ \end{document } . it means that the stokes shift in this solvent should decrease with decreasing temperature and indeed it is observed . figure 7 shows the temperature dependence of the experimental stokes shifts , vs , for c153 in all solvents.fig . 7stokes shift vs temperature for c153 in : clp ( filled circles ) , clh ( empty circles ) , ppn ( filled triangles ) , etoh ( empty triangles ) and tfetoh ( filled squares ) stokes shift vs temperature for c153 in : clp ( filled circles ) , clh ( empty circles ) , ppn ( filled triangles ) , etoh ( empty triangles ) and tfetoh ( filled squares ) the independence of vs of temperature in 1-chloroalkanes is unexpected as the emitting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } $ $ \end{document } state is more strongly stabilized than \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{fc } $ $ \end{document } due to the reorientational solvation of the solvent taking place when c153 is relaxed . the energy of this additional stabilizing interaction should increase with decreasing temperature , which in turn should lead to an increase in vs . reveal that the theoretical stokes shift in clp and clh changes only by 90 cm within the 120 k temperature interval studied in this work a value in the range of the error of the points in fig . 7 for c153 in 1-chloroalkanes . in ppn , etoh and tfetoh even a smaller vs change is expected from the onsager model . in contrast , clear vs ( t ) dependencies in these solvents are observed . in tfetoh vs(t ) in ppn and etoh the increase in vs must be related to an additional stabilization of c153 in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } $ $ \end{document } state , rising in energy with decreasing temperature . the difference between the expected positions of the c153 emission spectra in methanol , ppn and tfetoh and the experimental ones have been found to show temperature dependencies with the same slope signs as observed in fig . 7 for vs in the same two solvents . the analysis of the results given in and in this work is based on the continuum solvent onsager model , thus their reliability can be questioned by those who believe that this model fails in solvation description . additionally , we have found the absorption spectrum full width at half of the maximum ( fwhm ) to be higher than that of the emission spectrum in all solvents , at each temperature . according to such an observation can be a manifestation of a nonlinear dependence of the local solute potential on the solute dipole moment . this observation would however need a much deeper study , as absorption and emission spectra fwhm are strongly dependent on the frequency of the most active vibrational mode in a selected type of transition . our results , shown later , and these presented in reveal this frequency to be higher in the absorbing ground state , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_0^{rel } $ $ \end{document } , than in the emitting \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } $ $ \end{document}. nevertheless , the experimental vs(t ) dependence obtained for c153 in ppn compared to that found in 1-chloroalkanes gives a very strong argument for the presence of specific interactions between c153 in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } $ $ \end{document } and ppn molecules . thus , the results show that c153 can act as a hydrogen donor . the presence of this type of h - bonding , however , does not influence c153 f(t ) dependence , as can be deduced from results presented in fig . 5 . these results show that the protic character of the solvent has a significant impact on the f(t ) dependence , when compared to non - protic solvents ( clp as well ) . overall , the shortest lifetime is observed in the most protic tfetoh , longer in etoh , clh , and the longest in ppn . in alcohols a slight modulation of the linear f(t ) dependence can be observed , while in clh and ppn the values of f changes with temperature in a similar way as in clp , that is the slope of f(t ) changes in sign at a temperature in the range 260280 k. in both protic solvents a small modulation of f(t ) takes place in the same temperature range in which the change in sign of the f(t ) dependence occurs in clh and ppn . thus , we can assume that h - bonding with protic solvents dilutes c153 f(t ) dependence resulting from pure intramolecular deactivation observed in clh and ppn . non - negligible is also the retarded solvation , which however as shown for c153 in etoh is expected to lead to an increase in f . the f(t ) dependence in neat solvents cover a narrow range of f values as can be seen in fig . such subtle changes in f are also a manifestation of the narrow ranges in which f changes in all solvents . together these quantities gives the radiative , kf , and non - radiative , knr , rates . 8temperature dependencies of the radiative , ( a ) kf , and non - radiative , ( b ) knr , rates in clh ( filled circles ) , ppn ( empty circles ) , etoh ( filled triangles ) and tfetoh ( empty triangles ) temperature dependencies of the radiative , ( a ) kf , and non - radiative , ( b ) knr , rates in clh ( filled circles ) , ppn ( empty circles ) , etoh ( filled triangles ) and tfetoh ( empty triangles ) non - radiative deactivation in c153 is known to be governed by internal conversion . according to our knowledge no isc for this molecule has ever been observed . relative knr values at the same temperatures in all solvents indicate that the energy - gap law partly controls non - radiative deactivation . the order in which knr values increase at a selected temperature corresponds well to the energy of emission in the solvents , thus to the emission position . however , the energy - gap law predicts an exponential decay of knr with temperature rising . the temperature range in which measurements were made does not correspond to the tail of decaying knr(t ) dependence , see fig . 8 in . in etoh the non - exponentiality is evident in the low temperature range . however , in this solvent the retardation of the solvent relaxation is the source of the low - temperature knr(t ) dependence , as the emission spectrum is not shifted totally to the red , which in turn leads to a drop in knr at the lowest t. in clh and ppn knr(t ) slope sign changes at higher temperatures . similarly to what was observed for c153 in clp , this effect is in conflict with the energy - gap law . radiative rate kf(t ) dependencies have also similar features in all solvents . using them , the modulus squared emission transition dipole moments at subsequent temperatures were determined , as shown in fig . these values were found using the birks equation : 2\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \left| m \right|_{e \to g}^2 = \frac{{3h}}{{64{\pi^4}}}\frac{1}{{{n^3}}}{k_f}\widetilde{v}_f^ { - 3 } , $ $ \end{document}3\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \widetilde{v}_f^ { - 3 } = \frac{{\int { i(v){v^ { - 3}}dv } } } { { \int { i(v)dv } } } . 9modulus squared of fluorescence ( meg ) transition dipole moments in clh ( filled circles ) , ppn ( empty circles ) , etoh ( filled triangles ) and tfetoh ( empty triangles ) modulus squared of fluorescence ( meg ) transition dipole moments in clh ( filled circles ) , ppn ( empty circles ) , etoh ( filled triangles ) and tfetoh ( empty triangles ) now , we see that the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } \to s_0^{fc } $ $ \end{document } radiative transition probability differs in protic solvents from that in non - protic ones . once again we stress that the results at lower temperatures in alcohols ( especially in etoh ) are not reliable . except for ppn , the t dependencies in the other solvents are not as well defined as for f . it is connected to the noise in f and to the range of values in which f changes in a particular solvent . we would like to point out that f(t ) results are blurred by a much smaller relative noise , comparing to f(t ) , which was determined indirectly from the results obtained by two different instruments . however , in clh similarly as in ppn , we can see that meg starts to rise with t increasing from 183 k to end by falling at the highest temperature . it is also interesting to notice that except for the points at 323 k , meg follows similar changes ( increase / decrease ) in both protic solvents . it suggests that an additional modulation of meg may be present in both solvents , however it is out of reach of the resolution of our equipment . changes in meg indicate changes in the equilibrium distance between c153 s0 and s1 potential energy surfaces . to check whether steady - state emission of c153 in ppn and clh reflects such changes , the same model of emission spectrum was applied as in , described in . according to this model , the emission spectrum of the molecule in a homogeneous non - interacting solvent can be defined as ( mks):4\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ i(v ) = \sum\limits_{{n_1 } } { \sum\limits_{{n_2 } } { \ldots \sum\limits_{{n_n } } { { i_{{n_1}{n_2 } \ldots { n_n } } } } } } ( v ) $ $ \end{document}5\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { i_{{n_1}{n_2 } \ldots { n_{\text{n}}}}}\left ( \nu \right ) = { \left ( { \frac{{{e_{00 } } - \sum\limits_{{\text{j } } = 1}^{\text{n } } { { n_{\text{j}}}h{\nu_{\text{j } } } } } } { { { e_{00 } } } } } \right)^3}\prod\limits_{{\text{j } } = 1}^{\text{n } } { \left ( { \frac{{\xi_{\text{j}}^{{n_{\text{j}}}}}}{{{n_{\text{j } } } ! } } } \right ) } \exp \left [ { - 4\left ( { \ln ( 2 ) } \right){{\left ( { \frac{{h\nu - { e_{00 } } + \sum\limits_{{\text{j } } = 1}^{\text{n } } { { n_{\text{j}}}h{\nu_{\text{j } } } } } } { { \delta { \nu_{1/2 } } } } } \right)}^2 } } \right ] $ $ \end{document}6\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \xi_j } = { { 1 } \left/ { 2 } \right.}{\left ( { { \delta_j } } \right)^2 } $ $ \end{document } emission intensity is given in units of the number of quanta emitted per energy interval . the number of modes is truncated to n most active accepting vibrational modes in the ground state . e00 is the energy gap between the lowest levels of the fitted vibronic modes of s0 and s1 states . ( 2 , 3 ) has been limited to n = 1 , n1 = 6 . the symbol j is the j - th accepting mode frequency . as the modes number was set to unity , we use below a substitution j = a , while is the frequency at which the emission spectrum is measured , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta { \overline v_{1/2 } } $ $ \end{document } is the full width at half - maximum of the gaussian inhomogeneous broadening , and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline \xi_{\text{a } } } $ $ \end{document } is the huang - rhys factor related to the equilibrium bond distance difference , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline \delta_{\text{a } } } $ $ \end{document } , between the excited and ground states , that is the parameter we are interested in . ( 2 ) served as a fitting model of the emission spectra at subsequent temperatures . e00 , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline v_{\text{a } } } $ $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline \xi_{\text{a } } } $ $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta { \overline v_{1/2 } } $ $ \end{document } and a scaling a0 factor ( by which i( ) was multiplied ) were parameters of the fit . the overlines in the symbols indicates \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline v_{\text{a } } } $ $ \end{document } is in fact an average mode . the quality of the fit was estimated using the value and the errors of the fitted parameters values . similar results were obtained in both solvents , except for e00 which included a solvent induced shift , thus it was higher in clh than in ppn ( fig . there is a correlation between \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline v_{\text{a } } } $ $ \end{document}(t ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta { \overline v_{1/2 } } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline \xi_{\text{a } } } $ $ \end{document}(t ) dependencies , with significant changes in the same temperature range in which f(t ) is observed to change . at this stage it is not possible to deduce how much these correlations affect the results of the fit , thus , to what extent \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline v_{\text{a } } } $ $ \end{document}(t ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta { \overline v_{1/2 } } $ $ \end{document } and \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline \xi_{\text{a } } } $ $ \end{document}(t ) dependencies reflects a true physical effect . the similarity between \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline v_{\text{a } } } $ $ \end{document } , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta { \overline v_{1/2 } } $ $ \end{document } and the corresponding quantities in shows that the 1,150 cm ( in fig . however , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline \xi_{\text{a } } } $ $ \end{document}(t ) changes are hard to explain on the basis of a single mode deactivation , as they would indicate a rise and then a fall in equilibrium distance with decreasing temperature for c153 in clh . in ppn an additional preliminary fall in this distance would be expected . however , assuming also that other accepting modes , as the 810 cm and 360 cm discussed in , are active in the fluorescence transition a quite simple explanation of the temperature dependence of c153 radiative deactivation can be proposed.fig . 10temperature dependence of : ( a ) e00 , s1s0 energy gap for c153 in clh ( filled circles ) and ppn ( empty circles ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta { \overline v_{1/2 } } $ $ \end{document}-full width at half - maximum in clh ( filled squares ) and ppn ( empty squares ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline v_{\text{a } } } $ $ \end{document}-in clh ( filled triangles ) and ppn ( empty triangles ) ; ( b ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline \xi_{\text{a } } } $ $ \end{document}huang - rhys vibrational coupling factor in clh ( filled circles ) and ppn ( empty circles ) as obtained from fit of c153 emission spectra in both solvents to the model given in eqs . ( 46 ) temperature dependence of : ( a ) e00 , s1s0 energy gap for c153 in clh ( filled circles ) and ppn ( empty circles ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \delta { \overline v_{1/2 } } $ $ \end{document}-full width at half - maximum in clh ( filled squares ) and ppn ( empty squares ) , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline v_{\text{a } } } $ $ \end{document}-in clh ( filled triangles ) and ppn ( empty triangles ) ; ( b ) \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline \xi_{\text{a } } } $ $ \end{document}huang - rhys vibrational coupling factor in clh ( filled circles ) and ppn ( empty circles ) as obtained from fit of c153 emission spectra in both solvents to the model given in eqs . ( 46 ) both steady - state absorption ( fig . 2 ) and fluorescence results ( fig . 10 ) indicate that a change in the equilibrium distance \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline \delta_{\text{a } } } $ $ \end{document } occurs on decreasing temperature . according to the born - oppenheimer approximation the transition dipole moment between the excited ( ) and ground ( ) states is proportional to : 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { m_{e \to g } } = \left\langle { \psi \prime } \right|\widehat{\mu } \left| { \psi \prime \prime } \right\rangle \propto { p_{el}}\left ( { \overline r } \right ) \cdot \int { \psi \prime { * _ { vib}}\psi \prime { \prime_{vib}}dr } , $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { p_{el}}\left ( { \overline r } \right ) $ $ \end{document } is the purely electronic transition dipole moment , dependent on the r - centroid for the transition . the integral represents the overlap of the vibrational wavefunctions in both electronic states . assuming the harmonicity of the oscillators representing molecular vibrations and no changes in the frequency of a selected oscillator between electronic states involved in the transition , one can quickly check that for a selected vibration mode ( frequency ) and two selected members of this mode progression in s0 and s1 states the integral in eq . 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { m_{vib } } = \int { \psi \prime { * _ { vib}}\psi \prime { \prime_{vib}}dr } $ $ \end{document } can oscillate with \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline \delta_{\text{a } } } $ $ \end{document } changed in the way shown in fig . 11oscillation with change in equilibrium distance of the franck - condon integral of two s0 and s1 harmonic oscillator wavefunctions corresponding to the same vibrational mode and two different members of its progression oscillation with change in equilibrium distance of the franck - condon integral of two s0 and s1 harmonic oscillator wavefunctions corresponding to the same vibrational mode and two different members of its progression thus , changes in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline \delta_{\text{a } } } $ $ \end{document } can be the source of similar slight modulation in meg for c153 in etoh and tfetoh , or in ppn and clh . both cases correspond to two different vibrational modes , in alcohols a high frequency one while in ppn and clh a low frequency one . changes in the equilibrium distance must result from changes in the solute - solvent interaction energies . 8) for aprotic and protic solvents and from the values of f(t ) obtained for aqueous clh and ppn compared to the ones obtained for the same solvents when dried properly ( fig . ppn the decrease in t changes the equilibrium between free c153 molecules concentration and c153+h2o complexes in favor of the former ones . this is related to the fact water forms clusters of the size increasing with decreasing t , thus reducing the concentration of water molecules accessible for c153 to form complexes . the difference in meg and knr must be small for c153 and its complex with water and we observe an average result as the decay time . this is the case totally different from that of 4-ap which is efficiently quenched by water h - bonding , responsible for the decay times of free 4-ap and 4-ap - water complexes significantly different and separable through optimization routines . comparison of c153 meg values in protic and aprotic solvents indicates that c153 h - bonding of molecules of protic solvents lead to a change in the equilibrium distance a or / and to a slight change in s0 and s1 potential energy surfaces shapes . simultaneously , the steady - state absorption results show that the same interaction in the same solvents leads to an additional stabilization of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{fc } $ $ \end{document } state with respect to s0 , when compared to what is observed in aprotic solvents . in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } $ $ \end{document } state h - bonds formed by c153 with ppn change in energy which leads to an additional stabilization of the emitting state . 7 ) . on the other hand , the absorption position temperature dependencies ( fig . 3 ) and vs(t ) show that in protic tfetoh the energy of h - bonds formed with c153 decreases with decreasing temperature in the probe \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } $ $ \end{document } state , in contrast to what is observed for \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{fc } $ $ \end{document}. in hydrogen accepting ppn the h - bonds become stronger with decreasing t for c153 in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } $ $ \end{document } state . in etoh overall , we can conclude that the s0 and s1 potential energy surfaces undergo two displacements upon changing the solvent and temperature . horizontal displacement along the equilibrium distance axis induce changes in meg , f and f . vertical displacement in the energy scale changes the absorption and emission positions and knr , which thus affects f and f . thus , the induced part of the total dipole moment of the c153 molecule is modified as well . this in turn involves changes in h - bonds energies formed by the probe with tfetoh , etoh and ppn in s0 and s1 , of franck - condon and relaxed character . at this stage a direct indication of c153 sites / atoms responsible for the experimental effects observed is out of reach . however , on the basis of the steady - state results we can safely conclude that the reorientational relaxation of the solvent accompanying the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{fc } \to s_1^{rel } $ $ \end{document } transition induces a weakening of the h - bonds formed by c153 with protic solvents and a rise in energy of the h - bonds formed with hydrogen accepting solvents . this effects is amplified by a decrease in t and it can be understood as a result of a competition between specific and nonspecific interactions in tfetoh , and as a result of cooperation between them in ppn . one should keep in mind that in both s1 states the h - bond energy is higher than in s0 in protic solvents , while in ppn the h - bond energy in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{fc } $ $ \end{document } is the same as in s0 .

in a recent paper ( j fluoresc ( 2011 ) 21:15471557 ) a temperature induced modulation of coumarin 153 ( c153 ) fluorescence lifetime and quantum yield for the probe dissolved in the polar , nonspecifically interacting 1-chloropropane was reported . this modulation was also observed in temperature dependencies of the radiative and nonradiative rates . here , we show that the modulation is also observed in another 1-chloroalkane1-chlorohexane , as well as in hydrogen bonding propionitrile , ethanol and trifluoroethanol . change in the equilibrium distance between s0 an s1 potential energies surfaces was identified as the source of this modulation . this change is driven by temperature changes . it leads to a modulation of the fluorescence transition dipole moment and it is the primary source of the experimental effects observed . additionally , we have found that proticity of the solvent induces a rise in the fluorescence transition dipole moment , which leads to a shortening of the fluorescence lifetime . hydrogen bonds are formed by c153 also with hydrogen accepting solvents like propionitrile . we show that while such bonds do not affect the transition probability , they do change the s0 an s1 energy gap which in turn implies a change in non - radiative transition rate in a similar way as in protic solvents , as well as in the fluorescence spectrum position . finally , the influence of temperature on the energies of hydrogen bonds formed by c153 when acting as hydrogen donor or acceptor is reported .

Introduction Methods Results Steady-State Results Time-Resolved Results Discussion Conclusions

in many cases thermochromic shifts in absorption and emission reflect the temperature induced changes in refractive index , n , and electric permittivity , , of the solvent as observed by our group in for 4-aminophthalimide ( 4-ap ) and coumarin 153 ( c153 , scheme 1 ) dissolved in several polar non - protic 1-chloroalkanes , or by suppan et al . in [ 36 ] for several probes including 4-ap.scheme 1coumarin 153 ( c153 ) structure coumarin 153 ( c153 ) structure however , for a dye interacting specifically with the solvent molecules , temperature influence on the energy of this interaction can affect much more significantly the scale of thermochromic shifts than for a dye showing nonspecific interaction , related to n(t ) and (t ) dependencies . in the case of the dyes acting as hydrogen donors , a slightly rise in the stabilization was observed with decreasing temperature . to identify the origin of such temperature changes in h - bond energies , a preliminary study on the influence of temperature on absorption and emission , including fluorescence time - resolved measurements , was performed for c153 dissolved in 1-chloropropane ( clp ) . the purpose of this study was to determine the influence of temperature on the kinetics of c153 deactivation from s1 in nonspecifically interacting polar solvents . in order to compare new results with the previous ones obtained for clp , the absorption and emission spectra as well as quantum yield and fluorescence lifetime were measured for c153 dissolved in 1-chlorohexane ( clh ) in the temperature range of 183 k323 k. additionally , the same spectra and quantities were determined in propionitrile ( ppn)a hydrogen acceptor ( kamlet - taft polarity scale = 0 , = 0.4 [ 810 ] ) , in trifluoroethanol ( tfetoh)a hydrogen donor ( = 1.51 , = 0 ) and in ethanol ( etoh)a hydrogen donor and acceptor ( = 0.86 , = 0.75 ) . in the alcohols the dominant long component time ( filled circles ) of the double - exponential decay function fitted to the experimental decays is presented along with the single exponential decay time for tfetoh ( empty circles ) temperature dependencies of the fluorescence decay time for c153 in clh ( a ) , ppn ( b ) , etoh ( c ) and tfetoh ( d ) . in the alcohols the dominant long component time ( filled circles ) of the double - exponential decay function fitted to the experimental decays is presented along with the single exponential decay time for tfetoh ( empty circles ) there is a similarity in f(t ) dependencies in clh and ppn , as well as in etoh and tfetoh . these results , combined with f values obtained for c153 in etoh and tfetoh , show that the interaction of c153 with protic solvents leads to a shortening of f . finally , as mentioned earlier , the retarded solvation is responsible for the bi - exponential character of the fluorescence decays in the alcohols measured at the maximum of the steady - state fluorescence spectra . in the alcohols the dominant long component time ( filled circles ) of the double - exponential decay function fitted to the experimental decays is presented along with the single exponential decay time for tfetoh ( empty circles ) temperature dependencies of the fluorescence decay time for c153 in clh ( a ) , ppn ( b ) , etoh ( c ) and tfetoh ( d ) . in the alcohols the dominant long component time ( filled circles ) of the double - exponential decay function fitted to the experimental decays is presented along with the single exponential decay time for tfetoh ( empty circles ) there is a similarity in f(t ) dependencies in clh and ppn , as well as in etoh and tfetoh . these results , combined with f values obtained for c153 in etoh and tfetoh , show that the interaction of c153 with protic solvents leads to a shortening of f . finally , as mentioned earlier , the retarded solvation is responsible for the bi - exponential character of the fluorescence decays in the alcohols measured at the maximum of the steady - state fluorescence spectra . thermochromic shifts of c153 emission spectra reported in indicated that the probe forms h - bond with hydrogen accepting solvents , and that the change in energy of this bond , stimulated by probe excitation , increases with decreasing temperature , at least in the vibrationally and solvent relaxed first singlet excited state , \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } $ $ \end{document}. to evaluate in a similar way the possible influence of specific interactions on absorption peak positions 1 .1\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ \matrix { { \delta { v_{abs } } = 2\frac{{{\mu_g}\left ( { { \mu_g } - { \mu_e } } \right)}}{{{a^3 } } } \cdot \left ( { \frac{{\varepsilon - 1}}{{\varepsilon + 2 } } - \frac{{{n^2 } - 1}}{{{n^2 } + 2 } } } \right ) } \\ { + \frac{{\mu_g^2 - \mu_e^2}}{{{a^3 } } } \cdot \left ( { \frac{{{n^2 } - 1}}{{2{n^2 } + 2 } } } \right ) } \\ { + \frac{{6\mu_g^2\left ( { { a_g } - { a_e } } \right)}}{{{a^6 } } } \cdot { { \left ( { \frac{{\varepsilon - 1}}{{\varepsilon + 2 } } - \frac{{{n^2 } - 1}}{{{n^2 } + 2 } } } \right)}^2 } } \\ { + \frac{{3\left ( { { a_g } - { a_e } } \right)j{j_s}}}{{2hc{a^3}\left ( { j + { j_s } } \right ) } } \cdot \frac{{{n^2 } - 1}}{{{n^2 } + 2 } } } \\ } $ $ \end{document } above , g and e are c153 dipole moments in the ground and excited states , a is the onsager radius of the probe molecule , g and e are the ground and excited state probe polarizabilities , j and js are the probe and solvent ionization potentials and h and c have the usual meanings . both these observations indicate that ppn interacts only nonspecifically with c153 in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_0^{rel } $ $ \end{document } , or that the h - bond formed by ppn with c153 do not change in energy after c153 excitation to \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{fc } $ $ \end{document } , contrary to what was deduced from the emissive results for c153 \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } $ $ \end{document } in the same solvent . additionally , we have found the absorption spectrum full width at half of the maximum ( fwhm ) to be higher than that of the emission spectrum in all solvents , at each temperature . these results show that the protic character of the solvent has a significant impact on the f(t ) dependence , when compared to non - protic solvents ( clp as well ) . in alcohols a slight modulation of the linear f(t ) dependence can be observed , while in clh and ppn the values of f changes with temperature in a similar way as in clp , that is the slope of f(t ) changes in sign at a temperature in the range 260280 k. in both protic solvents a small modulation of f(t ) takes place in the same temperature range in which the change in sign of the f(t ) dependence occurs in clh and ppn . 8temperature dependencies of the radiative , ( a ) kf , and non - radiative , ( b ) knr , rates in clh ( filled circles ) , ppn ( empty circles ) , etoh ( filled triangles ) and tfetoh ( empty triangles ) temperature dependencies of the radiative , ( a ) kf , and non - radiative , ( b ) knr , rates in clh ( filled circles ) , ppn ( empty circles ) , etoh ( filled triangles ) and tfetoh ( empty triangles ) non - radiative deactivation in c153 is known to be governed by internal conversion . however , in this solvent the retardation of the solvent relaxation is the source of the low - temperature knr(t ) dependence , as the emission spectrum is not shifted totally to the red , which in turn leads to a drop in knr at the lowest t. in clh and ppn knr(t ) slope sign changes at higher temperatures . 9modulus squared of fluorescence ( meg ) transition dipole moments in clh ( filled circles ) , ppn ( empty circles ) , etoh ( filled triangles ) and tfetoh ( empty triangles ) modulus squared of fluorescence ( meg ) transition dipole moments in clh ( filled circles ) , ppn ( empty circles ) , etoh ( filled triangles ) and tfetoh ( empty triangles ) now , we see that the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } \to s_0^{fc } $ $ \end{document } radiative transition probability differs in protic solvents from that in non - protic ones . however , assuming also that other accepting modes , as the 810 cm and 360 cm discussed in , are active in the fluorescence transition a quite simple explanation of the temperature dependence of c153 radiative deactivation can be proposed.fig . 10 ) indicate that a change in the equilibrium distance \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { \overline \delta_{\text{a } } } $ $ \end{document } occurs on decreasing temperature . according to the born - oppenheimer approximation the transition dipole moment between the excited ( ) and ground ( ) states is proportional to : 7\documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { m_{e \to g } } = \left\langle { \psi \prime } \right|\widehat{\mu } \left| { \psi \prime \prime } \right\rangle \propto { p_{el}}\left ( { \overline r } \right ) \cdot \int { \psi \prime { * _ { vib}}\psi \prime { \prime_{vib}}dr } , $ $ \end{document}where \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ { p_{el}}\left ( { \overline r } \right ) $ $ \end{document } is the purely electronic transition dipole moment , dependent on the r - centroid for the transition . comparison of c153 meg values in protic and aprotic solvents indicates that c153 h - bonding of molecules of protic solvents lead to a change in the equilibrium distance a or / and to a slight change in s0 and s1 potential energy surfaces shapes . simultaneously , the steady - state absorption results show that the same interaction in the same solvents leads to an additional stabilization of the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{fc } $ $ \end{document } state with respect to s0 , when compared to what is observed in aprotic solvents . in \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{rel } $ $ \end{document } state h - bonds formed by c153 with ppn change in energy which leads to an additional stabilization of the emitting state . however , on the basis of the steady - state results we can safely conclude that the reorientational relaxation of the solvent accompanying the \documentclass[12pt]{minimal } \usepackage{amsmath } \usepackage{wasysym } \usepackage{amsfonts } \usepackage{amssymb } \usepackage{amsbsy } \usepackage{mathrsfs } \usepackage{upgreek } \setlength{\oddsidemargin}{-69pt } \begin{document}$$ s_1^{fc } \to s_1^{rel } $ $ \end{document } transition induces a weakening of the h - bonds formed by c153 with protic solvents and a rise in energy of the h - bonds formed with hydrogen accepting solvents .

ghrelin is a 28-amino - acid gastrointestinal peptide with appetite - stimulating , growth hormone - releasing and adipogenic properties [ 13 ] . it was originally characterized as the endogenous ligand for the hypothalamic - pituitary growth hormone secretagogue receptor type 1a ( ghsr1a ) , stimulating the anterior gland of pituitary to produce gh [ 13 ] . in fact , ghrelin is the third physiological regulator of endogenous gh secretion , along with hypothalamic gh releasing hormone and somatostatin . ghrelin is predominantly produced in the so - called x / a - like endocrine cells of gastric mucosa , and is subsequently released into bloodstream [ 4 , 5 ] . ghrelin - producing cells are mostly abundant in the oxyntic glands of gastric fundus [ 4 , 5 ] . given the widespread distribution of ghsr1a in the human body , ghrelin exerts pluripotent biological activities , affecting cardiovascular system , pancreatic endocrine function , gastrointestinal tract motility , gastric acid secretion , cell proliferation and metabolism . one of the most important actions of ghrelin is its regulatory role for long - term energy homeostasis and short - term food intake . ghrelin activates neuropeptide y ( npy ) and agouti - related protein ( agrp ) neurons in the hypothalamic arcuate nucleus , providing a central stimulus for increased food intake and reduced energy expenditure . intracerebroventricular administration of ghrelin in rodents and peripheral administration in humans has shown to promote weight gain , by reducing fat utilization and increasing food consumption [ 8 , 9 ] . it acts as a circulating orexigenic signal , and has been also implicated in preprandial hunger and meal initiation . were the first to show that plasma ghrelin levels increase nearly twofold immediately before feeding onset , and are strongly suppressed by food ingestion , falling to trough ( nadir ) levels within an hour after meal initiation . this pattern of secretion is interestingly reciprocal to that of insulin , which is preprandially low and increases gradually in the postabsorptive period . ghrelin secretion is typically up - regulated under conditions of chronic negative energy balance ( anorexia nervosa , heart failure cachexia ) , and down - regulated in the setting of sustained positive energy balance ( obesity ) . furthermore , obese subjects fail to exhibit the normal postprandial decline of plasma ghrelin concentrations , observed in normal weight individuals . the postmeal inhibition of gastric ghrelin production is proportional to energy load and is profoundly influenced by the meal 's macronutrient content [ 13 , 14 ] . in rodents and normal weight humans , the postprandial drop in ghrelin levels is more pronounced after carbohydrate ( cho ) meals than after protein- or fat - enriched diet manipulations [ 15 , 16 ] . the type of ingested macronutrient seems to affect differentially the magnitude and pattern of postprandial ghrelin suppression . whether it is the direct intraluminal contact of nutrients with gastric mucosa or the insulin - mediated metabolic response to nutrient ingestion more important for postprandial ghrelin suppression remains still controversial . there is currently growing evidence that ghrelin suppression does not require the presence of nutrients in either the stomach or the duodenum , but requires effective post - gastric and postabsorptive feedback mechanisms , possibly mediated by insulin and gastrointestinal hormones with anorexigenic potential . vagal activity , gastric emptying rate and postprandial increases of intestinal osmolarity are also active players in meal - induced ghrelin regulation [ 17 , 18 ] . despite the well - established stimulatory effect of ghrelin on appetite and eating behavior , little information is available regarding its relationship with fasting and postprandial energy expenditure in normal weight and obese humans . in rodents , ghrelin infusion promotes weight gain , both by increasing food intake and by decreasing energy expenditure and fat catabolism . this effect is primarily due to an increase in caloric intake and respiratory quotient ( rq ) , suggestive of a switch from fatty acid oxidation to glycolysis leading ultimately to fat deposition . st - pierre et al . examined the relationship between serum ghrelin and resting metabolic rate , thermic effect of food , fasting and postprandial rq , physical activity level and peak aerobic capacity in 65 lean young women . significant inverse correlations were reported between ghrelin , resting metabolic rate and thermic effect of food , persisting after adjustment for fat - free mass , fat mass and insulin levels . these results suggest that higher levels of ghrelin are associated with low levels of resting and postprandial thermogenesis , indicating that the metabolic effects of ghrelin may extend far beyond the regulation of satiety and substrate oxidation , serving as a biomarker for decreased energy expenditure in humans . on the other hand , the relationship between ghrelin and energy expenditure in obesity constitutes a matter of debate . in a study by marzullo et al . , the obese subjects with low resting energy expenditure ( impaired energy balance ) exhibited lower active ghrelin levels , compared with obese subjects with high energy expenditure , indicating that ghrelin secretion and activity might be decreased in cases of obesity with impaired energy expenditure , as part of an obesity - related compensatory mechanism . the present review aims to shed light on the underlying mechanisms of postprandial ghrelin regulation . in addition , a significant body of clinical and experimental data will be discussed , elucidating the macronutrient - specific effect of several isocaloric test meals on postprandial ghrelin levels . we searched pubmed and other electronic databases for high quality articles written in english , using the following search terms : ghrelin , macronutrients , carbohydrate , protein , fat and postprandial response . a recent study in humans demonstrated that ghrelin levels can be suppressed by sham feeding ( when nutrients are only smelled , chewed or tasted without being swallowed ) , as well as by actual feeding , indicating the importance of cephalic response to nutrient intake and supporting the role of vagal activity for the control of postprandial ghrelin secretion . the vagally mediated cephalic phase appears to have a major role in initiating the postprandial fall in ghrelin levels , which are thereafter maintained suppressed , by other as yet not entirely elucidated gastrointestinal or postabsorptive mechanisms , mediating the nutrient - related ghrelin response . the gastric phase alone appears to play no role in the regulation of ghrelin secretion , because neither gastric distension alone , nor activation of chemical nutrient - sensing mechanisms of gastric mucosa ( gastric chemosensitization ) can modulate ghrelin levels . in an interesting experiment in rats , intragastric infusion of glucose reduced ghrelin levels by approximately 50% , while water infusion had no effect . however , when gastric emptying was prevented through the inflation of a pyloric cuff ( gastric distension ) , glucose and water infusions were similarly ineffective to suppress ghrelin . these experimental findings indicate that gastric distension and chemosensation are both insufficient to induce a ghrelin response . prandial ghrelin regulation is probably mediated by intestinal signals generated downstream of treitz ligament , meaning that postgastric feedback is definitely required for an adequate inhibitory ghrelin response . in this intestinal phase of food ingestion , there seems to be a prominent macronutrient effect , determining the depth and duration of postprandial ghrelin suppression . the macronutrient - related patterns of ghrelin response imply that either the direct exposure of gastrointestinal mucosa to ingested nutrients , or the increased circulating levels of nutrients or other related hormones , can influence postprandial ghrelin levels in a macronutrient - specific manner [ 17 , 22 ] . candidate mediators involved in the regulation of postprandial ghrelin secretion are glucose , insulin , cholecystokinin ( cck ) , glucagon - like peptide 1 ( glp-1 ) , glucose - dependent insulinotropic polypeptide ( gip ) , peptide yy ( pyy ) , pancreatic polypeptide ( pp ) , oxyntomodulin and somatostatin ( ss ) . most of these molecules are gastrointestinal hormones , which delay gastric emptying and display insulinotropic and anorexigenic activities [ 18 , 21 ] . insulin and glucose are thought to be dynamic modulators of plasma ghrelin concentrations in rodents and humans [ 2325 ] . hyperglycemia and hyperinsulinemia tend to decrease , while hypoglycemia and insulin deficiency tend to increase circulating ghrelin levels . both intravenous and oral administration of glucose leads to a significant decline in circulating ghrelin , indicating that ghrelin secretion may be suppressed , at least in part , by an increased plasma glucose level in healthy humans [ 23 , 26 ] . for intravenous glucose administration in particular , mhlig et al . showed that glucose elicits a significant decrease of plasma ghrelin concentrations , whereas intravenous free fatty acid or arginine load does not affect circulating ghrelin levels . insulin - mediated glucose uptake and metabolism may also control postprandial ghrelin levels , while insulin sensitivity is considered to be an important determinant of postprandial ghrelin suppression . a great increase in plasma free fatty acids , as a result of constant intravenous lipid infusion , failed to suppress plasma ghrelin , while ghrelin decreased by almost 50% under hyperinsulinemic euglycemic clamp conditions . the effect of acute hyperinsulinemia on plasma ghrelin concentrations is still a matter of debate . clinical data regarding the existing interrelation between ghrelin and insulin are rather conflicting . according to the study of flanagan et al . , using a stepped hyperinsulinemic eu- , hypo- and hyperglycemic glucose clamp , insulin may suppress circulating ghrelin independently of glucose , although glucose might have an additional synergistic effect . in the same direction , murdolo et al . tested the hypothesis that insulin is the driving force for postprandial ghrelin suppression by comparing the effects of meal ingestion on plasma ghrelin levels between insulin - deficient patients with type 1 diabetes and healthy controls . the investigators concluded that insulin is essential for meal - induced plasma ghrelin suppression , commenting that severe insulin deficiency in uncontrolled type 1 diabetic subjects may partly explain the episodes of hyperphagia observed in these patients , through compromising postprandial ghrelin response . studies in rats have shown that insulin - induced hypoglycemia increases , instead of decreasing , ghrelin mrna levels in the gastric fundus , providing no evidence for a direct inhibitory action of insulin on ghrelin synthesis . however , in healthy humans , plasma ghrelin concentrations are decreased during insulin - induced hypoglycemia , suggesting species - specific differences between rodents and humans . since ghrelin - producing cells are closely associated with the capillary network of the lamina propria of gastric mucosa , their function might be under endocrine control . however , there is still no evidence for insulin receptors on the surface of ghrelin - producing cells . indirect pathways for insulin suppressive effects on ghrelin synthesis and secretion include activation of hypothalamic insulin receptors and modulation of the cellular flux of glucose or free fatty acids . there seems to be an interaction between nutrients and insulin in reducing circulating ghrelin levels . a greater insulin - induced glucose uptake by x / a - like cells might inhibit ghrelin synthesis and/or secretion . contrary to the findings mentioned above , caixs et al . reported that , unlike food intake , the subcutaneous administration of a short - acting insulin analog was not able to suppress ghrelin levels . in concordance with this study , schaller et al . concluded that meal - related ghrelin suppression is not directly regulated by glucose or insulin , since a reduction in ghrelin was observed only at supraphysiological insulin concentrations , while hyperglycemia did not decrease ghrelin at all . contrary to murdolo , spranger et al . observed a substantial postprandial decrease of plasma ghrelin in an insulin - deficient patient with type 1 diabetes following a carbohydrate challenge , while a subsequent bolus of subcutaneous short - acting insulin induced no further changes in circulating ghrelin . taking everything into consideration , glucose and insulin are unlikely to explain the entire postprandial ghrelin response , since ghrelin remains suppressed long after the normalization of glucose and insulin levels , and furthermore , because lipids tend to suppress ghrelin in the absence of substantial increases in glucose or insulin . other hormonal mediators ( such as cck , glp-1 and gip ) released in the postabsorptive phase in response to nutrient stimuli , appear to orchestrate the whole postmeal ghrelin response , enhancing the inhibitory effects of glucose and insulin , which are still remaining the principal contributors . the inverse temporal relationship between circulating concentrations of ghrelin and insulin , reported in a large number of clinical studies , substantiates the key regulatory role of insulin for ghrelin regulation [ 3638 ] . it would be also interesting to examine the role of leptin in postprandial ghrelin regulation , since ghrelin and leptin are part of a dynamic peripheral feedback system that regulates body weight and energy homeostasis by modulating satiety . data concerning the relationship between ghrelin and leptin at fasting and postprandial state are rather contradictory . in vitro studies have previously shown that leptin inhibits ghrelin production from the gastric mucosa , and leptin levels in humans appear to be inversely related to ghrelin concentrations . on the other hand , the interesting observation by cummings et al . that leptin and intermeal ghrelin levels display diurnal rhythms that are in phase with one another suggests that leptin and ghrelin might be coordinately regulated . the same study reported a subtle postprandial drop in leptin levels that may reflect meal - related regulation of gastric leptin , according to the investigators . the relationship between insulin and leptin is more clearly defined and may partially explain the ghrelin - leptin interrelationship . previous studies indicate that leptin secretion is regulated by insulin - mediated glucose metabolism , suggesting that insulin is a positive modulator of leptin concentrations . this is why consumption of high - fat meals and high - fructose beverages that produce smaller postprandial glucose and insulin responses compared with isocaloric high - carbohydrate meals , have shown to reduce 24-hour circulating leptin concentrations in humans . accepting that leptin and insulin are positively associated , it is conceivable that postprandial surges of insulin are related to increased leptin levels , and given the reciprocal relationship between insulin and ghrelin , a similar inverse relationship might be supported for leptin and ghrelin as well . because insulin and leptin function as key signals conveying information on energy intake and body fat stores to the central nervous system for the long - term regulation of food intake and energy homeostasis , it is possible that reduced insulin and leptin production , as well as increased ghrelin levels , contribute to increased energy intake , weight gain , and obesity in humans . in an interesting study by foster - schubert et al . , three different macronutrient preloads of equal caloric content , volume and energy density ( protein- , fat- , and cho - based beverages ) were compared for their relative efficacy to suppress total and acylated ( bioactive ) ghrelin levels . total ghrelin levels decreased significantly more after cho or protein ingestion than after lipids , while ghrelin 's nadir ( lowest ) levels were reached most rapidly after the cho - enriched preload ( within 99 minutes ) . for both acylated and total ghrelin concentrations , investigators observed marked and macronutrient - dependent differences during the first 3 hours versus the subsequent 3 hours of the postprandial study period . more specifically , they observed only after cho ingestion a marked rebound of acylated ghrelin to 37% above baseline levels , during the second 3 hours of the 6 hour post - ingestive period . this study reveals a previously unidentified pattern in the response of acylated and total ghrelin after cho ingestion . ghrelin levels decreased in the initial 3 hours , followed by a marked overshoot to above the pre - ingestion baseline during the second 3 hours . no such overshoot was observed after protein or lipid ingestion , both of which suppressed acylated and total ghrelin levels until study completion . these observations suggest very different effects of high cho meals in the early versus the later postabsorptive phase , indicating that ingested cho might prompt an early hunger rebound . such findings have important clinical implications for design of dietary regimens . the faster gastric emptying after cho ingestion , compared with lipids or proteins , can partly explain the strong and rapid postprandial ghrelin suppression in the first phase . the rapid removal of cho from stomach should cause a rapid , strong suppression of ghrelin levels , an effect that might be short lived , because these nutrients are quickly absorbed and metabolized . if insulin - mediated glucose disposal is more important for ghrelin suppression than mere insulin levels , the late post - cho ghrelin overshoot may result from reduced intracellular glucose metabolism , when glucose levels decreased below baseline . not all types of dietary cho are likely to have the same effect on postprandial ghrelin levels . in a study comparing high - glucose with high - fructose meals , the mean postprandial suppression of ghrelin was markedly attenuated and significantly less pronounced after consuming the high - fructose meals . fructose , unlike glucose , does not stimulate insulin secretion from pancreatic beta cells , presumably because of the low number of fructose transporters ( glut5 ) on beta cell membrane . intravenous fructose infusion increases only marginally circulating insulin concentrations , while ingested fructose is ineffective in eliciting postprandial insulin secretion . what is more , fructose does not increase insulin - mediated glucose metabolism or circulating leptin levels . given the key role of insulin for postprandial ghrelin suppression , ingested fructose suppresses ghrelin poorly . the failure of fructose to effectively suppress ghrelin ( impaired satiety ) , along with the reduced insulin and leptin concentrations , could lead to an increased caloric intake and ultimately contribute to obesity , during chronic consumption of diets high in fructose . cho - enriched test meals , containing both simple and complex carbohydrates , have been used in various clinical studies investigating the differential response of postprandial ghrelin to meals of different macronutrient composition . in all of them , and particularly in normal weight subjects , cho ingestion provoked a significant postprandial ghrelin decline by approximately 30% from baseline values within 2 hours after meal onset [ 37 , 38 , 4042 ] . a common finding in all these studies is the inverse correlation between postprandial ghrelin and insulin concentrations throughout the whole study period . while the suppressant effect of cho on ghrelin levels is well established and taken for granted , the biphasic pattern of ghrelin suppression after cho intake and the clinically meaningful distinction between glucose and fructose , are novel thought - provoking findings that warrant further investigation . the higher satiety associated with protein consumption may be at least partially mediated by a protein - induced prolonged postprandial ghrelin suppression . such a reduction in the orexigenic signal might delay the initiation of a subsequent feeding episode or lower hunger and energy intake . the prolonged suppression of ghrelin after protein intake might relate to the protracted emptying of proteins from stomach , causing a more sustained activation of post - gastric ghrelin - suppressing mechanisms . additional mechanisms that account for the significant satiating effect of dietary protein include the following : proteins have a larger thermic effect than cho or fat , since they can not be stored in the body , but need to be metabolized immediately . moreover , increased circulating concentrations of amino acids after protein intake stimulate hepatic gluconeogenesis preventing hypoglycemia , and thus promoting satiety . in rats fed on protein - enriched diets , intestinal gluconeogenesis last but not least , proteins stimulate the secretion of specific gastrointestinal peptides ( cck , glp-1 , gip ) that delay gastric emptying and increase satiety . in a study of three isoenergetic meals ( balanced , high - fat and high - protein ) consumed by healthy young women , acylated ghrelin fell significantly after ingestion of both balanced and high - protein meals , while ghrelin persisted at significantly lower levels than baseline for a longer duration , following the high - protein meal . apart from prolonging postprandial ghrelin suppression , liquid protein preloads have also shown to prolong the elevation of anorexigenic gastrointestinal hormones , such as cck and glp-1 . these responses are observed irrespective of the type of protein consumed ( soy , whey , or gluten ) . in support of this , lang et al . has demonstrated no effect of protein type ( egg albumin , casein , gelatin , soy protein , pea protein and wheat gluten ) on satiety , 24 hour energy intake and postprandial glucose and insulin concentrations . in a further randomized crossover study in healthy adult males , the high - protein meal maintained significantly lower ghrelin levels at 180 minutes compared with the high - cho and high - fat meals , indicating that dietary protein exhibits longer - term postprandial ghrelin suppression and enhanced satiety . according to blom et al . , the high - protein breakfast decreased postprandial ghrelin secretion more than did the high - cho breakfast . it also increased glucagon and cck , tended to increase gip and glp-1 , and decreased gastric emptying rate , without affecting however ad libitum energy intake . despite the accumulating evidence supporting the satiating and ghrelin - suppressing capacity of dietary protein , there have been a few studies suggesting that protein ingestion stimulates , instead of suppressing , ghrelin levels [ 38 , 47 ] , while an additional study indicated that the satiating effect of protein is practically unrelated to postprandial ghrelin secretion . ingested lipids appear to suppress the orexigenic hormone ghrelin less effectively than do cho or protein . the relatively weak ability of this macronutrient to suppress ghrelin can be attributed to the poor stimulation of insulin secretion by lipids as well as to the lower osmolarity of lipid meals and beverages . however , lipids contribute fewer osmolar units compared with an isocaloric consumption of cho or proteins . in a study by pavlatos et al . , total ghrelin levels did not decline significantly after a fat - rich meal in normal weight women , as opposed to an isoenergetic protein - rich meal . in a similarly designed study by tentolouris et al . , fat consumption has also displayed a diminished capacity to induce satiety . in this study , the effect of two isocaloric test meals ( one rich in cho and one rich in fat ) on postprandial active ghrelin concentrations was comparatively evaluated in lean and obese women . after the fat - rich meal , active ghrelin levels were not significantly suppressed , even in the lean participants . the investigators conclude that increased fat intake might promote obesity not only through its high caloric content and adverse metabolic effects , but also through its failure to suppress postprandial hunger . reported a different ( more delayed ) time pattern of ghrelin suppression after fat ingestion , compared with cho . more specifically , the fat - rich meal decreased plasma ghrelin levels , but the nadir was reached towards the end of the study period , namely at 180 minutes . the potential impact of varying fatty acid composition ( saturated , monounsaturated and polyunsaturated fat ) on postprandial ghrelin response has been only scarcely investigated . in a relevant double - blind crossover study , researchers assessed two high - fat test meals , one with a high saturated to unsaturated fat ratio ( 70/30 ) and the other with a low ratio ( 55/45 ) , and concluded that increasing saturated fat consumption had no deleterious effects on fasting and postprandial plasma ghrelin concentrations . bmi , body fat and indices of central fat distribution are inversely associated with fasting plasma ghrelin concentrations . a large number of clinical studies have shown that obese subjects tend to display lower total and acylated ghrelin levels in the fasting state compared with normal weight individuals . this finding appears to be an appropriate compensatory response , so that obese individuals will not get any fatter and lean individuals will not get any thinner ( adaptive mechanism for prevention of obesity and cachexia resp . ) . it has been proposed that the sustained positive energy balance observed in obesity suppresses maximally circulating ghrelin levels , and thus limits flexibility for further short - term feeding regulation . the impaired cholinergic ( vagal ) regulation of postprandial drop in ghrelin concentrations might be also responsible for the dysregulated ghrelin control in obese subjects . furthermore , obese subjects are often insulin - resistant and thus hyperinsulinemic , and insulin is a well established inhibitory signal for ghrelin secretion . to the best of our knowledge , the differential rate and magnitude of preprandial rise in ghrelin levels has not been comparatively evaluated in lean and obese individuals . as already mentioned , it is widely accepted that obese subjects exhibit significantly lower fasting ghrelin concentrations than lean , but whether the rate of preprandial ghrelin increase is actually differentiated between lean and obese subjects has been scarcely addressed . in fact , most of the studies that used frequent blood sampling protocols in order to assess the diurnal plasma ghrelin profile in subjects of varying bmi ( preprandial and postprandial hormonal alterations ) , reported no specific bmi - related differences between lean and obese participants in terms of preprandial rate of ghrelin increase . another important aspect of ghrelin regulation in obese subjects is the blunted postprandial ghrelin response . this means that obese subjects have low ghrelin levels preprandially , but postprandial ghrelin secretion is not sufficiently suppressed , suggesting a severe defect in ghrelin - induced satiety mechanisms , which makes them feel still hungry , even though they have just completed their meal have shown that neither a protein- nor a fat - rich meal was able to elicit a significant acute ghrelin response in obese women . in the same direction , tentolouris et al . reported that a high - cho meal ( with a well established ghrelin - suppressing potential in lean individuals ) was also insufficient to suppress postprandial active ghrelin levels in obese women , indicating a considerable secretory and possibly satiety impairment in these subjects . another interesting conclusion of the same study was that , the leaner a person is , the higher his fasting ghrelin is , and the steepest its postprandial decline . this means that a lean subject feels quite hungry before meals , but afterwards feels easily satiated . apart from ghrelin , additional hormonal factors that contribute to this auto - regulation of body weight homeostasis in normal weight subjects include glp-1 , gip , and pyy , which delay gastric emptying , induce satiety and prevent hyperphagia , and are significantly more functional in lean subjects compared with the obese . however , as a person gains weight , this autoregulatory effect appears to become severely compromised . an obese subject can not experience postprandial fullness , independently of the macronutrient composition of his meal . the fact that lean subjects display higher fasting ghrelin levels than obese does not necessarily mean that they also consume greater amounts of food . on the contrary , food intake is most likely to be increased in obese subjects , because of the blunted postprandial ghrelin response , as described above . besides , the effect of ghrelin on hunger and satiety sensations is not necessarily translated into alterations in ad libitum energy intake , as shown by the study of erdmann et al . . an additional study by druce et al . showed that low - dose infusion of ghrelin increased ad libitum energy intake at a buffet meal only in the obese group , and not in the lean , indicating that obese people are highly sensitive to the appetite - stimulating effects of ghrelin , even when the circulating ghrelin is low . as a result , the absolute difference of fasting ghrelin levels between lean and obese subjects is not a major determinant of subsequent food intake , since other factors such as endogenous sensitivity to circulating ghrelin , ghrelin activity and postprandial ghrelin changes are thought to play an important role , as well . additional factors that can in part explain the suppressed basal ghrelin levels in obese subjects include hyperleptinaemia and increased circulating levels of il-1b ( interleukin 1b ) , since both leptin and il-1b are thought to inhibit ghrelin secretion . hyperleptinaemia is observed frequently in obesity due to leptin resistance , and high levels of il-1b and other inflammatory mediators are also a common finding in patients with obesity and metabolic syndrome . concerning the blunted postprandial ghrelin response in obese subjects , the impaired post - meal elevation of gastrointestinal hormones with anorexigenic and insulinomimetic properties , such as glp-1 , gip and pyy , has been implicated as an additional significant contributor . as far as insulin resistance is concerned , its role for ghrelin regulation is different in fasting and postprandial state . in fasting , insulin resistance and thus hyperinsulinemia lead to decreased fasting ghrelin levels . fasting plasma ghrelin concentrations are lower in insulin - resistant obese adults , compared with equally obese individuals with relatively higher insulin sensitivity . on the other hand , postabsorptive insulin resistance and impaired intracellular insulin signaling lead to inadequately suppressed and thus increased levels of ghrelin , since insulin sensitivity is regarded as prerequisite for sufficient postprandial ghrelin suppression . the macronutrient - specific effect of meals on postprandial ghrelin levels has interesting implications only in normal weight individuals . in the obese population , where obese individuals with metabolic syndrome elicited no differences in plasma ghrelin or feelings of hunger and satiety , after consuming two high - cho meals producing different insulin responses ( whole - grain rye bread and wheat bread ) . despite the different insulin response , ghrelin levels did not change in obese patients in response to either type of bread meals . in addition , ghrelin levels did not correlate with insulin or glucose , indicating that regulation of ghrelin might be altered in obese patients with metabolic syndrome independently of insulin . an additional study by moran et al . revealed a dysregulation of ghrelin homeostasis in overweight women with polycystic ovary syndrome ( pcos ) , suggesting that women with pcos exhibit similar ghrelin abnormalities with obese women ( down - regulated fasting ghrelin , blunted postprandial ghrelin suppression ) , and this disorder was not differentially affected by diet macronutrient composition . diet - induced weight loss , contrary to gastric bypass surgery where ghrelin levels remain dramatically decreased , has shown to elevate fasting ghrelin levels and normalize postprandial ghrelin response . this means that when a person loses a significant amount of weight by diet he might feel a greater preprandial desire to eat , but his postprandial satiety is significantly improved . the greater sensitivity to vagal stimulation after weight loss may result in a more pronounced drop in postprandial ghrelin levels , in addition to the improvement in insulin sensitivity , which is a major determinant of postprandial ghrelin suppression . romon et al . reported that diet - induced weight reduction preferentially improves ghrelin response to a high - cho meal , compared with a high - fat meal , indicating that weight loss might selectively improve the response of ghrelin to carbohydrate . zimmermann et al . addressed the interesting question , whether acute ethanol ingestion affects ghrelin secretion . ghrelin declined significantly within 15 minutes after alcohol drinking , fell to a minimum of 66% of baseline at 75 minutes and remained suppressed until the last sample at 2 hours . given that alcohol seems to acutely attenuate circulating ghrelin levels and is also known for its satiating power , one might expect from alcohol to promote weight loss . however , its considerable caloric density and its detrimental overall health effects should not be overlooked . as far as smoking is concerned , in an interesting study by kokkinos et al . , acute cigarette smoking induced no significant suppression of post - smoking ghrelin in habitual smokers , possibly desensitized to any possible effect of smoking on ghrelin , through prolonged nicotine exposure . on the other hand , there was a progressive decline of ghrelin in non - smokers , reaching its nadir 60 minutes after smoking . fasting total ghrelin levels were not significantly different between smokers and non - smokers , indicating that smoking is unlikely to exert a long - term anorectic effect in smoking populations . the significant decrease in circulating ghrelin after smoking cessation , reported by lee et al . , provides further evidence for lack of correlation between smoking status and suppressed plasma ghrelin concentrations . many clinical studies have used isoenergetic test meals ( protein- , fat- and cho - rich ) in order to examine the relative efficacy of each macronutrient to suppress postprandial ghrelin . even though the overall concept in these studies is common , the experimental design ( meal composition , measured parameters , blood sampling intervals , duration of post - ingestive period ) is slightly or moderately different . this discrepancy may be in part responsible for heterogeneity in findings . trying to delineate the central message behind all these divergent data , carbohydrate appears to be the most effective macronutrient in terms of postprandial ghrelin suppression , possibly because of its glucose - elevating and insulin - secreting effect . however , recent data indicate that cho ingestion may provoke a delayed ghrelin rebound in the later postabsorptive period , questioning the role of cho - rich meals in weight loss dietary approaches . fructose - enriched meals display a poor ghrelin - suppressing capacity , promoting increased caloric intake , weight gain and obesity under conditions of chronic consumption . protein induces prolonged ghrelin suppression and elevation of gut - derived anorexigenic hormones that delay gastric emptying regardless of the type of protein consumed . however , the influence of solid forms of protein ( turkey , pork ) on postprandial ghrelin levels may require assessment over a longer period of time than 3 - 4 hours , since slow gastric emptying delays postprandial ghrelin nadir . as far as fat is concerned , it appears to be the least potent ghrelin - suppressant , even in normal weight subjects . some studies have shown that fat decreases ghrelin concentrations , but later or more weakly than other macronutrients . at the same time , other studies report that fatty meals have absolutely no effect on postprandial ghrelin levels . in obese subjects , postprandial ghrelin response is blunted , and the macronutrient effect on ghrelin levels appears to be rather neutral . however , weight loss restores ghrelin response and leads to a significant improvement of ghrelin - mediated appetite regulation . from now on , it would be interesting to evaluate the long - term effect of macronutrient - enriched diet manipulations on fasting and postprandial ghrelin levels . additional parameters that could possibly influence ghrelin response and should be further investigated are food form and viscosity ( liquid , solid , semi - solid products ) , portion size and meal duration .

ghrelin is a powerful orexigenic gut hormone with growth hormone releasing activity . it plays a pivotal role for long - term energy balance and short - term food intake . it is also recognized as a potent signal for meal initiation . ghrelin levels rise sharply before feeding onset , and are strongly suppressed by food ingestion . postprandial ghrelin response is totally macronutrient specific in normal weight subjects , but is rather independent of macronutrient composition in obese . in rodents and lean individuals , isoenergetic meals of different macronutrient content suppress ghrelin to a variable extent . carbohydrate appears to be the most effective macronutrient for ghrelin suppression , because of its rapid absorption and insulin - secreting effect . protein induces prolonged ghrelin suppression and is considered to be the most satiating macronutrient . fat , on the other hand , exhibits rather weak and insufficient ghrelin - suppressing capacity . the principal mediators involved in meal - induced ghrelin regulation are glucose , insulin , gastrointestinal hormones released in the postabsorptive phase , vagal activity , gastric emptying rate , and postprandial alterations in intestinal osmolarity .

1. Introduction 2. Underlying Pathophysiology of Postprandial Ghrelin Regulation 3. Carbohydrate Ingestion and Postprandial Ghrelin Response 4. Protein Ingestion and Postprandial Ghrelin Response 5. Fat Ingestion and Postprandial Ghrelin Response 6. Effect of BMI on Nutrient-Related Ghrelin Regulation 7. Acute Effect of Ethanol and Smoking on Plasma Ghrelin Levels 8. Summary, Conclusions, and Perspectives

ghrelin is a 28-amino - acid gastrointestinal peptide with appetite - stimulating , growth hormone - releasing and adipogenic properties [ 13 ] . ghrelin is predominantly produced in the so - called x / a - like endocrine cells of gastric mucosa , and is subsequently released into bloodstream [ 4 , 5 ] . given the widespread distribution of ghsr1a in the human body , ghrelin exerts pluripotent biological activities , affecting cardiovascular system , pancreatic endocrine function , gastrointestinal tract motility , gastric acid secretion , cell proliferation and metabolism . one of the most important actions of ghrelin is its regulatory role for long - term energy homeostasis and short - term food intake . ghrelin activates neuropeptide y ( npy ) and agouti - related protein ( agrp ) neurons in the hypothalamic arcuate nucleus , providing a central stimulus for increased food intake and reduced energy expenditure . it acts as a circulating orexigenic signal , and has been also implicated in preprandial hunger and meal initiation . were the first to show that plasma ghrelin levels increase nearly twofold immediately before feeding onset , and are strongly suppressed by food ingestion , falling to trough ( nadir ) levels within an hour after meal initiation . this pattern of secretion is interestingly reciprocal to that of insulin , which is preprandially low and increases gradually in the postabsorptive period . ghrelin secretion is typically up - regulated under conditions of chronic negative energy balance ( anorexia nervosa , heart failure cachexia ) , and down - regulated in the setting of sustained positive energy balance ( obesity ) . in rodents and normal weight humans , the postprandial drop in ghrelin levels is more pronounced after carbohydrate ( cho ) meals than after protein- or fat - enriched diet manipulations [ 15 , 16 ] . the type of ingested macronutrient seems to affect differentially the magnitude and pattern of postprandial ghrelin suppression . whether it is the direct intraluminal contact of nutrients with gastric mucosa or the insulin - mediated metabolic response to nutrient ingestion more important for postprandial ghrelin suppression remains still controversial . there is currently growing evidence that ghrelin suppression does not require the presence of nutrients in either the stomach or the duodenum , but requires effective post - gastric and postabsorptive feedback mechanisms , possibly mediated by insulin and gastrointestinal hormones with anorexigenic potential . vagal activity , gastric emptying rate and postprandial increases of intestinal osmolarity are also active players in meal - induced ghrelin regulation [ 17 , 18 ] . despite the well - established stimulatory effect of ghrelin on appetite and eating behavior , little information is available regarding its relationship with fasting and postprandial energy expenditure in normal weight and obese humans . in rodents , ghrelin infusion promotes weight gain , both by increasing food intake and by decreasing energy expenditure and fat catabolism . examined the relationship between serum ghrelin and resting metabolic rate , thermic effect of food , fasting and postprandial rq , physical activity level and peak aerobic capacity in 65 lean young women . on the other hand , the relationship between ghrelin and energy expenditure in obesity constitutes a matter of debate . the present review aims to shed light on the underlying mechanisms of postprandial ghrelin regulation . a recent study in humans demonstrated that ghrelin levels can be suppressed by sham feeding ( when nutrients are only smelled , chewed or tasted without being swallowed ) , as well as by actual feeding , indicating the importance of cephalic response to nutrient intake and supporting the role of vagal activity for the control of postprandial ghrelin secretion . the vagally mediated cephalic phase appears to have a major role in initiating the postprandial fall in ghrelin levels , which are thereafter maintained suppressed , by other as yet not entirely elucidated gastrointestinal or postabsorptive mechanisms , mediating the nutrient - related ghrelin response . the gastric phase alone appears to play no role in the regulation of ghrelin secretion , because neither gastric distension alone , nor activation of chemical nutrient - sensing mechanisms of gastric mucosa ( gastric chemosensitization ) can modulate ghrelin levels . in this intestinal phase of food ingestion , there seems to be a prominent macronutrient effect , determining the depth and duration of postprandial ghrelin suppression . the macronutrient - related patterns of ghrelin response imply that either the direct exposure of gastrointestinal mucosa to ingested nutrients , or the increased circulating levels of nutrients or other related hormones , can influence postprandial ghrelin levels in a macronutrient - specific manner [ 17 , 22 ] . candidate mediators involved in the regulation of postprandial ghrelin secretion are glucose , insulin , cholecystokinin ( cck ) , glucagon - like peptide 1 ( glp-1 ) , glucose - dependent insulinotropic polypeptide ( gip ) , peptide yy ( pyy ) , pancreatic polypeptide ( pp ) , oxyntomodulin and somatostatin ( ss ) . most of these molecules are gastrointestinal hormones , which delay gastric emptying and display insulinotropic and anorexigenic activities [ 18 , 21 ] . insulin and glucose are thought to be dynamic modulators of plasma ghrelin concentrations in rodents and humans [ 2325 ] . hyperglycemia and hyperinsulinemia tend to decrease , while hypoglycemia and insulin deficiency tend to increase circulating ghrelin levels . insulin - mediated glucose uptake and metabolism may also control postprandial ghrelin levels , while insulin sensitivity is considered to be an important determinant of postprandial ghrelin suppression . , using a stepped hyperinsulinemic eu- , hypo- and hyperglycemic glucose clamp , insulin may suppress circulating ghrelin independently of glucose , although glucose might have an additional synergistic effect . tested the hypothesis that insulin is the driving force for postprandial ghrelin suppression by comparing the effects of meal ingestion on plasma ghrelin levels between insulin - deficient patients with type 1 diabetes and healthy controls . the investigators concluded that insulin is essential for meal - induced plasma ghrelin suppression , commenting that severe insulin deficiency in uncontrolled type 1 diabetic subjects may partly explain the episodes of hyperphagia observed in these patients , through compromising postprandial ghrelin response . studies in rats have shown that insulin - induced hypoglycemia increases , instead of decreasing , ghrelin mrna levels in the gastric fundus , providing no evidence for a direct inhibitory action of insulin on ghrelin synthesis . however , in healthy humans , plasma ghrelin concentrations are decreased during insulin - induced hypoglycemia , suggesting species - specific differences between rodents and humans . however , there is still no evidence for insulin receptors on the surface of ghrelin - producing cells . there seems to be an interaction between nutrients and insulin in reducing circulating ghrelin levels . reported that , unlike food intake , the subcutaneous administration of a short - acting insulin analog was not able to suppress ghrelin levels . concluded that meal - related ghrelin suppression is not directly regulated by glucose or insulin , since a reduction in ghrelin was observed only at supraphysiological insulin concentrations , while hyperglycemia did not decrease ghrelin at all . taking everything into consideration , glucose and insulin are unlikely to explain the entire postprandial ghrelin response , since ghrelin remains suppressed long after the normalization of glucose and insulin levels , and furthermore , because lipids tend to suppress ghrelin in the absence of substantial increases in glucose or insulin . other hormonal mediators ( such as cck , glp-1 and gip ) released in the postabsorptive phase in response to nutrient stimuli , appear to orchestrate the whole postmeal ghrelin response , enhancing the inhibitory effects of glucose and insulin , which are still remaining the principal contributors . the inverse temporal relationship between circulating concentrations of ghrelin and insulin , reported in a large number of clinical studies , substantiates the key regulatory role of insulin for ghrelin regulation [ 3638 ] . on the other hand , the interesting observation by cummings et al . previous studies indicate that leptin secretion is regulated by insulin - mediated glucose metabolism , suggesting that insulin is a positive modulator of leptin concentrations . accepting that leptin and insulin are positively associated , it is conceivable that postprandial surges of insulin are related to increased leptin levels , and given the reciprocal relationship between insulin and ghrelin , a similar inverse relationship might be supported for leptin and ghrelin as well . because insulin and leptin function as key signals conveying information on energy intake and body fat stores to the central nervous system for the long - term regulation of food intake and energy homeostasis , it is possible that reduced insulin and leptin production , as well as increased ghrelin levels , contribute to increased energy intake , weight gain , and obesity in humans . , three different macronutrient preloads of equal caloric content , volume and energy density ( protein- , fat- , and cho - based beverages ) were compared for their relative efficacy to suppress total and acylated ( bioactive ) ghrelin levels . these observations suggest very different effects of high cho meals in the early versus the later postabsorptive phase , indicating that ingested cho might prompt an early hunger rebound . the faster gastric emptying after cho ingestion , compared with lipids or proteins , can partly explain the strong and rapid postprandial ghrelin suppression in the first phase . if insulin - mediated glucose disposal is more important for ghrelin suppression than mere insulin levels , the late post - cho ghrelin overshoot may result from reduced intracellular glucose metabolism , when glucose levels decreased below baseline . given the key role of insulin for postprandial ghrelin suppression , ingested fructose suppresses ghrelin poorly . cho - enriched test meals , containing both simple and complex carbohydrates , have been used in various clinical studies investigating the differential response of postprandial ghrelin to meals of different macronutrient composition . in all of them , and particularly in normal weight subjects , cho ingestion provoked a significant postprandial ghrelin decline by approximately 30% from baseline values within 2 hours after meal onset [ 37 , 38 , 4042 ] . the higher satiety associated with protein consumption may be at least partially mediated by a protein - induced prolonged postprandial ghrelin suppression . the prolonged suppression of ghrelin after protein intake might relate to the protracted emptying of proteins from stomach , causing a more sustained activation of post - gastric ghrelin - suppressing mechanisms . additional mechanisms that account for the significant satiating effect of dietary protein include the following : proteins have a larger thermic effect than cho or fat , since they can not be stored in the body , but need to be metabolized immediately . apart from prolonging postprandial ghrelin suppression , liquid protein preloads have also shown to prolong the elevation of anorexigenic gastrointestinal hormones , such as cck and glp-1 . in a further randomized crossover study in healthy adult males , the high - protein meal maintained significantly lower ghrelin levels at 180 minutes compared with the high - cho and high - fat meals , indicating that dietary protein exhibits longer - term postprandial ghrelin suppression and enhanced satiety . it also increased glucagon and cck , tended to increase gip and glp-1 , and decreased gastric emptying rate , without affecting however ad libitum energy intake . despite the accumulating evidence supporting the satiating and ghrelin - suppressing capacity of dietary protein , there have been a few studies suggesting that protein ingestion stimulates , instead of suppressing , ghrelin levels [ 38 , 47 ] , while an additional study indicated that the satiating effect of protein is practically unrelated to postprandial ghrelin secretion . , total ghrelin levels did not decline significantly after a fat - rich meal in normal weight women , as opposed to an isoenergetic protein - rich meal . more specifically , the fat - rich meal decreased plasma ghrelin levels , but the nadir was reached towards the end of the study period , namely at 180 minutes . in a relevant double - blind crossover study , researchers assessed two high - fat test meals , one with a high saturated to unsaturated fat ratio ( 70/30 ) and the other with a low ratio ( 55/45 ) , and concluded that increasing saturated fat consumption had no deleterious effects on fasting and postprandial plasma ghrelin concentrations . a large number of clinical studies have shown that obese subjects tend to display lower total and acylated ghrelin levels in the fasting state compared with normal weight individuals . this finding appears to be an appropriate compensatory response , so that obese individuals will not get any fatter and lean individuals will not get any thinner ( adaptive mechanism for prevention of obesity and cachexia resp . ) it has been proposed that the sustained positive energy balance observed in obesity suppresses maximally circulating ghrelin levels , and thus limits flexibility for further short - term feeding regulation . furthermore , obese subjects are often insulin - resistant and thus hyperinsulinemic , and insulin is a well established inhibitory signal for ghrelin secretion . as already mentioned , it is widely accepted that obese subjects exhibit significantly lower fasting ghrelin concentrations than lean , but whether the rate of preprandial ghrelin increase is actually differentiated between lean and obese subjects has been scarcely addressed . another important aspect of ghrelin regulation in obese subjects is the blunted postprandial ghrelin response . this means that obese subjects have low ghrelin levels preprandially , but postprandial ghrelin secretion is not sufficiently suppressed , suggesting a severe defect in ghrelin - induced satiety mechanisms , which makes them feel still hungry , even though they have just completed their meal have shown that neither a protein- nor a fat - rich meal was able to elicit a significant acute ghrelin response in obese women . reported that a high - cho meal ( with a well established ghrelin - suppressing potential in lean individuals ) was also insufficient to suppress postprandial active ghrelin levels in obese women , indicating a considerable secretory and possibly satiety impairment in these subjects . apart from ghrelin , additional hormonal factors that contribute to this auto - regulation of body weight homeostasis in normal weight subjects include glp-1 , gip , and pyy , which delay gastric emptying , induce satiety and prevent hyperphagia , and are significantly more functional in lean subjects compared with the obese . however , as a person gains weight , this autoregulatory effect appears to become severely compromised . on the contrary , food intake is most likely to be increased in obese subjects , because of the blunted postprandial ghrelin response , as described above . showed that low - dose infusion of ghrelin increased ad libitum energy intake at a buffet meal only in the obese group , and not in the lean , indicating that obese people are highly sensitive to the appetite - stimulating effects of ghrelin , even when the circulating ghrelin is low . as a result , the absolute difference of fasting ghrelin levels between lean and obese subjects is not a major determinant of subsequent food intake , since other factors such as endogenous sensitivity to circulating ghrelin , ghrelin activity and postprandial ghrelin changes are thought to play an important role , as well . additional factors that can in part explain the suppressed basal ghrelin levels in obese subjects include hyperleptinaemia and increased circulating levels of il-1b ( interleukin 1b ) , since both leptin and il-1b are thought to inhibit ghrelin secretion . concerning the blunted postprandial ghrelin response in obese subjects , the impaired post - meal elevation of gastrointestinal hormones with anorexigenic and insulinomimetic properties , such as glp-1 , gip and pyy , has been implicated as an additional significant contributor . as far as insulin resistance is concerned , its role for ghrelin regulation is different in fasting and postprandial state . on the other hand , postabsorptive insulin resistance and impaired intracellular insulin signaling lead to inadequately suppressed and thus increased levels of ghrelin , since insulin sensitivity is regarded as prerequisite for sufficient postprandial ghrelin suppression . the macronutrient - specific effect of meals on postprandial ghrelin levels has interesting implications only in normal weight individuals . in addition , ghrelin levels did not correlate with insulin or glucose , indicating that regulation of ghrelin might be altered in obese patients with metabolic syndrome independently of insulin . revealed a dysregulation of ghrelin homeostasis in overweight women with polycystic ovary syndrome ( pcos ) , suggesting that women with pcos exhibit similar ghrelin abnormalities with obese women ( down - regulated fasting ghrelin , blunted postprandial ghrelin suppression ) , and this disorder was not differentially affected by diet macronutrient composition . diet - induced weight loss , contrary to gastric bypass surgery where ghrelin levels remain dramatically decreased , has shown to elevate fasting ghrelin levels and normalize postprandial ghrelin response . the greater sensitivity to vagal stimulation after weight loss may result in a more pronounced drop in postprandial ghrelin levels , in addition to the improvement in insulin sensitivity , which is a major determinant of postprandial ghrelin suppression . reported that diet - induced weight reduction preferentially improves ghrelin response to a high - cho meal , compared with a high - fat meal , indicating that weight loss might selectively improve the response of ghrelin to carbohydrate . given that alcohol seems to acutely attenuate circulating ghrelin levels and is also known for its satiating power , one might expect from alcohol to promote weight loss . on the other hand , there was a progressive decline of ghrelin in non - smokers , reaching its nadir 60 minutes after smoking . fasting total ghrelin levels were not significantly different between smokers and non - smokers , indicating that smoking is unlikely to exert a long - term anorectic effect in smoking populations . trying to delineate the central message behind all these divergent data , carbohydrate appears to be the most effective macronutrient in terms of postprandial ghrelin suppression , possibly because of its glucose - elevating and insulin - secreting effect . fructose - enriched meals display a poor ghrelin - suppressing capacity , promoting increased caloric intake , weight gain and obesity under conditions of chronic consumption . protein induces prolonged ghrelin suppression and elevation of gut - derived anorexigenic hormones that delay gastric emptying regardless of the type of protein consumed . however , the influence of solid forms of protein ( turkey , pork ) on postprandial ghrelin levels may require assessment over a longer period of time than 3 - 4 hours , since slow gastric emptying delays postprandial ghrelin nadir . as far as fat is concerned , it appears to be the least potent ghrelin - suppressant , even in normal weight subjects . in obese subjects , postprandial ghrelin response is blunted , and the macronutrient effect on ghrelin levels appears to be rather neutral . however , weight loss restores ghrelin response and leads to a significant improvement of ghrelin - mediated appetite regulation . from now on , it would be interesting to evaluate the long - term effect of macronutrient - enriched diet manipulations on fasting and postprandial ghrelin levels .

gastric cancer is one of the most common malignancies worldwide . despite decreasing incidence rates in western industrialized countries , gastric carcinoma remains in the top ten causes of tumor - related death in germany . the incidence rate of stomach cancer in poland , also decreasing , is similar to that of germany , with 16.9 cases per 100,000 population ( 19931997 ) . although surgery remains the only curative treatment , the type of operative treatment used for gastric cancer varies geographically . increased survival may depend on early detection made possible with endoscopy , surgical resection including appropriate lymph node dissection , and improvements in perioperative care . differing survival rates have been reported for patients receiving surgical intervention in japan versus those in western industrialized countries [ 5 , 6 ] . however , for patients with gastric cancer , there exists no published prognostic comparison between eastern and western europe . the purpose of this study was to compare the characteristics of gastric cancer patients and the prognostic impact between two surgical departments : one in gdansk ( poland ) , and the other in cologne ( germany ) . there were 117 patients ( 90% of all patients with gastric cancer ) in gdansk ( department of surgical oncology , medical university of gdansk [ poland ] ) , and 130 patients ( 85% of all patients with gastric cancer ) in cologne ( department of general , visceral and cancer surgery , university of cologne [ germany ] ) . inclusion criteria were : histopathologically confirmed gastric cancer ( from gastroscopy ) , no signs of metastases in preoperative assessment , and operable tumor . in all patients , the preoperative assessment of gastric cancer included not only gastroscopy but also standard laboratory tests , chest x - ray . besides , in case of doubt the patients underwent laparoscopy ( in cologne ) , and a few patients had peritoneal cytology assessment performed . the comorbidity of a patient was classified according the american society of anesthesiologists physical status : asa classification [ 7 , 8 ] . for both centers the asa grade assessment was reevaluated by one doctor according to the patient s medical history . cardiac evaluation was mostly based on reviewing the patient s medical history and assessing the coexistence of coronary disease , arterial hypertension , diabetes mellitus , and the new york heart association ( nyha ) score . respiratory system evaluation included analysis of the patient s medical history , medical examination of the patient , and chest x - ray . patients who had a prior history of a respiratory disorder had spirometry performed and its result had an impact on the final asa score . in cologne , the guidelines of the german society of surgery were used to evaluate indications for therapy and the selection of the surgical procedure [ 9 , 10 ] . summarized , these guidelines are as follows : endoscopic mucosal resection and submucosal dissection are indicated in superficial cancer confined to the mucosa with special characteristics ( t1a / no ulcer / g1,2/laurn intestinal / l0/v0/tumor size < 2 cm ) . in all other cases total gastrectomy or distal subtotal gastric resection are indicated , the latter in cases of tumors located in the distal two - thirds of the stomach . carcinoma in the cardia or upper third of the stomach , classified according to siewert et al . as type ii or iii tumors , can be treated by an extended total gastrectomy with a transhiatal resection of the distal esophagus and lad of the lower mediastinum and the abdominal d2 compartment . extended organ resections are only indicated in cases where r0 resection is possible . in gdansk , the main goal of surgical intervention was the complete removal of tumor . indications for performing either subtotal gastrectomy , total gastrectomy , or extended total gastrectomy were based on tumor location , histological type , and trial for achieving surgical tumor - free margins . splenectomy was carried out after iatrogenic stimulus or when there was apparent direct infiltration of tumor . reconstruction of the digestive tract was performed with either billroth ii reconstruction ( after subtotal gastrectomy ) , or with the roux - en - y reconstruction technique . resected specimens were routinely fixed in 5% phosphate - buffered formalin and embedded in paraffin . histopathological examination of all resected specimens consisted of a thorough and standardized evaluation of tumor stage , residual tumor ( r ) category , grading , and number of resected and infiltrated lymph nodes . postoperative staging was done according to the 6th edition of the international union against cancer ( uicc)-tnm classification of malignant tumors . gastric lymph nodes were documented according to the classification of the japanese research society of gastric cancer ( jrsgc ) , with lymph node groups 113 . lesions were further classified and graded in accordance with the who recommendations , the laurn classification , and tumor differentiation ( g1g4 ) . descriptive analysis included the frequency of nominal parameters , the median with the lower quartile ( lq ) and upper quartile ( uq ) for numeric variables ( ordinal or asymmetric distribution ) , and the mean for numeric variables with normal distribution . univariate analysis was calculated for tables using statistics with the yates correction or fisher s exact test if necessary . the mann more than 90% of the patients had a follow - up duration longer than four years . univariate survival analysis was conducted according to kaplan meier , and survival curves were compared with the log rank test . in addition , the hazard ratio with the 95% confidence interval ( 95% ci ) was calculated . multivariate analysis was performed by the cox regression method , using the backward option , meaning : first enter all variables into the model and then remove nonsignificant variables sequentially . assuming that the hazard function for gdansk was different from that for cologne over time , we stratified according to center . the spss for windows ( version 17.0 ) application ( spss , chicago il , usa ) was used . for graphical presentation of the survival curve , the statistical program medcalc for windows version 11.4 ( there were 117 patients ( 90% of all patients with gastric cancer ) in gdansk ( department of surgical oncology , medical university of gdansk [ poland ] ) , and 130 patients ( 85% of all patients with gastric cancer ) in cologne ( department of general , visceral and cancer surgery , university of cologne [ germany ] ) . inclusion criteria were : histopathologically confirmed gastric cancer ( from gastroscopy ) , no signs of metastases in preoperative assessment , and operable tumor . in all patients , the preoperative assessment of gastric cancer included not only gastroscopy but also standard laboratory tests , chest x - ray . besides , in case of doubt the patients underwent laparoscopy ( in cologne ) , and a few patients had peritoneal cytology assessment performed . the comorbidity of a patient was classified according the american society of anesthesiologists physical status : asa classification [ 7 , 8 ] . for both centers the asa grade assessment was reevaluated by one doctor according to the patient s medical history . cardiac evaluation was mostly based on reviewing the patient s medical history and assessing the coexistence of coronary disease , arterial hypertension , diabetes mellitus , and the new york heart association ( nyha ) score . respiratory system evaluation included analysis of the patient s medical history , medical examination of the patient , and chest x - ray . patients who had a prior history of a respiratory disorder had spirometry performed and its result had an impact on the final asa score . in cologne , the guidelines of the german society of surgery were used to evaluate indications for therapy and the selection of the surgical procedure [ 9 , 10 ] . summarized , these guidelines are as follows : endoscopic mucosal resection and submucosal dissection are indicated in superficial cancer confined to the mucosa with special characteristics ( t1a / no ulcer / g1,2/laurn intestinal / l0/v0/tumor size < 2 cm ) . in all other cases total gastrectomy or distal subtotal gastric resection are indicated , the latter in cases of tumors located in the distal two - thirds of the stomach . carcinoma in the cardia or upper third of the stomach , classified according to siewert et al . as type ii or iii tumors , can be treated by an extended total gastrectomy with a transhiatal resection of the distal esophagus and lad of the lower mediastinum and the abdominal d2 compartment . extended organ resections are only indicated in cases where r0 resection is possible . in gdansk , the main goal of surgical intervention was the complete removal of tumor . indications for performing either subtotal gastrectomy , total gastrectomy , or extended total gastrectomy were based on tumor location , histological type , and trial for achieving surgical tumor - free margins . splenectomy was carried out after iatrogenic stimulus or when there was apparent direct infiltration of tumor . reconstruction of the digestive tract was performed with either billroth ii reconstruction ( after subtotal gastrectomy ) , or with the roux - en - y reconstruction technique . resected specimens were routinely fixed in 5% phosphate - buffered formalin and embedded in paraffin . histopathological examination of all resected specimens consisted of a thorough and standardized evaluation of tumor stage , residual tumor ( r ) category , grading , and number of resected and infiltrated lymph nodes . postoperative staging was done according to the 6th edition of the international union against cancer ( uicc)-tnm classification of malignant tumors . gastric lymph nodes were documented according to the classification of the japanese research society of gastric cancer ( jrsgc ) , with lymph node groups 113 . lesions were further classified and graded in accordance with the who recommendations , the laurn classification , and tumor differentiation ( g1g4 ) . descriptive analysis included the frequency of nominal parameters , the median with the lower quartile ( lq ) and upper quartile ( uq ) for numeric variables ( ordinal or asymmetric distribution ) , and the mean for numeric variables with normal distribution . univariate analysis was calculated for tables using statistics with the yates correction or fisher s exact test if necessary . the mann more than 90% of the patients had a follow - up duration longer than four years . univariate survival analysis was conducted according to kaplan meier , and survival curves were compared with the log rank test . in addition , the hazard ratio with the 95% confidence interval ( 95% ci ) was calculated . multivariate analysis was performed by the cox regression method , using the backward option , meaning : first enter all variables into the model and then remove nonsignificant variables sequentially . assuming that the hazard function for gdansk was different from that for cologne over time , we stratified according to center . the spss for windows ( version 17.0 ) application ( spss , chicago il , usa ) was used . for graphical presentation of the survival curve , the statistical program medcalc for windows version 11.4 ( the proportion of females was significantly ( p = 0.019 ) higher in the polish group ( 41.9% ) compared to the german group ( 27.7% ) . patients from gdansk were younger , with a median age of 63.9 years compared to those from germany ( median 66.8 years ) ; however , the difference was not statistically significant ( p = 0.212 ) . cologne patients had significantly ( p < 0.001 ) more severe comorbidities ( asa iii and iv ) than patients in gdansk . the demographic characteristics are shown in table 1.table 1demographic and histopathological data from patients treated surgically for gastric carcinoma from gdansk ( poland ) and cologne ( germany)featuregdansk , poland ( n = 117)cologne , germany ( n = 130)p valuedemographicsgender males68 ( 58.1%)94 ( 72.3%)0.019 females49 ( 41.9%)36 ( 27.7%)median age ( min max ) all patients63.9 years ( 2688 years)66.8 years ( 3187 years)0.212 males63.3 years66.7 years0.063 females67.8 years67.0 years0.972comorbidities asa i and ii77 ( 65.5%)51 ( 39.0%)<0.001 asa iii and iv40 ( 34.5%)79 ( 61.0%)histopathologytumor localization upper third43 ( 36.8%)59 ( 45.4%)0.356 middle third41 ( 35.0%)37 ( 28.5% ) lower third33 ( 28.2%)34 ( 26.2%)type of histopathology ( who ) adenocarcinoma100 ( 85.3%)82 ( 63.2%)<0.001 signet - ring cell carcinoma16 ( 13.7%)45 ( 34.4% ) other histology1 ( 1.0%)3 ( 2.4%)pt category pt16 ( 4.8%)29 ( 22.3%)<0.001 pt222 ( 19.0%)28 ( 21.5% ) pt372 ( 61.9%)37 ( 28.5% ) pt417 ( 14.3%)36 ( 27.7%)pn category pn037 ( 31.3%)44 ( 33.8%)0.339 pn142 ( 35.7%)41 ( 31.5% ) pn229 ( 25.0%)26 ( 20.0% ) pn39 ( 8.0%)19 ( 14.6%)pm category pm0100 ( 85.6%)93 ( 71.5%)0.009 pm117 ( 14.4%)37 ( 28.5%)asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases demographic and histopathological data from patients treated surgically for gastric carcinoma from gdansk ( poland ) and cologne ( germany ) asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases the tumor was located in the upper third of the stomach for nearly half of the patients in both clinics . there was no significant difference in tumor localization . signet - ring cell carcinoma histology was diagnosed significantly ( p < 0.001 ) more often in patients from cologne ( 34.4% ) versus those from gdansk ( 13.7% ) . in contrast , the polish patients showed significantly ( p < 0.001 ) more advanced pt categories than the german patients . at a rate of only 5% , early cancer was a rare diagnosis in gdansk , in contrast to the cologne group , where the frequency was 22% ( p the 30- and 90-day mortality rates did not differ between the clinics , at 0.9 and 5.9% in gdansk versus 2.3 and 4.6% in cologne.table 2surgical treatment of patients with gastric cancer from gdansk ( poland ) and cologne ( germany)featuregdansk , poland ( n = 117)cologne , germany ( n = 130)p valuetype of resection subtotal gastrectomy14 ( 11.8%)17 ( 13.4%)<0.001 total gastrectomy82 ( 70.0%)40 ( 30.7% ) extended gastrectomy20 ( 17.3%)36 ( 27.6% ) transhiatal extended gastrectomy0 ( 0%)36 ( 27.6% ) others1 ( 0.9%)1 ( 0.8%)splenectomy splenectomy during gastrectomy67 ( 57.1%)26 ( 19.8%)<0.001r0 resection r0103 ( 87.7%)119 ( 91.5%)0.389 r1/r214 ( 12.3%)11 ( 8.5%)lymphadenectomy d199 ( 84.8%)14 ( 10.6%)<0.001 d218 ( 15.2%)116 ( 89.4%)number of resected lymph nodes median ( min max)13.0 ( 047)33.5 ( 382)<0.001number of metastatic lymph nodes median ( min max)3.0 ( 041)3.0 ( 043)0.948r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection group surgical treatment of patients with gastric cancer from gdansk ( poland ) and cologne ( germany ) r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection group univariate analysis identified no significant difference in prognosis between the two surgical departments ( table 3 ) . the estimated 5-year overall survival rate for patients from gdansk was 28.3% and that for patients from cologne was 40.3% ( p = 0.056 ) , with a hazard ratio of 0.71 ( 95% ci 0.541.01 ) ( fig . 1 ) . in the univariate analysis performed separately for each center , the following prognostic factors were identified as significant for both centers : clear surgical resection margin ( r0 category ) , pt , pn , and pm category ( p < 0.001 for each ) . in cologne , there was improved survival in younger ( < 60 years ) versus older patients ( 60 years ) ( p = 0.021 ) . for patients from gdansk , tumor localization ( p = 0.012 ) , histological tumor type ( p = 0.001 ) , and type of lymphadenectomy performed ( p = 0.031 ) had prognostic relevance . the following parameters did not influence survival in either clinic : gender , comorbidity ( asa i and ii vs. iii and iv ) , type of gastric resection , and splenectomy.table 3univariate survival analysis comparing the impact of demographic and histopathological factors between 117 patients with gastric cancer from gdansk ( poland ) and 130 patients from cologne ( germany)univariate survival analysis : 5 year survival rategdansk , poland ( n = 117 ) ( % ) p valuecologne , germany ( n = 130 ) ( % ) p valuep value between the two centersgender male30.10.38842.30.9620.168 female20.435.20.336comorbidities asa i and ii26.50.69147.30.1020.017 asa iii and iv30.736.10.781tumor localization upper third32.90.01242.10.5240.325 middle third14.632.10.202 lower third40.147.10.529histopathological classification ( who ) adenocarcinoma ( except for signet - ring cell cancer)32.20.00139.90.5240.237 signet - ring cell cancer0.039.90.007pt category pt1<0.00164.4<0.001 pt245.246.70.928 pt318.735.80.271 pt40.018.70.201pn category pn059.5<0.00163.1<0.0010.592 pn120.444.40.079 pn20.013.40.079 pn30.013.00.916pm category pm032.0<0.00153.9<0.0010.013 pm10.08.50.175r category r031.6<0.00141.8<0.0010.179 r1/r2000.107type of gastric resection subtotal resection44.00.11650.40.8290.752 total gastrectomy29.934.10.437 extended gastrectomy15.824.00.314 transhiatal extended gastrectomy52.2lymphadenectomy d125.10.03119.20.1650.804 d247.142.80.521asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases , r category : r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection groupfig . 1comparison of survival curves of gastric cancer patients from gdansk ( poland ) and from cologne ( germany ) [ p = 0.056 ; hazard ratio 0.71 ( 95% confidence interval [ ci ] 0.541.01 ) ] univariate survival analysis comparing the impact of demographic and histopathological factors between 117 patients with gastric cancer from gdansk ( poland ) and 130 patients from cologne ( germany ) asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases , r category : r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection group comparison of survival curves of gastric cancer patients from gdansk ( poland ) and from cologne ( germany ) [ p = 0.056 ; hazard ratio 0.71 ( 95% confidence interval [ ci ] 0.541.01 ) ] on comparison of the clinics , parameters responsible for the better prognosis in cologne were healthy baseline status ( asa i or ii ) , signet - ring cell carcinoma histology , and absence of distant metastasis ( table 3 ) . the most relevant prognostic factors from the univariate survival analysis were used for multivariate analysis : pt , pn , pm , and r category . in addition , the parameters age ( continuous variable ) , gender , comorbidity , tumor localization , histology , and the number of resected lymph nodes ( continuous variable ) were included . the established independent prognostic factors , pt , pn , pm , and r category , were confirmed with our analysis . patient age was an independent prognostic factor , with a hazard ratio of 1.022 for each subsequent year . as well , each additional resected lymph node improved the prognosis.table 4multivariate survival analysis of independent prognostic factors for patients with surgically treated gastric carcinomafeaturehazard ratio95% cip valueage each year1.0221.0050.012pt category0.002 pt1pt21.450.643.300.379 pt1pt32.831.345.930.006 pt1pt43.191.437.130.005pn category<0.001 pn0pn12.361.374.080.002 pn0pn23.081.735.49<0.001 pn0pn38.824.1018.98<0.001number of resected lns each ln0.9790.970.990.005pm category pm0pm11.811.073.060.027r category r0r1/r22.421.324.450.004ci confidence interval , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases , ln lymph node , r category : r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection ( according to the 6th edition of the international union against cancer ( uicc)-tnm classification of malignant tumors ) multivariate survival analysis of independent prognostic factors for patients with surgically treated gastric carcinoma ci confidence interval , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases , ln lymph node , r category : r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection ( according to the 6th edition of the international union against cancer ( uicc)-tnm classification of malignant tumors ) the proportion of females was significantly ( p = 0.019 ) higher in the polish group ( 41.9% ) compared to the german group ( 27.7% ) . patients from gdansk were younger , with a median age of 63.9 years compared to those from germany ( median 66.8 years ) ; however , the difference was not statistically significant ( p = 0.212 ) . cologne patients had significantly ( p < 0.001 ) more severe comorbidities ( asa iii and iv ) than patients in gdansk . the demographic characteristics are shown in table 1.table 1demographic and histopathological data from patients treated surgically for gastric carcinoma from gdansk ( poland ) and cologne ( germany)featuregdansk , poland ( n = 117)cologne , germany ( n = 130)p valuedemographicsgender males68 ( 58.1%)94 ( 72.3%)0.019 females49 ( 41.9%)36 ( 27.7%)median age ( min max ) all patients63.9 years ( 2688 years)66.8 years ( 3187 years)0.212 males63.3 years66.7 years0.063 females67.8 years67.0 years0.972comorbidities asa i and ii77 ( 65.5%)51 ( 39.0%)<0.001 asa iii and iv40 ( 34.5%)79 ( 61.0%)histopathologytumor localization upper third43 ( 36.8%)59 ( 45.4%)0.356 middle third41 ( 35.0%)37 ( 28.5% ) lower third33 ( 28.2%)34 ( 26.2%)type of histopathology ( who ) adenocarcinoma100 ( 85.3%)82 ( 63.2%)<0.001 signet - ring cell carcinoma16 ( 13.7%)45 ( 34.4% ) other histology1 ( 1.0%)3 ( 2.4%)pt category pt16 ( 4.8%)29 ( 22.3%)<0.001 pt222 ( 19.0%)28 ( 21.5% ) pt372 ( 61.9%)37 ( 28.5% ) pt417 ( 14.3%)36 ( 27.7%)pn category pn037 ( 31.3%)44 ( 33.8%)0.339 pn142 ( 35.7%)41 ( 31.5% ) pn229 ( 25.0%)26 ( 20.0% ) pn39 ( 8.0%)19 ( 14.6%)pm category pm0100 ( 85.6%)93 ( 71.5%)0.009 pm117 ( 14.4%)37 ( 28.5%)asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases demographic and histopathological data from patients treated surgically for gastric carcinoma from gdansk ( poland ) and cologne ( germany ) asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases the tumor was located in the upper third of the stomach for nearly half of the patients in both clinics . signet - ring cell carcinoma histology was diagnosed significantly ( p < 0.001 ) more often in patients from cologne ( 34.4% ) versus those from gdansk ( 13.7% ) . in contrast , the polish patients showed significantly ( p < 0.001 ) more advanced pt categories than the german patients . at a rate of only 5% , early cancer was a rare diagnosis in gdansk , in contrast to the cologne group , where the frequency was 22% ( p < 0.001 ) . the 30- and 90-day mortality rates did not differ between the clinics , at 0.9 and 5.9% in gdansk versus 2.3 and 4.6% in cologne.table 2surgical treatment of patients with gastric cancer from gdansk ( poland ) and cologne ( germany)featuregdansk , poland ( n = 117)cologne , germany ( n = 130)p valuetype of resection subtotal gastrectomy14 ( 11.8%)17 ( 13.4%)<0.001 total gastrectomy82 ( 70.0%)40 ( 30.7% ) extended gastrectomy20 ( 17.3%)36 ( 27.6% ) transhiatal extended gastrectomy0 ( 0%)36 ( 27.6% ) others1 ( 0.9%)1 ( 0.8%)splenectomy splenectomy during gastrectomy67 ( 57.1%)26 ( 19.8%)<0.001r0 resection r0103 ( 87.7%)119 ( 91.5%)0.389 r1/r214 ( 12.3%)11 ( 8.5%)lymphadenectomy d199 ( 84.8%)14 ( 10.6%)<0.001 d218 ( 15.2%)116 ( 89.4%)number of resected lymph nodes median ( min max)13.0 ( 047)33.5 ( 382)<0.001number of metastatic lymph nodes median ( min max)3.0 ( 041)3.0 ( 043)0.948r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection group surgical treatment of patients with gastric cancer from gdansk ( poland ) and cologne ( germany ) r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection group univariate analysis identified no significant difference in prognosis between the two surgical departments ( table 3 ) . the estimated 5-year overall survival rate for patients from gdansk was 28.3% and that for patients from cologne was 40.3% ( p = 0.056 ) , with a hazard ratio of 0.71 ( 95% ci 0.541.01 ) ( fig . 1 ) . in the univariate analysis performed separately for each center , the following prognostic factors were identified as significant for both centers : clear surgical resection margin ( r0 category ) , pt , pn , and pm category ( p < 0.001 for each ) . in cologne , there was improved survival in younger ( < 60 years ) versus older patients ( 60 years ) ( p = 0.021 ) . for patients from gdansk , tumor localization ( p = 0.012 ) , histological tumor type ( p = 0.001 ) , and type of lymphadenectomy performed ( p = 0.031 ) had prognostic relevance . the following parameters did not influence survival in either clinic : gender , comorbidity ( asa i and ii vs. iii and iv ) , type of gastric resection , and splenectomy.table 3univariate survival analysis comparing the impact of demographic and histopathological factors between 117 patients with gastric cancer from gdansk ( poland ) and 130 patients from cologne ( germany)univariate survival analysis : 5 year survival rategdansk , poland ( n = 117 ) ( % ) p valuecologne , germany ( n = 130 ) ( % ) p valuep value between the two centersgender male30.10.38842.30.9620.168 female20.435.20.336comorbidities asa i and ii26.50.69147.30.1020.017 asa iii and iv30.736.10.781tumor localization upper third32.90.01242.10.5240.325 middle third14.632.10.202 lower third40.147.10.529histopathological classification ( who ) adenocarcinoma ( except for signet - ring cell cancer)32.20.00139.90.5240.237 signet - ring cell cancer0.039.90.007pt category pt1<0.00164.4<0.001 pt245.246.70.928 pt318.735.80.271 pt40.018.70.201pn category pn059.5<0.00163.1<0.0010.592 pn120.444.40.079 pn20.013.40.079 pn30.013.00.916pm category pm032.0<0.00153.9<0.0010.013 pm10.08.50.175r category r031.6<0.00141.8<0.0010.179 r1/r2000.107type of gastric resection subtotal resection44.00.11650.40.8290.752 total gastrectomy29.934.10.437 extended gastrectomy15.824.00.314 transhiatal extended gastrectomy52.2lymphadenectomy d125.10.03119.20.1650.804 d247.142.80.521asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases , r category : r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection groupfig . 1comparison of survival curves of gastric cancer patients from gdansk ( poland ) and from cologne ( germany ) [ p = 0.056 ; hazard ratio 0.71 ( 95% confidence interval [ ci ] 0.541.01 ) ] univariate survival analysis comparing the impact of demographic and histopathological factors between 117 patients with gastric cancer from gdansk ( poland ) and 130 patients from cologne ( germany ) asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases , r category : r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection group comparison of survival curves of gastric cancer patients from gdansk ( poland ) and from cologne ( germany ) [ p = 0.056 ; hazard ratio 0.71 ( 95% confidence interval [ ci ] 0.541.01 ) ] on comparison of the clinics , parameters responsible for the better prognosis in cologne were healthy baseline status ( asa i or ii ) , signet - ring cell carcinoma histology , and absence of distant metastasis ( table 3 ) . the most relevant prognostic factors from the univariate survival analysis were used for multivariate analysis : pt , pn , pm , and r category . in addition , the parameters age ( continuous variable ) , gender , comorbidity , tumor localization , histology , and the number of resected lymph nodes ( continuous variable ) were included . the established independent prognostic factors , pt , pn , pm , and r category , were confirmed with our analysis . patient age was an independent prognostic factor , with a hazard ratio of 1.022 for each subsequent year . as well , each additional resected lymph node improved the prognosis.table 4multivariate survival analysis of independent prognostic factors for patients with surgically treated gastric carcinomafeaturehazard ratio95% cip valueage each year1.0221.0050.012pt category0.002 pt1pt21.450.643.300.379 pt1pt32.831.345.930.006 pt1pt43.191.437.130.005pn category<0.001 pn0pn12.361.374.080.002 pn0pn23.081.735.49<0.001 pn0pn38.824.1018.98<0.001number of resected lns each ln0.9790.970.990.005pm category pm0pm11.811.073.060.027r category r0r1/r22.421.324.450.004ci confidence interval , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases , ln lymph node , r category : r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection ( according to the 6th edition of the international union against cancer ( uicc)-tnm classification of malignant tumors ) multivariate survival analysis of independent prognostic factors for patients with surgically treated gastric carcinoma ci confidence interval , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases , ln lymph node , r category : r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection ( according to the 6th edition of the international union against cancer ( uicc)-tnm classification of malignant tumors ) not only did demographic and tumor characteristics vary , but also the indications for choice of therapy and the radicality of surgical intervention performed varied . despite these differences , established independent prognostic factors for gastric carcinoma , such as depth of tumor infiltration , extent of lymph node metastasis , and presence of distant metastasis , as well as r0 resection , the importance of these prognostic factors for the comparison between nations has already been reported in studies comparing japan and western industrialized nations [ 5 , 6 ] . depth of tumor infiltration and the extent of metastasis , as well as the possibility for an r0 resection selected from suitable surgical procedures , are clinical features that patients possess before entering a clinic . in contrast are the frequency of successful r0 resections and the extent of lymphadenectomies performed , which are determined at least partially by the operating surgeon . in terms of patient - related factors , there were more women in gdansk than in cologne who were subjected to surgery for gastric carcinoma . it is possible that this represents a selection bias , because the incidence of gastric carcinoma in women in the two countries does not differ . although the age distribution at the two clinics did not differ , presentation of the illness later in life does decrease life expectancy . age also correlates with the severity of comorbidities , a fact that could explain why more severe comorbidity appears to have no prognostic relevance . furthermore , the parameter chosen here , the asa classification , is a nonspecific measure to estimate prognosis . the main difference in clinicopathological features was seen in the depth of tumor invasion into the gastric wall . this correlates with data published by the polish gastric cancer study group and the german gastric cancer study , for gdansk and cologne , respectively [ 1921 ] . we believe that the observed differences in tumor stage frequency are secondary to varying schools of thought in german society and polish society , as well as differences in national health policies . there is probably less access to gastroscopy in poland than in germany , and perhaps polish patients do not immediately visit general practitioners with the first symptoms of gastric disease . although there were differences between the clinics in the tumor stage assessed at time of operation , the tumor stage distribution among our patients was nothing like that observed in japan , where 5665% of all gastric cancers are stage i . this suggests that all european union countries must take more care to achieve earlier diagnosis . surgeon - dependent factors include the selection of surgical therapy and the radicality of the tumor resection performed . in germany , different indications were used by the two clinics to determine the types of resection and lymphadenectomy performed and the need for splenectomy , as well as the number of resected lymph nodes . . found similar results for patients with adenocarcinoma of the esophagus or gastric cardia in an international study assessing the impact of the extent of surgical resection . an extended lymph node dissection is thought to provide more appropriate pathological staging and better regional disease control , as well as possible survival advantages compared to limited lymph node dissection . two recent prospective , randomized european trials ( in the netherlands and the united kingdom ) were designed to evaluate whether extended lymphadenectomy improves overall survival . however , the results of both studies were influenced by increased postoperative morbidity / mortality rates associated with increased rates of splenectomy and pancreatectomy in patients undergoing d2-dissection [ 2629 ] . in a review study , mcculloch et al . concluded that the question of extended versus limited lymph node dissection has not yet been decided . the fact that 57% of gdansk patients had splenectomy performed during gastrectomy came out as an unanticipated observation . it was related to the conviction that removal of the lymph nodes located in the splenic hilum may be a prognostic marker . several studies have concluded that the disadvantages of splenectomy outweigh the prognostic benefits for patients with gastric cancer [ 22 , 31 , 32 ] . the results of our analysis convinced the gdansk surgical team to change the strategy of management and to improve the surgical quality despite differences in the frequencies of various prognostic factors between polish and german gastric carcinoma patients , established prognostic factors such as the depth of tumor infiltration , the existence of lymph node or distant metastasis , and complete removal of the tumor were verified by our results . of interest is the simultaneous emergence , at the two study centers , of the number of removed lymph nodes as an independent prognostic factor .

backgroundalthough studies comparing the surgical treatment of gastric carcinoma in japan and western industrialized countries have revealed differing survival rates , no studies to date have been performed comparing western and eastern europe . this study aimed to compare demographics and surgical practice as well as the related prognostic impact on gastric cancer patients treated in poland and germany.methodsthis retrospective study included gastric cancer patients treated between 1999 and 2004 by surgical departments in gdansk ( poland ) and cologne ( germany ) . univariate and multivariate analyses of demographic , histopathological , surgical , and prognostic data were performed.resultsincluded were 117 patients from gdansk and 130 patients from cologne . the cologne patients showed higher incidence rates of serious comorbidity , pt1 cancer , and distant metastasis than those from gdansk . indications for and frequency of selected surgical procedures differed significantly . d2-lymphadenectomy was performed in 89% of the cologne patients , while d1-lymphadenectomy was done for 85% of the gdansk patients . univariate analysis yielded a 5-year survival rate of 28.3% for the gdansk patients , and 40.3% for the cologne patients ( p = 0.056 ) . independent prognostic factors were pt category ( p = 0.002 ) , pn category ( p < 0.001 ) , pm category ( p = 0.027 ) , residual tumor ( r ) category ( p = 0.004 ) , age ( p = 0.012 ) , and number of resected lymph nodes ( p = 0.005).conclusionssignificant differences of clinical and surgical parameters exist between gastric cancer patients treated in poland and germany . in addition to established independent prognostic factors , we found that survival improved with each additionally resected lymph node .

Introduction Patients and methods Patients Surgical procedures Histopathology Statistical analysis Results Demographics Histopathology Surgical treatment Prognosis Discussion Conclusions

the purpose of this study was to compare the characteristics of gastric cancer patients and the prognostic impact between two surgical departments : one in gdansk ( poland ) , and the other in cologne ( germany ) . the demographic characteristics are shown in table 1.table 1demographic and histopathological data from patients treated surgically for gastric carcinoma from gdansk ( poland ) and cologne ( germany)featuregdansk , poland ( n = 117)cologne , germany ( n = 130)p valuedemographicsgender males68 ( 58.1%)94 ( 72.3%)0.019 females49 ( 41.9%)36 ( 27.7%)median age ( min max ) all patients63.9 years ( 2688 years)66.8 years ( 3187 years)0.212 males63.3 years66.7 years0.063 females67.8 years67.0 years0.972comorbidities asa i and ii77 ( 65.5%)51 ( 39.0%)<0.001 asa iii and iv40 ( 34.5%)79 ( 61.0%)histopathologytumor localization upper third43 ( 36.8%)59 ( 45.4%)0.356 middle third41 ( 35.0%)37 ( 28.5% ) lower third33 ( 28.2%)34 ( 26.2%)type of histopathology ( who ) adenocarcinoma100 ( 85.3%)82 ( 63.2%)<0.001 signet - ring cell carcinoma16 ( 13.7%)45 ( 34.4% ) other histology1 ( 1.0%)3 ( 2.4%)pt category pt16 ( 4.8%)29 ( 22.3%)<0.001 pt222 ( 19.0%)28 ( 21.5% ) pt372 ( 61.9%)37 ( 28.5% ) pt417 ( 14.3%)36 ( 27.7%)pn category pn037 ( 31.3%)44 ( 33.8%)0.339 pn142 ( 35.7%)41 ( 31.5% ) pn229 ( 25.0%)26 ( 20.0% ) pn39 ( 8.0%)19 ( 14.6%)pm category pm0100 ( 85.6%)93 ( 71.5%)0.009 pm117 ( 14.4%)37 ( 28.5%)asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases demographic and histopathological data from patients treated surgically for gastric carcinoma from gdansk ( poland ) and cologne ( germany ) asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases the tumor was located in the upper third of the stomach for nearly half of the patients in both clinics . at a rate of only 5% , early cancer was a rare diagnosis in gdansk , in contrast to the cologne group , where the frequency was 22% ( p the 30- and 90-day mortality rates did not differ between the clinics , at 0.9 and 5.9% in gdansk versus 2.3 and 4.6% in cologne.table 2surgical treatment of patients with gastric cancer from gdansk ( poland ) and cologne ( germany)featuregdansk , poland ( n = 117)cologne , germany ( n = 130)p valuetype of resection subtotal gastrectomy14 ( 11.8%)17 ( 13.4%)<0.001 total gastrectomy82 ( 70.0%)40 ( 30.7% ) extended gastrectomy20 ( 17.3%)36 ( 27.6% ) transhiatal extended gastrectomy0 ( 0%)36 ( 27.6% ) others1 ( 0.9%)1 ( 0.8%)splenectomy splenectomy during gastrectomy67 ( 57.1%)26 ( 19.8%)<0.001r0 resection r0103 ( 87.7%)119 ( 91.5%)0.389 r1/r214 ( 12.3%)11 ( 8.5%)lymphadenectomy d199 ( 84.8%)14 ( 10.6%)<0.001 d218 ( 15.2%)116 ( 89.4%)number of resected lymph nodes median ( min max)13.0 ( 047)33.5 ( 382)<0.001number of metastatic lymph nodes median ( min max)3.0 ( 041)3.0 ( 043)0.948r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection group surgical treatment of patients with gastric cancer from gdansk ( poland ) and cologne ( germany ) r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection group univariate analysis identified no significant difference in prognosis between the two surgical departments ( table 3 ) . the following parameters did not influence survival in either clinic : gender , comorbidity ( asa i and ii vs. iii and iv ) , type of gastric resection , and splenectomy.table 3univariate survival analysis comparing the impact of demographic and histopathological factors between 117 patients with gastric cancer from gdansk ( poland ) and 130 patients from cologne ( germany)univariate survival analysis : 5 year survival rategdansk , poland ( n = 117 ) ( % ) p valuecologne , germany ( n = 130 ) ( % ) p valuep value between the two centersgender male30.10.38842.30.9620.168 female20.435.20.336comorbidities asa i and ii26.50.69147.30.1020.017 asa iii and iv30.736.10.781tumor localization upper third32.90.01242.10.5240.325 middle third14.632.10.202 lower third40.147.10.529histopathological classification ( who ) adenocarcinoma ( except for signet - ring cell cancer)32.20.00139.90.5240.237 signet - ring cell cancer0.039.90.007pt category pt1<0.00164.4<0.001 pt245.246.70.928 pt318.735.80.271 pt40.018.70.201pn category pn059.5<0.00163.1<0.0010.592 pn120.444.40.079 pn20.013.40.079 pn30.013.00.916pm category pm032.0<0.00153.9<0.0010.013 pm10.08.50.175r category r031.6<0.00141.8<0.0010.179 r1/r2000.107type of gastric resection subtotal resection44.00.11650.40.8290.752 total gastrectomy29.934.10.437 extended gastrectomy15.824.00.314 transhiatal extended gastrectomy52.2lymphadenectomy d125.10.03119.20.1650.804 d247.142.80.521asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases , r category : r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection groupfig . 1comparison of survival curves of gastric cancer patients from gdansk ( poland ) and from cologne ( germany ) [ p = 0.056 ; hazard ratio 0.71 ( 95% confidence interval [ ci ] 0.541.01 ) ] univariate survival analysis comparing the impact of demographic and histopathological factors between 117 patients with gastric cancer from gdansk ( poland ) and 130 patients from cologne ( germany ) asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases , r category : r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection group comparison of survival curves of gastric cancer patients from gdansk ( poland ) and from cologne ( germany ) [ p = 0.056 ; hazard ratio 0.71 ( 95% confidence interval [ ci ] 0.541.01 ) ] on comparison of the clinics , parameters responsible for the better prognosis in cologne were healthy baseline status ( asa i or ii ) , signet - ring cell carcinoma histology , and absence of distant metastasis ( table 3 ) . the demographic characteristics are shown in table 1.table 1demographic and histopathological data from patients treated surgically for gastric carcinoma from gdansk ( poland ) and cologne ( germany)featuregdansk , poland ( n = 117)cologne , germany ( n = 130)p valuedemographicsgender males68 ( 58.1%)94 ( 72.3%)0.019 females49 ( 41.9%)36 ( 27.7%)median age ( min max ) all patients63.9 years ( 2688 years)66.8 years ( 3187 years)0.212 males63.3 years66.7 years0.063 females67.8 years67.0 years0.972comorbidities asa i and ii77 ( 65.5%)51 ( 39.0%)<0.001 asa iii and iv40 ( 34.5%)79 ( 61.0%)histopathologytumor localization upper third43 ( 36.8%)59 ( 45.4%)0.356 middle third41 ( 35.0%)37 ( 28.5% ) lower third33 ( 28.2%)34 ( 26.2%)type of histopathology ( who ) adenocarcinoma100 ( 85.3%)82 ( 63.2%)<0.001 signet - ring cell carcinoma16 ( 13.7%)45 ( 34.4% ) other histology1 ( 1.0%)3 ( 2.4%)pt category pt16 ( 4.8%)29 ( 22.3%)<0.001 pt222 ( 19.0%)28 ( 21.5% ) pt372 ( 61.9%)37 ( 28.5% ) pt417 ( 14.3%)36 ( 27.7%)pn category pn037 ( 31.3%)44 ( 33.8%)0.339 pn142 ( 35.7%)41 ( 31.5% ) pn229 ( 25.0%)26 ( 20.0% ) pn39 ( 8.0%)19 ( 14.6%)pm category pm0100 ( 85.6%)93 ( 71.5%)0.009 pm117 ( 14.4%)37 ( 28.5%)asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases demographic and histopathological data from patients treated surgically for gastric carcinoma from gdansk ( poland ) and cologne ( germany ) asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases the tumor was located in the upper third of the stomach for nearly half of the patients in both clinics . the 30- and 90-day mortality rates did not differ between the clinics , at 0.9 and 5.9% in gdansk versus 2.3 and 4.6% in cologne.table 2surgical treatment of patients with gastric cancer from gdansk ( poland ) and cologne ( germany)featuregdansk , poland ( n = 117)cologne , germany ( n = 130)p valuetype of resection subtotal gastrectomy14 ( 11.8%)17 ( 13.4%)<0.001 total gastrectomy82 ( 70.0%)40 ( 30.7% ) extended gastrectomy20 ( 17.3%)36 ( 27.6% ) transhiatal extended gastrectomy0 ( 0%)36 ( 27.6% ) others1 ( 0.9%)1 ( 0.8%)splenectomy splenectomy during gastrectomy67 ( 57.1%)26 ( 19.8%)<0.001r0 resection r0103 ( 87.7%)119 ( 91.5%)0.389 r1/r214 ( 12.3%)11 ( 8.5%)lymphadenectomy d199 ( 84.8%)14 ( 10.6%)<0.001 d218 ( 15.2%)116 ( 89.4%)number of resected lymph nodes median ( min max)13.0 ( 047)33.5 ( 382)<0.001number of metastatic lymph nodes median ( min max)3.0 ( 041)3.0 ( 043)0.948r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection group surgical treatment of patients with gastric cancer from gdansk ( poland ) and cologne ( germany ) r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection group univariate analysis identified no significant difference in prognosis between the two surgical departments ( table 3 ) . the following parameters did not influence survival in either clinic : gender , comorbidity ( asa i and ii vs. iii and iv ) , type of gastric resection , and splenectomy.table 3univariate survival analysis comparing the impact of demographic and histopathological factors between 117 patients with gastric cancer from gdansk ( poland ) and 130 patients from cologne ( germany)univariate survival analysis : 5 year survival rategdansk , poland ( n = 117 ) ( % ) p valuecologne , germany ( n = 130 ) ( % ) p valuep value between the two centersgender male30.10.38842.30.9620.168 female20.435.20.336comorbidities asa i and ii26.50.69147.30.1020.017 asa iii and iv30.736.10.781tumor localization upper third32.90.01242.10.5240.325 middle third14.632.10.202 lower third40.147.10.529histopathological classification ( who ) adenocarcinoma ( except for signet - ring cell cancer)32.20.00139.90.5240.237 signet - ring cell cancer0.039.90.007pt category pt1<0.00164.4<0.001 pt245.246.70.928 pt318.735.80.271 pt40.018.70.201pn category pn059.5<0.00163.1<0.0010.592 pn120.444.40.079 pn20.013.40.079 pn30.013.00.916pm category pm032.0<0.00153.9<0.0010.013 pm10.08.50.175r category r031.6<0.00141.8<0.0010.179 r1/r2000.107type of gastric resection subtotal resection44.00.11650.40.8290.752 total gastrectomy29.934.10.437 extended gastrectomy15.824.00.314 transhiatal extended gastrectomy52.2lymphadenectomy d125.10.03119.20.1650.804 d247.142.80.521asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases , r category : r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection groupfig . 1comparison of survival curves of gastric cancer patients from gdansk ( poland ) and from cologne ( germany ) [ p = 0.056 ; hazard ratio 0.71 ( 95% confidence interval [ ci ] 0.541.01 ) ] univariate survival analysis comparing the impact of demographic and histopathological factors between 117 patients with gastric cancer from gdansk ( poland ) and 130 patients from cologne ( germany ) asa anesthesia risk according to american society of anesthesiologists [ 7 , 8 ] , pt tumor infiltration depth into gastric wall , pn lymph node status , pm0 no distant metastases , pm1 distant metastases , r category : r0 no residual tumor resection , r1 microscopic residual tumor resection , r2 macroscopic residual tumor resection , d1 limited lymph node dissection group , d2 extended lymph node dissection group comparison of survival curves of gastric cancer patients from gdansk ( poland ) and from cologne ( germany ) [ p = 0.056 ; hazard ratio 0.71 ( 95% confidence interval [ ci ] 0.541.01 ) ] on comparison of the clinics , parameters responsible for the better prognosis in cologne were healthy baseline status ( asa i or ii ) , signet - ring cell carcinoma histology , and absence of distant metastasis ( table 3 ) .

outofhospital cardiac arrest ( ohca ) in children is an uncommon event , with incidence rates between 3.0 and 9.0 per 100 000/year.1 recent populationbased studies of children with ohca showed survival rates ranging from 4.7% to 16%.2 , 3 , 4 , 5 , 6 , 7 children with ohca and initial shockable rhythms ( ventricular fibrillation [ vf ] and pulseless ventricular tachycardia ) have improved outcomes when compared to patients with initial nonshockable rhythm ( pulseless electrical activity [ pea ] and asystole).8 , 9 , 10 however , the proportion of initial shockable rhythms in children with ohca is very low , ranging from 2.0% to 19.8%,2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 with the higher incidence among adolescents . defibrillation for shockable rhythms is widely applied and has been strongly emphasized in recent guidelines for pediatric cardiopulmonary resuscitation ( cpr).11 , 12 , 13 , 14 in children who experienced inhospital cardiac arrest , the survival rate with subsequent defibrillation was substantially lower than that of patients with initial shockable rhythms and in those with an initial nonshockable rhythm with no followup shock delivery.15 , 16 however , in children with ohca , it remains unclear as to whether shock delivery for subsequent shockable rhythm following an initial nonshockable rhythm is associated with improved outcomes . in the present study of children with ohca and initial nonshockable rhythms , we examined whether improved neurological outcomes are associated with shock delivery for a presumed subsequent shockable rhythm , when compared to those who did not receive shock delivery . this investigation was a nationwide populationbased observational study in japan of children with ohca for whom resuscitation had been attempted between january 1 , 2005 and december 31 , 2012 . cardiac arrest was defined as the cessation of cardiac mechanical activity and was confirmed by the absence of signs of circulation . the cause of arrest was presumed to be cardiac , with the exception of those cases that showed evidence of external causes ( eg , drowning , foreign body obstruction , hanging , mechanical suffocation , trauma , and accidental hypothermia ) , respiratory diseases , cerebrovascular diseases , malignant tumors , or any other noncardiac cause . the determination of the cause as noncardiac or cardiac was made by the physicians in charge in collaboration with the emergency medical services ( ems ) personnel . this study was approved by the ethics committee of kanazawa university . according to guidelines in japan,17 informed consent from each patient to use secondary data such as that in this anonymous database is unnecessary . japan has 127 million residents in an area of 378 000 km , approximately two thirds of which is uninhabited mountainous terrain.18 municipal governments in japan provide ems through 800 fire stations with dispatch centers.19 the fire and disaster management agency ( fdma ) of japan supervises the nationwide ems system . during the study period , all ems providers performed cpr according to guidelines of the japan resuscitation council20 , 21 and the american heart association.22 emergency lifesaving technicians ( elsts ) , who are ems providers , are allowed to provide several resuscitation therapies , including use of an automated external defibrillator ( aed ) , insertion of an airway adjunct , establishment of peripheral intravenous access , and administration of ringer 's lactate solution.19 , 23 however , only specially trained elsts receiving online physician instruction are permitted to insert a tracheal tube and administer intravenous epinephrine in the field . because ems personnel in japan are legally prohibited from terminating resuscitation in the field ( except in specific situations such as decapitation , incineration , decomposition , rigor mortis , or dependent cyanosis ) , most patients with ohca undergo cpr by ems providers and are subsequently transported to the hospital . when ems providers arrived at the scene , initiation of cpr and initial rhythm assessment through aed were generally performed simultaneously . when initial nonshockable rhythm was identified , rhythm analysis was performed every 2 minutes by aed during cpr . in january 2005 , the fdma launched a prospective , populationbased , observational study including all ohca patients who received ems in japan.19 ems personnel at each center recorded ohca patient data in cooperation with the physician in charge , using an utsteinstyle template.24 the data were transferred to their fire stations and then integrated into the registry system on the fdma database server . all data were stored in the nationwide database developed by the fdma for public use . the fdma gave permission to analyze this database and provided all anonymous data to our research group . the main variables included in the data set were as follows : sex , age , cause of arrest , bystander witness status , bystanderwitnessed category ( such as a family member , a layperson other than family , or ems personnel ) , initially identified cardiac rhythm , presence and type of bystander cpr ( compression only or compression with ventilation ) , use of aed ( either by the public or by ems providers ) , epinephrine administration , advanced airway management , time variables ( collapse , emergency call , vehicle arrival , and cpr initiation ) , prehospital return of spontaneous circulation ( rosc ) , 1month survival , and neurological outcomes 1 month after cardiac arrest . the neurological outcome was defined using the cerebral performance category ( cpc ) scale : category 1 , good cerebral performance ; category 2 , moderate cerebral disability ; category 3 , severe cerebral disability ; category 4 , coma or vegetative state ; and category 5 , death.24 the cpc categorization was determined by the physician in charge . the primary study endpoint was 1month survival with favorable neurological outcome ( defined as a cpc score of 1 or 2 ) . the wilcoxon and kruskal wallis tests for continuous variables and the chisquare test for categorical variables were performed to compare the characteristics or outcomes of the cohorts . we further analyzed multivariate logistic regression models in order to clarify the relationship between subsequent shockable rhythm and outcomes . multivariate logistic regression analyses including 7 variables were performed to assess the factors associated with increased odds of prehospital rosc , 1month survival , and 1month cpc 1 or 2 for all eligible patients . the potential prehospital confounders for the analytic model were selected based on biological plausibility and data from previous studies . independent variables included age , bystanderwitnessed arrest ( yes or no ) , initial cardiac rhythm ( pea or asystole ) , presumed cardiac etiology ( yes or no ) , prehospital epinephrine administration ( yes or no ) , use of advanced airway management ( yes or no ) , and subsequent treated shockable rhythm ( yes or no ) . the calltoresponse time was calculated as the time from receipt of the call to the time of vehicle arrival at the scene . shockdelivery time was defined as the time interval between initiation of cpr by ems personnel and the first emsadministered shock . outcomes of patients with subsequent treated shockable rhythm were compared according to shockdelivery time and classified into 3 groups : 0 to 9 , 10 to 19 , and 20 to 59 minutes . moreover , after dividing patients into 2 groups according to age ( age < 7 years or age 717 years for elementary to high school children in japan ) , the outcomes in the 2 groups were compared according to the presence of subsequent treated shockable rhythm and shockdelivery time . continuous variables are expressed as median with interquartile range ( iqr ) 1 to 3 . an estimate of effect size and variability , odds ratios ( ors ) or proportion of outcomes with 95% cis were used . all statistical analyses were performed using the jmp statistical package ( version 11 pro ; sas institute inc . , this investigation was a nationwide populationbased observational study in japan of children with ohca for whom resuscitation had been attempted between january 1 , 2005 and december 31 , 2012 . cardiac arrest was defined as the cessation of cardiac mechanical activity and was confirmed by the absence of signs of circulation . the cause of arrest was presumed to be cardiac , with the exception of those cases that showed evidence of external causes ( eg , drowning , foreign body obstruction , hanging , mechanical suffocation , trauma , and accidental hypothermia ) , respiratory diseases , cerebrovascular diseases , malignant tumors , or any other noncardiac cause . the determination of the cause as noncardiac or cardiac was made by the physicians in charge in collaboration with the emergency medical services ( ems ) personnel . this study was approved by the ethics committee of kanazawa university . according to guidelines in japan,17 informed consent from each patient to use secondary data such as that in this anonymous database is unnecessary . japan has 127 million residents in an area of 378 000 km , approximately two thirds of which is uninhabited mountainous terrain.18 municipal governments in japan provide ems through 800 fire stations with dispatch centers.19 the fire and disaster management agency ( fdma ) of japan supervises the nationwide ems system . during the study period , all ems providers performed cpr according to guidelines of the japan resuscitation council20 , 21 and the american heart association.22 emergency lifesaving technicians ( elsts ) , who are ems providers , are allowed to provide several resuscitation therapies , including use of an automated external defibrillator ( aed ) , insertion of an airway adjunct , establishment of peripheral intravenous access , and administration of ringer 's lactate solution.19 , 23 however , only specially trained elsts receiving online physician instruction are permitted to insert a tracheal tube and administer intravenous epinephrine in the field . because ems personnel in japan are legally prohibited from terminating resuscitation in the field ( except in specific situations such as decapitation , incineration , decomposition , rigor mortis , or dependent cyanosis ) , most patients with ohca undergo cpr by ems providers and are subsequently transported to the hospital . when ems providers arrived at the scene , initiation of cpr and initial rhythm assessment through aed were generally performed simultaneously . when initial nonshockable rhythm was identified , rhythm analysis was performed every 2 minutes by aed during cpr . in january 2005 , the fdma launched a prospective , populationbased , observational study including all ohca patients who received ems in japan.19 ems personnel at each center recorded ohca patient data in cooperation with the physician in charge , using an utsteinstyle template.24 the data were transferred to their fire stations and then integrated into the registry system on the fdma database server . all data were stored in the nationwide database developed by the fdma for public use . the fdma gave permission to analyze this database and provided all anonymous data to our research group . the main variables included in the data set were as follows : sex , age , cause of arrest , bystander witness status , bystanderwitnessed category ( such as a family member , a layperson other than family , or ems personnel ) , initially identified cardiac rhythm , presence and type of bystander cpr ( compression only or compression with ventilation ) , use of aed ( either by the public or by ems providers ) , epinephrine administration , advanced airway management , time variables ( collapse , emergency call , vehicle arrival , and cpr initiation ) , prehospital return of spontaneous circulation ( rosc ) , 1month survival , and neurological outcomes 1 month after cardiac arrest . the neurological outcome was defined using the cerebral performance category ( cpc ) scale : category 1 , good cerebral performance ; category 2 , moderate cerebral disability ; category 3 , severe cerebral disability ; category 4 , coma or vegetative state ; and category 5 , death.24 the cpc categorization was determined by the physician in charge . the primary study endpoint was 1month survival with favorable neurological outcome ( defined as a cpc score of 1 or 2 ) . the wilcoxon and kruskal wallis tests for continuous variables and the chisquare test for categorical variables were performed to compare the characteristics or outcomes of the cohorts . we further analyzed multivariate logistic regression models in order to clarify the relationship between subsequent shockable rhythm and outcomes . multivariate logistic regression analyses including 7 variables were performed to assess the factors associated with increased odds of prehospital rosc , 1month survival , and 1month cpc 1 or 2 for all eligible patients . the potential prehospital confounders for the analytic model were selected based on biological plausibility and data from previous studies . independent variables included age , bystanderwitnessed arrest ( yes or no ) , initial cardiac rhythm ( pea or asystole ) , presumed cardiac etiology ( yes or no ) , prehospital epinephrine administration ( yes or no ) , use of advanced airway management ( yes or no ) , and subsequent treated shockable rhythm ( yes or no ) . the calltoresponse time was calculated as the time from receipt of the call to the time of vehicle arrival at the scene . shockdelivery time was defined as the time interval between initiation of cpr by ems personnel and the first emsadministered shock . outcomes of patients with subsequent treated shockable rhythm were compared according to shockdelivery time and classified into 3 groups : 0 to 9 , 10 to 19 , and 20 to 59 minutes . the cochranarmitage trend test was applied to analyze these data . moreover , after dividing patients into 2 groups according to age ( age < 7 years or age 717 years for elementary to high school children in japan ) , the outcomes in the 2 groups were compared according to the presence of subsequent treated shockable rhythm and shockdelivery time . continuous variables are expressed as median with interquartile range ( iqr ) 1 to 3 . an estimate of effect size and variability , odds ratios ( ors ) or proportion of outcomes with 95% cis were used . all statistical analyses were performed using the jmp statistical package ( version 11 pro ; sas institute inc . , during the 8year study period , 925 288 patients were documented in the database . patients with initial shockable rhythms , those aged 18 years , and those with unknown initial rhythm or unknown 1month outcome were excluded , so a total of 12 402 ( 1.34% of the total patients in the database ) children ( aged < 18 years ) with initial nonshockable rhythms were enrolled in this study . figure 1 shows a flow diagram depicting the inclusion and exclusion criteria for subjects in the present study . the overall prehospital rosc , 1month survival , and 1month cpc 1 or 2 rates were 3.7% ( n=461 ) , 7.7% ( n=953 ) , and 1.3% ( n=166 ) , respectively . patients were divided into 2 cohorts : subsequent treated shockable rhythm ( yes ; n=239 ) and subsequent treated shockable rhythm ( no ; n=12 163 ) . those patients with initial nonshockable rhythm who converted to shockable rhythms were identified by shocks delivered later in the course of resuscitation ; these were assigned to the subsequent treated shockable rhythm ( yes ) cohort . thus , the delivery of subsequent shocks was used as a surrogate maker for conversion to a shockable rhythm . conversely , the subsequent treated shockable rhythm ( no ) cohort was composed of those patients who did not receive any shocks during resuscitation . design of the patient selection process . cpc indicates cerebral performance category ; ems , emergency medical services ; rosc , return of spontaneous circulation . table 1 shows the baseline characteristics and results of the analyses of the 2 cohorts . age , rates of bystanderwitnessed arrest , initial pea , presumed cardiac etiology , epinephrine administration , and advanced airway management were significantly higher in the subsequent treated shockable rhythm ( yes ) cohort when compared to the subsequent treated shockable rhythm ( no ) cohort . the subsequent treated shockable rhythm ( yes ) cohort had significantly higher rates of prehospital rosc , 1month survival , and 1month cpc 1 or 2 than the subsequent treated shockable rhythm ( no ) cohort ( 13.8% vs 3.5% , 15.9% vs 7.5% , and 4.6% vs 1.3% , respectively , p<0.001 ; figure 2 ) . baseline characteristics of the study cohorts according to subsequent shockable rhythm values are reported as number ( % ) , unless indicated otherwise . time from the initiation of cpr by emergency medical services personnel to the first shock delivery . table 2 shows the results of multivariate logistic regression analyses to determine factors associated with outcomes in all participants . subsequent shockable rhythm was significantly associated with increased odds of prehospital rosc ( adjusted or , 2.77 ; 95% ci , 1.814.12 ) , 1month survival ( adjusted or , 2.30 ; 95% ci , 1.563.29 ) , and 1month cpc 1 or 2 ( adjusted or , 2.90 ; 95% ci , 1.425.36 ) . bystanderwitnessed arrest and initial pea were significantly associated with improved 1month survival and 1month cpc 1 or 2 . results of multivariate logistic regression analyses for variables associated with outcomes in all participants cpc indicates cerebral performance category ; or , odds ratio ; rosc , return of spontaneous circulation . figure 3 shows the outcomes stratified by shockdelivery time in the subsequent treated shockable rhythm ( yes ) cohort . rates of 1month survival and 1month cpc 1 or 2 decreased significantly as time to shock delivery increased ( 1month survival : 23.5% for 09 minutes , 22.0% for 1019 minutes , and 9.2% for 2059 minutes ; p=0.01 [ for trend ] ; 1month cpc 1 or 2 : 17.7% for 09 minutes , 7.3% for 1019 minutes , and 0% for 2059 minutes ; p<0.001 [ for trend ] ) . outcomes stratified by shockdelivery time in the subsequent treated shockable rhythm ( yes ) cohort . table 3 compares the characteristics and outcomes according to age group . the proportion of male sex , bystanderwitnessed arrest , initial pea , epinephrine administration , advanced airway management , and subsequent treated shockable rhythm were significantly higher in the group aged 7 to 17 years when compared with the group aged < 7 years . the group aged 7 to 17 years had a significantly higher rate of prehospital rosc compared to the group aged < 7 years ( 5.8% vs 2.7% ; p<0.001 ) . however , no significant differences were found in the rates of 1month survival and 1month cpc 1 or 2 between the 2 groups ( 1month survival : 8.0% for those aged < 7 years vs 7.1% for those aged 717 years ; p=0.08 ; 1month cpc 1 or 2 : 1.3% for those aged < 7 years vs 1.4% for those aged 717 years ; p=0.62 ) . characteristics and outcomes of study patients according to age group values are reported as number ( % ) , unless indicated otherwise . cpc indicates cerebral performance category ; cpr , cardiopulmonary resuscitation ; iqr , interquartile range ; rosc , return of spontaneous circulation . time from the initiation of cardiopulmonary resuscitation by emergency medical services personnel to the first shock delivery . the group aged 7 to 17 years , outcomes in the subsequent treated shockable rhythm ( yes ) cohort were significantly better than for those in the subsequent treated shockable rhythm ( no ) cohort ( 17.9% vs 5.3% for prehospital rosc , 18.5% vs 6.6% for 1month survival , and 6.0% vs 1.2% for 1month cpc 1 or 2 , respectively ; p<0.001 ) . however , in the group aged < 7 years , no significant differences were found between the 2 cohorts . table 4 shows the agestratified outcomes according to shockdelivery time in the subsequent treated shockable rhythm ( yes ) cohort ( n=235 ) . rates of 1month survival and 1month cpc 1 or 2 in the group aged 7 to 17 years decreased significantly as time to shock delivery increased ( 1month survival : 26.7% for 09 minutes , 26.0% for 1019 minutes , and 9.6% for 2059 minutes ; p for trend=0.02 ; 1month cpc 1 or 2 : 20.0% for 09 minutes , 9.1% for 1019 minutes , and 0% for 2059 minutes ; p for trend < 0.01 ) . however , significant differences were not found in the group aged < 7 years . agestratified outcomes according to shockdelivery time in the subsequent treated shockable rhythm ( yes ) cohort values are reported as number ( % ) , unless indicated otherwise . time from the initiation of cardiopulmonary resuscitation by emergency medical services personnel to the first shock delivery . the present study of emstreated ohca children with initial nonshockable rhythms in japan shows that subsequent treated shockable rhythm is significantly associated with improved prehospital rosc , 1month survival , and 1month survival with favorable neurological outcomes , when compared with no subsequent treated shockable rhythm . in patients with subsequent treated shockable rhythm , 1month survival and 1month survival with favorable neurological outcomes decreased as time to shock delivery increased . moreover , these findings were only applicable to older children ( aged 717 years for elementary to high school children in japan ) . the present study results are inconsistent with those of a previous pediatric inhospital cardiac arrest study.16 in 2006 , samson et al16 demonstrated that the proportion of survival to hospital discharge in pediatric inhospital cardiac arrest with subsequent shockable rhythm was significantly lower than that for those who did not convert to shockable rhythms ( 11% vs 27% ; adjusted or , 3.8 ; 95% ci , 1.87.6 ) . they surmised that a delay in diagnosis of subsequent shockable rhythm , adverse effects of resuscitative interventions , and severity of the underlying myocardial condition might have contributed to these results . unlike in samson et al 's study , we enrolled only patients with ohca and a subsequent shockable rhythm who were treated with shock delivery into the subsequent treated shockable rhythm ( yes ) cohort . therefore , our study could both underestimate the frequency of development of subsequent shockable rhythm and overestimate favorable outcomes ( survival and/or cpc 1 or 2 ) . moreover , most patients with inhospital cardiac arrest are usually in a monitored setting and received shocks more quickly . actually , the interval to first attempted defibrillation in samson et al 's study was clearly shorter than the subsequent shockdelivery time in our study ( median [ iqr ] , 0 [ 03 ] vs 19 minutes [ 1426 ] ; p<0.01 ) . furthermore , cpr duration for patients with subsequent shockable rhythm was significantly longer than for those with no shockable rhythm in samson et al 's study ( median [ iqr ] , 30 [ 1553 ] vs 20 minutes [ 953 ] ) . accordingly , the differences in outcomes between the present study of ohca and samson et al 's study of inhospital cardiac arrest may be explained simply by the cpr duration . however , we could not analyze cpr duration because of lack of data for inhospital cpr time . samson et al 's study of inhospital cardiac arrest16 indicated that rates of survival to hospital discharge after initial shockable rhythm were significantly higher than those after subsequent shockable rhythm : 35% versus 11% ; adjusted or ( 95% ci ) , 2.6 ( 1.25.8 ) . table 5 compares the outcomes in patients with initial shockable rhythm ( n=637 ; figure 1 ) and those with subsequent treated shockable rhythm ( n=239 ) in the present study . rates of favorable outcomes in patients with initial shockable rhythm were significantly higher than in those with subsequent treated shockable rhythm ( 34.9% vs 15.9% for 1month survival , 23.4% vs 4.6% for 1month cpc 1 or 2 ; p<0.001 ) . patients with initial shockable rhythm had significant positive adjusted ors for favorable outcomes compared to those with subsequent treated shockable rhythm ( 1month survival : 2.23 [ 95% ci , 1.493.39 ] ; 1month cpc 1 or 2 : 4.30 [ 95% ci , 2.318.79 ] ; p<0.001 ) . these results were consistent with those in samson et al 's study.16 moreover , in the present study , the finding that patients with initial shockable rhythm had 1month outcomes superior to those with subsequent treated shockable rhythm was also observed when analyzed by age group ( aged < 7 years or aged 717 years ) . outcomes in patients with initial shockable rhythm and those with subsequent treated shockable rhythm values are reported as number ( % ) , unless indicated otherwise . adjusted variables for potential confounders were included 7 variables : age , bystanderwitnessed arrest , bystander cardiopulmonary resuscitation , initial cardiac rhythm , presumed cardiac etiology , epinephrine administration , and advanced airway management . in adult patients who experienced ohca , hallstrom et al25 noted a significant low adjusted or of 0.18 for survival to hospital discharge in patients with subsequent shockable rhythms relative to those who did not receive shocks for nonshockable rhythms . thomas et al26 recently reported that increased survival to hospital discharge for ohca patients was not associated with subsequent shockable rhythm and shock delivery during ems resuscitation efforts ( adjusted or , 0.88 ; 95% ci , 0.601.30 ) . however , other studies of ohca adults27 , 28 , 29 , 30 , 31 demonstrated that subsequent treated shockable rhythm was associated with improved outcomes compared with no shock delivery after an initial nonshockable rhythm . notably , goto et al27 showed that subsequent shockable rhythm was significantly associated with increased adjusted odds of 1month survival with favorable neurological outcomes when the shockdelivery time was < 20 minutes . the present study on children with ohca is consistent with those adult ohca studies ( table 2 ) . earlier shock delivery ( shockdelivery time < 20 minutes ) in children with ohca and subsequent treated shockable rhythm , particularly in patients aged 7 to 17 years , was found to be superior to later shock delivery , as demonstrated by 1month survival with favorable neurological outcomes ( figure 3 ; table 4 ) . however , we were unable to show the benefit of early shock delivery in patients with subsequent treated shockable rhythm on neurological outcomes in the multivariate logistic regression model , because an insufficient number of cases prevented further risk adjustment for outcomes . in the present study , prehospital epinephrine administration was independently associated with increased odds of prehospital rosc . however , it was independently associated with decreased odds of 1month survival and 1month cpc 1 or 2 ( table 2 ) . moreover , advanced airway management was not associated with prehospital rosc , 1month survival , or 1month cpc 1 or 2 . these results are consistent with those from previous studies on pediatric ohcas.32 , 33 eilevstjnn et al34 demonstrated that cardiac rhythms before subsequent shockable rhythm were related to the outcomes ( rosc ) . they found that pea before shockable rhythm had a significantly higher rosc rate than that of asystole . their results were consistent with our previous study for adults27 and the present study for children . moreover , they reported that median slope , which represents the average steepness of the vf waveform reflecting both the amplitude and frequency of the rhythm , might be a useful tool for predicting outcomes . this lack of data was associated with our study design of a retrospective record review . therefore , prospective studies are required to analyze the subsequent vf waveform and clarify the effect of subsequent shockable rhythm and shock delivery on outcomes in children with ohca first , we could not exclude patients who received shocks for the wrong indication or for unrecognized initial shockable rhythms attributed to electrical misreading when an aed was used while transporting a patient35 and/or when cpr was provided for a child with ohca,36 which would result in an overestimation of favorable outcomes . in addition , our study population included only those patients with initial nonshockable rhythm who had a subsequent shockable rhythm that was treated with shock delivery . if a significant number of patients who developed a subsequent shockable rhythm never received a shock , their exclusion would mean that survival in our study population is falsely high . second , our registry database lacked detailed data to permit further risk adjustment for outcomes : for example , comorbid disease of patients , location where the ohca occurred , years of experience as a member of ems personnel , the degree of regional differences among ems centers,37 , 38 inhospital medication , and the availability of specialists in emergency care ( cardiologists and/or pediatric physicians ) . regarding regional differences in outcomes postohca in japan , previous studies suggested there were regional disparities in prehospital care and inhospital postresuscitation care.37 , 38 lowspending regions had significantly worse neurological outcomes postohca compared to mediumspending or highspending regions.38 moreover , although ems providers delivered a shock according to guidelines during the study period,20 , 21 , 22 precise energy dose , shock frequency , and defibrillation mode ( monophasic or biphasic ) were unknown ; therefore , we could not analyze those data in the present study . third , though a uniform data collection procedure based on the utsteinstyle guidelines for reporting cardiac arrest , a large sample size , and a populationbased design was used , we can not exclude the possibility of uncontrolled confounders . fourth , as with all epidemiological studies , the integrity , validity , and ascertainment bias of the data were potential limitations . fifth , we should note that caution must be exercised when generalizing these results to other countries or ems systems . finally , because we lacked precise data on the causes of cardiac arrest , it is possible that cardiac arrest in some patients may have been caused by sudden infant death syndrome , trauma , or respiratory disease.39 first , we could not exclude patients who received shocks for the wrong indication or for unrecognized initial shockable rhythms attributed to electrical misreading when an aed was used while transporting a patient35 and/or when cpr was provided for a child with ohca,36 which would result in an overestimation of favorable outcomes . in addition , our study population included only those patients with initial nonshockable rhythm who had a subsequent shockable rhythm that was treated with shock delivery . if a significant number of patients who developed a subsequent shockable rhythm never received a shock , their exclusion would mean that survival in our study population is falsely high . second , our registry database lacked detailed data to permit further risk adjustment for outcomes : for example , comorbid disease of patients , location where the ohca occurred , years of experience as a member of ems personnel , the degree of regional differences among ems centers,37 , 38 inhospital medication , and the availability of specialists in emergency care ( cardiologists and/or pediatric physicians ) . regarding regional differences in outcomes postohca in japan , previous studies suggested there were regional disparities in prehospital care and inhospital postresuscitation care.37 , 38 lowspending regions had significantly worse neurological outcomes postohca compared to mediumspending or highspending regions.38 moreover , although ems providers delivered a shock according to guidelines during the study period,20 , 21 , 22 precise energy dose , shock frequency , and defibrillation mode ( monophasic or biphasic ) were unknown ; therefore , we could not analyze those data in the present study . third , though a uniform data collection procedure based on the utsteinstyle guidelines for reporting cardiac arrest , a large sample size , and a populationbased design was used , we can not exclude the possibility of uncontrolled confounders . fourth , as with all epidemiological studies , the integrity , validity , and ascertainment bias of the data were potential limitations . fifth , we should note that caution must be exercised when generalizing these results to other countries or ems systems . finally , because we lacked precise data on the causes of cardiac arrest , it is possible that cardiac arrest in some patients may have been caused by sudden infant death syndrome , trauma , or respiratory disease.39 in children with ohca and initial nonshockable rhythms , subsequent treated shockable rhythm during ems resuscitation efforts was significantly associated with improved prehospital rosc , 1month survival , and 1month survival with favorable neurological outcomes . in addition , in the cohort of older children ( aged 717 years ) with subsequent treated shockable rhythm , 1month survival and 1month survival with favorable neurological outcomes decreased as time to shock delivery increased . this work was supported by the japan society for the promotion of science ( kakenhi grant no . : 15k08543 ) , which had no role in the design and implementation of the study , analysis and interpretation of the data , or approval of the manuscript .

backgroundthe effect of a subsequent treated shockable rhythm during cardiopulmonary resuscitation on the outcome of children who suffer outofhospital cardiac arrest with initial nonshockable rhythm is unclear . we hypothesized that subsequent treated shockable rhythm in children with outofhospital cardiac arrest would improve survival with favorable neurological outcomes ( cerebral performance category scale 12).methods and resultsfrom the alljapan utstein registry , we analyzed the records of 12 402 children ( aged < 18 years ) with outofhospital cardiac arrest and initial nonshockable rhythms . patients were divided into 2 cohorts : subsequent treated shockable rhythm ( yes ; n=239 ) and subsequent treated shockable rhythm ( no ; n=12 163 ) . the rate of 1month cerebral performance category 1 to 2 in the subsequent treated shockable rhythm ( yes ) cohort was significantly higher when compared to the subsequent treated shockable rhythm ( no ) cohort ( 4.6% [ 11 of 239 ] vs 1.3% [ 155 of 12 163 ] ; adjusted odds ratio , 2.90 ; 95% ci , 1.425.36 ; all p<0.001 ) . in the subsequent treated shockable rhythm ( yes ) cohort , the rate of 1month cerebral performance category 1 to 2 decreased significantly as time to shock delivery increased ( 17.7% [ 3 of 17 ] for patients with shockdelivery time 09 minutes , 7.3% [ 8 of 109 ] for 1019 minutes , and 0% [ 0 of 109 ] for 2059 minutes ; p<0.001 [ for trend ] ) . agestratified outcomes showed no significant differences between the 2 cohorts in the group aged < 7 years old : 1.3% versus 1.4% , p=0.62.conclusionsin children with outofhospital cardiac arrest and initial nonshockable rhythms , subsequent treated shockable rhythm was associated with improved 1month survival with favorable neurological outcomes . in the cohort of older children ( 717 years ) , these outcomes worsened as time to shock delivery increased .

Introduction Methods Study Design Study Setting Data Collection and Quality Control Study Endpoints Statistical Analysis Results Discussion Study Limitations Conclusions Sources of Funding Disclosures

outofhospital cardiac arrest ( ohca ) in children is an uncommon event , with incidence rates between 3.0 and 9.0 per 100 000/year.1 recent populationbased studies of children with ohca showed survival rates ranging from 4.7% to 16%.2 , 3 , 4 , 5 , 6 , 7 children with ohca and initial shockable rhythms ( ventricular fibrillation [ vf ] and pulseless ventricular tachycardia ) have improved outcomes when compared to patients with initial nonshockable rhythm ( pulseless electrical activity [ pea ] and asystole).8 , 9 , 10 however , the proportion of initial shockable rhythms in children with ohca is very low , ranging from 2.0% to 19.8%,2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 with the higher incidence among adolescents . defibrillation for shockable rhythms is widely applied and has been strongly emphasized in recent guidelines for pediatric cardiopulmonary resuscitation ( cpr).11 , 12 , 13 , 14 in children who experienced inhospital cardiac arrest , the survival rate with subsequent defibrillation was substantially lower than that of patients with initial shockable rhythms and in those with an initial nonshockable rhythm with no followup shock delivery.15 , 16 however , in children with ohca , it remains unclear as to whether shock delivery for subsequent shockable rhythm following an initial nonshockable rhythm is associated with improved outcomes . in the present study of children with ohca and initial nonshockable rhythms , we examined whether improved neurological outcomes are associated with shock delivery for a presumed subsequent shockable rhythm , when compared to those who did not receive shock delivery . because ems personnel in japan are legally prohibited from terminating resuscitation in the field ( except in specific situations such as decapitation , incineration , decomposition , rigor mortis , or dependent cyanosis ) , most patients with ohca undergo cpr by ems providers and are subsequently transported to the hospital . the main variables included in the data set were as follows : sex , age , cause of arrest , bystander witness status , bystanderwitnessed category ( such as a family member , a layperson other than family , or ems personnel ) , initially identified cardiac rhythm , presence and type of bystander cpr ( compression only or compression with ventilation ) , use of aed ( either by the public or by ems providers ) , epinephrine administration , advanced airway management , time variables ( collapse , emergency call , vehicle arrival , and cpr initiation ) , prehospital return of spontaneous circulation ( rosc ) , 1month survival , and neurological outcomes 1 month after cardiac arrest . the primary study endpoint was 1month survival with favorable neurological outcome ( defined as a cpc score of 1 or 2 ) . independent variables included age , bystanderwitnessed arrest ( yes or no ) , initial cardiac rhythm ( pea or asystole ) , presumed cardiac etiology ( yes or no ) , prehospital epinephrine administration ( yes or no ) , use of advanced airway management ( yes or no ) , and subsequent treated shockable rhythm ( yes or no ) . outcomes of patients with subsequent treated shockable rhythm were compared according to shockdelivery time and classified into 3 groups : 0 to 9 , 10 to 19 , and 20 to 59 minutes . moreover , after dividing patients into 2 groups according to age ( age < 7 years or age 717 years for elementary to high school children in japan ) , the outcomes in the 2 groups were compared according to the presence of subsequent treated shockable rhythm and shockdelivery time . during the study period , all ems providers performed cpr according to guidelines of the japan resuscitation council20 , 21 and the american heart association.22 emergency lifesaving technicians ( elsts ) , who are ems providers , are allowed to provide several resuscitation therapies , including use of an automated external defibrillator ( aed ) , insertion of an airway adjunct , establishment of peripheral intravenous access , and administration of ringer 's lactate solution.19 , 23 however , only specially trained elsts receiving online physician instruction are permitted to insert a tracheal tube and administer intravenous epinephrine in the field . because ems personnel in japan are legally prohibited from terminating resuscitation in the field ( except in specific situations such as decapitation , incineration , decomposition , rigor mortis , or dependent cyanosis ) , most patients with ohca undergo cpr by ems providers and are subsequently transported to the hospital . the main variables included in the data set were as follows : sex , age , cause of arrest , bystander witness status , bystanderwitnessed category ( such as a family member , a layperson other than family , or ems personnel ) , initially identified cardiac rhythm , presence and type of bystander cpr ( compression only or compression with ventilation ) , use of aed ( either by the public or by ems providers ) , epinephrine administration , advanced airway management , time variables ( collapse , emergency call , vehicle arrival , and cpr initiation ) , prehospital return of spontaneous circulation ( rosc ) , 1month survival , and neurological outcomes 1 month after cardiac arrest . the primary study endpoint was 1month survival with favorable neurological outcome ( defined as a cpc score of 1 or 2 ) . independent variables included age , bystanderwitnessed arrest ( yes or no ) , initial cardiac rhythm ( pea or asystole ) , presumed cardiac etiology ( yes or no ) , prehospital epinephrine administration ( yes or no ) , use of advanced airway management ( yes or no ) , and subsequent treated shockable rhythm ( yes or no ) . outcomes of patients with subsequent treated shockable rhythm were compared according to shockdelivery time and classified into 3 groups : 0 to 9 , 10 to 19 , and 20 to 59 minutes . moreover , after dividing patients into 2 groups according to age ( age < 7 years or age 717 years for elementary to high school children in japan ) , the outcomes in the 2 groups were compared according to the presence of subsequent treated shockable rhythm and shockdelivery time . patients with initial shockable rhythms , those aged 18 years , and those with unknown initial rhythm or unknown 1month outcome were excluded , so a total of 12 402 ( 1.34% of the total patients in the database ) children ( aged < 18 years ) with initial nonshockable rhythms were enrolled in this study . the overall prehospital rosc , 1month survival , and 1month cpc 1 or 2 rates were 3.7% ( n=461 ) , 7.7% ( n=953 ) , and 1.3% ( n=166 ) , respectively . patients were divided into 2 cohorts : subsequent treated shockable rhythm ( yes ; n=239 ) and subsequent treated shockable rhythm ( no ; n=12 163 ) . those patients with initial nonshockable rhythm who converted to shockable rhythms were identified by shocks delivered later in the course of resuscitation ; these were assigned to the subsequent treated shockable rhythm ( yes ) cohort . conversely , the subsequent treated shockable rhythm ( no ) cohort was composed of those patients who did not receive any shocks during resuscitation . age , rates of bystanderwitnessed arrest , initial pea , presumed cardiac etiology , epinephrine administration , and advanced airway management were significantly higher in the subsequent treated shockable rhythm ( yes ) cohort when compared to the subsequent treated shockable rhythm ( no ) cohort . the subsequent treated shockable rhythm ( yes ) cohort had significantly higher rates of prehospital rosc , 1month survival , and 1month cpc 1 or 2 than the subsequent treated shockable rhythm ( no ) cohort ( 13.8% vs 3.5% , 15.9% vs 7.5% , and 4.6% vs 1.3% , respectively , p<0.001 ; figure 2 ) . subsequent shockable rhythm was significantly associated with increased odds of prehospital rosc ( adjusted or , 2.77 ; 95% ci , 1.814.12 ) , 1month survival ( adjusted or , 2.30 ; 95% ci , 1.563.29 ) , and 1month cpc 1 or 2 ( adjusted or , 2.90 ; 95% ci , 1.425.36 ) . bystanderwitnessed arrest and initial pea were significantly associated with improved 1month survival and 1month cpc 1 or 2 . results of multivariate logistic regression analyses for variables associated with outcomes in all participants cpc indicates cerebral performance category ; or , odds ratio ; rosc , return of spontaneous circulation . figure 3 shows the outcomes stratified by shockdelivery time in the subsequent treated shockable rhythm ( yes ) cohort . rates of 1month survival and 1month cpc 1 or 2 decreased significantly as time to shock delivery increased ( 1month survival : 23.5% for 09 minutes , 22.0% for 1019 minutes , and 9.2% for 2059 minutes ; p=0.01 [ for trend ] ; 1month cpc 1 or 2 : 17.7% for 09 minutes , 7.3% for 1019 minutes , and 0% for 2059 minutes ; p<0.001 [ for trend ] ) . outcomes stratified by shockdelivery time in the subsequent treated shockable rhythm ( yes ) cohort . the proportion of male sex , bystanderwitnessed arrest , initial pea , epinephrine administration , advanced airway management , and subsequent treated shockable rhythm were significantly higher in the group aged 7 to 17 years when compared with the group aged < 7 years . the group aged 7 to 17 years had a significantly higher rate of prehospital rosc compared to the group aged < 7 years ( 5.8% vs 2.7% ; p<0.001 ) . however , no significant differences were found in the rates of 1month survival and 1month cpc 1 or 2 between the 2 groups ( 1month survival : 8.0% for those aged < 7 years vs 7.1% for those aged 717 years ; p=0.08 ; 1month cpc 1 or 2 : 1.3% for those aged < 7 years vs 1.4% for those aged 717 years ; p=0.62 ) . the group aged 7 to 17 years , outcomes in the subsequent treated shockable rhythm ( yes ) cohort were significantly better than for those in the subsequent treated shockable rhythm ( no ) cohort ( 17.9% vs 5.3% for prehospital rosc , 18.5% vs 6.6% for 1month survival , and 6.0% vs 1.2% for 1month cpc 1 or 2 , respectively ; p<0.001 ) . however , in the group aged < 7 years , no significant differences were found between the 2 cohorts . table 4 shows the agestratified outcomes according to shockdelivery time in the subsequent treated shockable rhythm ( yes ) cohort ( n=235 ) . rates of 1month survival and 1month cpc 1 or 2 in the group aged 7 to 17 years decreased significantly as time to shock delivery increased ( 1month survival : 26.7% for 09 minutes , 26.0% for 1019 minutes , and 9.6% for 2059 minutes ; p for trend=0.02 ; 1month cpc 1 or 2 : 20.0% for 09 minutes , 9.1% for 1019 minutes , and 0% for 2059 minutes ; p for trend < 0.01 ) . however , significant differences were not found in the group aged < 7 years . agestratified outcomes according to shockdelivery time in the subsequent treated shockable rhythm ( yes ) cohort values are reported as number ( % ) , unless indicated otherwise . the present study of emstreated ohca children with initial nonshockable rhythms in japan shows that subsequent treated shockable rhythm is significantly associated with improved prehospital rosc , 1month survival , and 1month survival with favorable neurological outcomes , when compared with no subsequent treated shockable rhythm . in patients with subsequent treated shockable rhythm , 1month survival and 1month survival with favorable neurological outcomes decreased as time to shock delivery increased . moreover , these findings were only applicable to older children ( aged 717 years for elementary to high school children in japan ) . the present study results are inconsistent with those of a previous pediatric inhospital cardiac arrest study.16 in 2006 , samson et al16 demonstrated that the proportion of survival to hospital discharge in pediatric inhospital cardiac arrest with subsequent shockable rhythm was significantly lower than that for those who did not convert to shockable rhythms ( 11% vs 27% ; adjusted or , 3.8 ; 95% ci , 1.87.6 ) . unlike in samson et al 's study , we enrolled only patients with ohca and a subsequent shockable rhythm who were treated with shock delivery into the subsequent treated shockable rhythm ( yes ) cohort . actually , the interval to first attempted defibrillation in samson et al 's study was clearly shorter than the subsequent shockdelivery time in our study ( median [ iqr ] , 0 [ 03 ] vs 19 minutes [ 1426 ] ; p<0.01 ) . furthermore , cpr duration for patients with subsequent shockable rhythm was significantly longer than for those with no shockable rhythm in samson et al 's study ( median [ iqr ] , 30 [ 1553 ] vs 20 minutes [ 953 ] ) . samson et al 's study of inhospital cardiac arrest16 indicated that rates of survival to hospital discharge after initial shockable rhythm were significantly higher than those after subsequent shockable rhythm : 35% versus 11% ; adjusted or ( 95% ci ) , 2.6 ( 1.25.8 ) . table 5 compares the outcomes in patients with initial shockable rhythm ( n=637 ; figure 1 ) and those with subsequent treated shockable rhythm ( n=239 ) in the present study . rates of favorable outcomes in patients with initial shockable rhythm were significantly higher than in those with subsequent treated shockable rhythm ( 34.9% vs 15.9% for 1month survival , 23.4% vs 4.6% for 1month cpc 1 or 2 ; p<0.001 ) . patients with initial shockable rhythm had significant positive adjusted ors for favorable outcomes compared to those with subsequent treated shockable rhythm ( 1month survival : 2.23 [ 95% ci , 1.493.39 ] ; 1month cpc 1 or 2 : 4.30 [ 95% ci , 2.318.79 ] ; p<0.001 ) . these results were consistent with those in samson et al 's study.16 moreover , in the present study , the finding that patients with initial shockable rhythm had 1month outcomes superior to those with subsequent treated shockable rhythm was also observed when analyzed by age group ( aged < 7 years or aged 717 years ) . outcomes in patients with initial shockable rhythm and those with subsequent treated shockable rhythm values are reported as number ( % ) , unless indicated otherwise . thomas et al26 recently reported that increased survival to hospital discharge for ohca patients was not associated with subsequent shockable rhythm and shock delivery during ems resuscitation efforts ( adjusted or , 0.88 ; 95% ci , 0.601.30 ) . however , other studies of ohca adults27 , 28 , 29 , 30 , 31 demonstrated that subsequent treated shockable rhythm was associated with improved outcomes compared with no shock delivery after an initial nonshockable rhythm . notably , goto et al27 showed that subsequent shockable rhythm was significantly associated with increased adjusted odds of 1month survival with favorable neurological outcomes when the shockdelivery time was < 20 minutes . earlier shock delivery ( shockdelivery time < 20 minutes ) in children with ohca and subsequent treated shockable rhythm , particularly in patients aged 7 to 17 years , was found to be superior to later shock delivery , as demonstrated by 1month survival with favorable neurological outcomes ( figure 3 ; table 4 ) . however , we were unable to show the benefit of early shock delivery in patients with subsequent treated shockable rhythm on neurological outcomes in the multivariate logistic regression model , because an insufficient number of cases prevented further risk adjustment for outcomes . therefore , prospective studies are required to analyze the subsequent vf waveform and clarify the effect of subsequent shockable rhythm and shock delivery on outcomes in children with ohca first , we could not exclude patients who received shocks for the wrong indication or for unrecognized initial shockable rhythms attributed to electrical misreading when an aed was used while transporting a patient35 and/or when cpr was provided for a child with ohca,36 which would result in an overestimation of favorable outcomes . in addition , our study population included only those patients with initial nonshockable rhythm who had a subsequent shockable rhythm that was treated with shock delivery . regarding regional differences in outcomes postohca in japan , previous studies suggested there were regional disparities in prehospital care and inhospital postresuscitation care.37 , 38 lowspending regions had significantly worse neurological outcomes postohca compared to mediumspending or highspending regions.38 moreover , although ems providers delivered a shock according to guidelines during the study period,20 , 21 , 22 precise energy dose , shock frequency , and defibrillation mode ( monophasic or biphasic ) were unknown ; therefore , we could not analyze those data in the present study . third , though a uniform data collection procedure based on the utsteinstyle guidelines for reporting cardiac arrest , a large sample size , and a populationbased design was used , we can not exclude the possibility of uncontrolled confounders . in addition , our study population included only those patients with initial nonshockable rhythm who had a subsequent shockable rhythm that was treated with shock delivery . regarding regional differences in outcomes postohca in japan , previous studies suggested there were regional disparities in prehospital care and inhospital postresuscitation care.37 , 38 lowspending regions had significantly worse neurological outcomes postohca compared to mediumspending or highspending regions.38 moreover , although ems providers delivered a shock according to guidelines during the study period,20 , 21 , 22 precise energy dose , shock frequency , and defibrillation mode ( monophasic or biphasic ) were unknown ; therefore , we could not analyze those data in the present study . third , though a uniform data collection procedure based on the utsteinstyle guidelines for reporting cardiac arrest , a large sample size , and a populationbased design was used , we can not exclude the possibility of uncontrolled confounders . finally , because we lacked precise data on the causes of cardiac arrest , it is possible that cardiac arrest in some patients may have been caused by sudden infant death syndrome , trauma , or respiratory disease.39 in children with ohca and initial nonshockable rhythms , subsequent treated shockable rhythm during ems resuscitation efforts was significantly associated with improved prehospital rosc , 1month survival , and 1month survival with favorable neurological outcomes . in addition , in the cohort of older children ( aged 717 years ) with subsequent treated shockable rhythm , 1month survival and 1month survival with favorable neurological outcomes decreased as time to shock delivery increased .

neurological disorders pose a great challenge to healthcare in developing countries , as limited resources and manpower are not enough to tackle the increasing burden . although brain tumor is not a frequent neurological disorder , still it contributes significantly to morbidity and is no longer rare in clinical practice [ 2 , 3 ] . brain tumors can present challenging medical problems , and effective medical management would result in decreased morbidity and mortality and improved quality of life . brain tumors are generally classified as either primary ( originating from within the brain cavity ) or secondary ( originating elsewhere in the body , and then spread to the brain ) . the most common types of primary brain tumors are gliomas and meningiomas , constituting , respectively , 60% and 20% of all intracranial tumors ( tumors within the brain ) in adults . treatment varies from one tumor type to the other and often involves a combination of surgery , radiotherapy , and chemotherapy . meningiomas are almost always benign tumors and have good prognosis after surgery , whereas gliomas being malignant tumors comprise multidisciplinary approach and have relatively poorer prognosis . hence it is very essential to differentiate between the two tumor types especially when gliomas are peripherally placed and imitate a meningioma [ 4 , 5 ] . the introduction of imaging techniques into clinical practice has been among the most important of all advances in the care of patients with brain tumors . the potential advantage of advanced magnetic resonance imaging ( mri ) methods is that they permit more uniform sampling than heterogeneous biopsies , and that , they can be applied repeatedly to monitor therapy success . diffusion weighted - magnetic resonance imaging ( dw - mri ) is an imaging technique based on the sensitivity of magnetic resonance ( mr ) to microscopic mobility of water molecules within tissues , and provides image contrast that is dependent on the molecular motion of water , which may be substantially altered by disease . dw - mri probes water molecular diffusion over distances that correspond to typical cell sizes , and this diffusion is impeded by membranes ( structures that are an integral part of the cell - architecture ) . of the histologic features for tumor analysis , cellularity has been the target of quantitative assessment with dw - mri . the rationale of using diffusion imaging to quantify cellularity is based on the principle that water diffusivity within the extracellular compartment is inversely related to the content and attenuation of the constituents of the intracellular space . with increasing cell density therefore , tumors with higher cellularity show increased signal intensity on diffusion weighted ( dw ) images , in contrast to the tumors with low cellularity that presents greater signal intensity loss . quantification of the amount of diffusion of water molecules in terms of signal intensity variations found within the brain tumors can thereby provide information about the histologic composition and biologic aggressiveness of the tumor . as a result , the variation in the signal intensity observed on dw images for the different types of brain tumors makes the technique helpful in the differential diagnosis of these tumors . quantification of the amount of diffusion of water molecules found within and adjacent to brain tumors in the biological tissues has been carried out in the past by means of an apparent diffusion coefficient ( adc ) that provides information about the tumor cellularity . the higher the tumor cellularity , the lower the adc because of decreased water diffusivity caused by a relative reduction in extracellular space for the proton to move about . although the adc is thought to be inversely correlated with tumor cellularity , and hence tumor grade , its clinical effect remains limited as studies indicate a substantial overlap in the regional adcs between gliomas of differing grades [ 11 , 12 ] . investigators have correlated the adc values with the cellularity of glial and nonglial brain tumors , such as meningioma . others have found that in the case of intracranial tumors , high signal intensity on dw - mri had low adc values , and further , signal intensities and adc values appeared significantly different among the tumor types . additionally , studies have found that adc values are useful for the differentiation of some human brain tumors , and that water diffusivity and the adc values could be reliably used for tumor differentiation . although there are several reports in the past that discuss the application of adc values in tumor analysis [ 1723 ] , there are studies that report contradictory findings [ 2427 ] . most of the previous studies [ 18 , 2830 ] are based on selected region of interests ( rois ) placed on a representative section of the tumor for analysis . it is widely known that a single glioma , especially if high grade , may demonstrate a wide spectrum of histologic features ; in turn , the adcs within a given glioma can vary widely between different regions of that tumor . therefore , an adc derived from regional rois will likely underestimate the heterogeneity of the tumor cellularity . moreover , the selection of a localized area within a tumor can be subjective and prone to sampling bias . this sampling bias may explain the discrepant results among previous articles [ 30 , 32 , 33 ] for the ability of dw - mri to distinguish high- from low - grade gliomas . moreover , these methods depend on correct identification of the measured structure by the operator , and the choice of location , number , and size of the roi areas is operator dependent . this can lead to site - selection bias , which limits the reproducibility of results [ 34 , 35 ] . a more objective approach than placement of regional rois would be to analyze the entire volume of the tumor to provide quantitative information about the tissue characteristics and heterogeneity of the whole tumor . by encompassing the whole tumor , potential sampling bias would be largely eliminated , and the tumor areas with different diffusion characteristics would be reflected , meaning that all elements of the tumor that contribute to group differences would be analyzed . the adcs of glioma and meningioma are related to tumor cellularity and thereby dw - mri and adcs can provide information useful to diagnose these brain tumors that can not be obtained with conventional mri . although there are reports that qualitatively describe the signal intensity characteristics of glioma and meningioma on dw images [ 12 , 13 , 36 ] , quantifying the signal intensity on dw images with whole tumor volume for these tumors is not much reported in literature . however , visual assessment of the relative tissue signal attenuation on dw images is being applied in the clinical practice for tumor detection , tumor characterization , and the evaluation of treatment response in subjects . the purpose of the present work was to evaluate the role of dw - mri in the examination and classification of brain tumors , namely , glioma and meningioma . an attempt is made to explore the usefulness of the signal intensity characteristics on dw images to differentiate between subjects with gliomas and subjects with meningiomas , based on the entire tumor volume data . the signal intensity variations on dw images are quantified to identify the different levels of changes taking place in the subjects with tumor and further applied in the differential diagnosis of the tumors . brain tumors are a heterogeneous group of neoplasms , each with its own biology , treatment , and prognosis . dw - mri allows noninvasive examination of tumor cellularity since cells constitute barriers that restrict microscopically the motion of the water molecules within the tissue . increased cellularity in the tumor tissues restricts the regular water movement compared to normal brain tissues and vice versa . in this sense , the diffusion of water molecules across the tumor as compared to the diffusion in normal brain is expected be different , depicting different diffusivity , depending on the tumor cellularity . thereby , a homogeneous signal intensity variation is observed in the normal region of the brain as compared to the region of tumor , wherein there is a heterogeneous signal intensity variation . understanding the heterogeneity of individual tumors may prove to be useful in the diagnosis and classification of tumors . the variation in the signal intensity of different types of brain tumors on dw images yields qualitative and quantitative information that provides unique insight into tumor characteristics . it is observed that the signal intensity of gliomas on dw images is variable ( hyper- , iso- , or hypointense ) . the higher grade gliomas contain greater cellularity and fewer extracellular spaces . in the tissue microenvironment , water diffusion is more constrained , and tissues consequently display greater signal intensity on dw images and low signal intensity on adc maps [ 18 , 20 ] . thereby by carrying out dw - mri studies and determining signal intensity variations across the brain tumor area it is possible to distinguish different regions within the tumor and also differentiate between the tumor types . further , differentiation between these tumor types can play a significant role in planning of therapy and also evaluating response to therapy . the clinical data is obtained from ragavs diagnostic and research center , bangalore , india , and vikram hospital , bangalore , india . the ethics approval is obtained from the committee of clinical research at the ragavs diagnostic and research center and vikram hospital to carry out the investigations on the clinical data provided . the study group includes 20 cases ( 14 males , 06 females ; mean age , 55 years ; age range , 3686 years ) with clinically proven glioma and 12 cases ( 04 males , 08 females ; mean age , 59 years ; age range , 5078 years ) with clinically proven meningioma . all the subjects underwent clinical mr imaging with 1.5 t symphony maestro class mr scanning system from siemens . dw - mri was performed by using a multisection , single - shot , spin - echo , echo - planar pulse sequence with the following parameters : repetition time [ tr ] = 3200 ms , echo time [ te ] = 94 ms , acquisition matrix = 128 128 , field of view [ fov ] = 230 mm 230 mm , and diffusion gradient value of b = 1000 s / m along 19 axial slices , 5 mm thick slice , and intersection gap of 1.5 mm . for all the subjects with tumor considered in the study , we confirmed the appearance of tumor by ruling out the possibility of bright intensity due to t2 shine through effects . this was done by verifying the appearance of hyperintensities on dw images and concomitant - reduced adcs relative to the contralateral normal brain in their initial mri studies . dw - mri technique makes it to visualize the altered rates of water diffusion , by producing a high signal intensity appearance in the area of tumor , compared to the normal brain tissues . consequently , on examining the spatial intensity variation distribution on dw images for the subjects with tumors , it is observed that the signal intensity is not uniformly distributed over the entire dw image . there are abrupt jumps in signal intensity in the area of tumor in contrast to the other healthy areas of the brain , where the signal intensity distribution is almost uniform . the gradient of an image measures the rate of intensity changes taking place within the image . the magnitude of the gradient at a particular point within the image provides information on how the image intensity is changing at that point , leading to a higher value for the higher rate of intensity changes and a smaller value for uniform intensity changes . dw images of the subjects with brain tumors , showing bright imaging appearance in the areas of tumor , would thereby lead to higher values for intensity gradients in contrast to the other healthy areas of the brain . in the present work , we have made an attempt to investigate the water molecule diffusion patterns producing signal intensity variations on dw images in the area of tumor and , further , quantify the amount of signal intensity variations using sig parameter . the axial dw images of the subjects with brain tumor , obtained in dicom format , are converted to bmp format with intensities scaled to fit the conventional range of 0255 . this is done to reduce the complexity in the image manipulation algorithms and to achieve speed up in processing of the images . the axial dw image from each tumor subject is divided into six areas for the convenience of analysis as shown in figure 1 . each subject with brain tumor is represented by a set of dw images in the axial plane taken at different axial levels ( from 1 to 19 ) . the subject with brain tumor presents abrupt changes in the signal intensities in one ( or more ) of the six areas on the affected axial slices . the axial slices that indicate such abrupt changes in the signal intensities are selected for image analysis . for each axial slice selected from the tumor subject , to carry out image analysis an roi , i(x , y ) , is manually drawn to cover the bright region in the particular brain area , representing the region of tumor . as a control for signal intensity , another roi of the same size is placed in the same ( mirror image ) location in the contralateral hemisphere . the rois on each side are further divided automatically ( using adobe photo - shop cs3 ) into smaller subregions , f(x , y ) , corresponding to an image size represented by ( m n ) pixels . the typical values for m and n additionally , extra care is taken while choosing the subregions ( especially on the tumor side ) to avoid any errors that may be caused due to edge effects ( change in intensity along the periphery of tumor ) . the roi and the corresponding subregions on the tumor side and on the contralateral normal side for a subject with glioma ( area 6 , axial slice 13 ) are shown in figure 2 . each and every subregion of the roi on the tumor side is analysed for the variation in signal intensities observed in that subregion . the nature of signal intensity changes in the subregion provides information about the water diffusion characteristics and thereby provides information about cellular density and tumor behavior . given that water diffusion has directionality , in order to take care of the orientation dependent contrast ( diffusion anisotropy ) , the sig value for each subregion , f(x , y ) , is evaluated using the robinson compass operator , comprising eight kernels , as shown in figure 3 . the set of 8 kernels are convolved with the subregion , f(x , y ) , to obtain the sigs along the corresponding directions . gs , gse , ge , gne , gn , gnw , gw , and gsw , represent gradients along south , south - east , east , north - east , north , north - west , west , and south - west directions , respectively . the magnitude of the maximum change in signal intensities can be evaluated as the maximum value gained for the sig parameter , subsequent to applying all the eight kernels to the pixel neighborhood [ 37 , 38 ] . the maximum response from all the 8 kernels ( sigmax ) is given by ( 1)sigmax=max{gs , gse , ge , gne , gn , gnw , gw , gsw}. the above procedure is repeated for all the subregions of the roi on the tumor side . the subregion of the roi that results in the highest sigmax value is the subregion that exhibits highest cellular density and is chosen as the resultant sig value for that axial dw slice . the resultant sig value so obtained for the axial dw slice indicates whether there is any sudden change in the signal intensity level in any of the subregions of the roi for that axial slice . subsequently , the subregions of the ( mirror ) roi on the contralateral hemisphere are considered for image analysis . the set of 8 kernels are convolved with each subregion to obtain the sigs along the corresponding directions . however in case of contralateral normal hemisphere , by taking the maximum response from the 8 kernels ( as on tumor side ) , we observed that the sig values were irregular over the normal hemisphere . they were considerably high especially at the regions of sulci ( depression or fissure in the surface of the brain ) and gyri ( elevated convolutions on the surfaces of the cerebral hemispheres ) . as an alternative , the minimum response from all the 8 kernels ( sigmin ) is taken as the resulting sig value for the subregion on the contralateral hemisphere , as it best represents the normal side of the brain ( region with uniform signal intensity variation ) . eventually , sigmin values are obtained for all the subregions of the roi on the contralateral hemisphere , and the lowest of all the sigmin values is chosen as the resultant sig value for the axial dw slice . the above procedure is repeated for all the subsets of axial dw slices selected for image analysis from the tumor subject . to take into consideration the effect of the presence of tumor on multiple axial dw slices of the subject , the average of the resultant sig values evaluated across all the axial dw slices similarly , the average of the resultant sig values from the contralateral hemisphere is evaluated . the decisive sig value on the tumor side is compared with the corresponding decisive sig value on the contralateral hemisphere , of the same subject , for quantification of the results using relative sig ( rsig ) value . the rsig value for each tumor subject is evaluated using ( 2)rsig=(sig on contralateral hemispheresig on tumor side hhh.sig on contralateral hemisphere ) (sig on contralateral hemisphere)1 . the results are tested for statistical significant difference between the sig values obtained from the subjects with tumor and their contralateral counterpart and also for the rsig values across the two tumor types . the analysis is carried out at 99% confidence level ( p < 0.01 ) using the significance of difference between the means using student 's t - distribution . the axial dw image of a subject showing tumor ( glioma ) in area 5 , axial slice 12 , is shown in figure 4 . the plot of the spatial variation of image intensity distribution of the subregion of the roi ( area 5 , axial slice 12 ) resulting in maximum sig value ( resultant sig on tumor side ) , for the subject shown in figure 4 , is shown in figure 5 . the plot of the spatial variation of image intensity distribution of the subregion on the contralateral hemisphere resulting in minimum sig value ( resultant sig on contralateral side ) for the same subject is shown in figure 6 . it is observed from figure 5 that the spatial intensity variation distribution of the subregion on the tumor side has abrupt jumps in contrast to the spatial intensity variation distribution of the subregion on the contralateral hemisphere , which is almost unvarying as shown in figure 6 . the nonuniform image intensity distribution in the subregion on the tumor side results in a higher value for the sig parameter ( 108 ) . in contrast , the uniform intensity distribution in the subregion on the contralateral hemisphere results in a considerable smaller value for the sig parameter ( 8) . the resultant sig value evaluated from the subregion of the roi on the tumor side indicates the maximum change in the signal intensity variation across the roi on the axial dw slice . the plot of the variation in the maximum sig values ( decisive sig value on tumor side ) on the glioma side and the minimum sig values ( decisive sig values on contralateral side ) on the contralateral hemisphere , for all the 20 subjects with glioma , is shown in figure 7 . similarly , the plot of the variation in the decisive sig values on the meningioma side and the corresponding decisive sig values on the contralateral hemisphere , for all the 12 subjects with meningioma , is shown in figure 8 . statistical study is carried out by using the excel program and spss 17.0 software package . the analysis is performed at 99% confidence level ( p < 0.01 ) using the significance of difference between the means using student 's t distribution . the decisive sig values for the subjects with glioma and meningioma are statistically compared to the corresponding values from the contralateral hemisphere . the difference in the decisive sig values for the tumor subjects compared to their contralateral hemisphere are highly significant ( p < 0.01 ) in the regions of the brain , where there was a high incidence of tumor ( glioma / meningioma ) . results suggest that the decisive sig values obtained from dw images for the subjects with glioma or meningioma can be used to clearly differentiate the tumor subjects from the normal subjects . the results of analysis for the subjects with glioma and meningioma considered in the present study are summarized in table 1 . the range of the decisive sig values obtained for the subjects with glioma and meningioma and their corresponding decisive sig values on the contralateral hemisphere are listed . also the range of the rsig values evaluated across the subjects with glioma and meningioma is listed . the plot of the variation in the mean rsig values across the subjects with glioma and meningioma ( last column of table 1 ) is shown in figure 9 . it is observed that the mean rsig value obtained for the subjects with glioma ( 16.87 4.69 ) is notably higher in comparison to the mean rsig value for the subjects with meningioma ( 7.94 1.54 ) . the statistical significant difference between the rsig values obtained across the two tumor types is tested . statistical study is carried out at 99% confidence level ( p < 0.01 ) using the significance of difference between the means using student 's t distribution . it is observed that the mean rsig value is significantly different ( p < 0.01 ) across the subjects with glioma and meningioma ( as listed in the last column of table 1 ) . our study is primarily based on quantifying the signal intensity variations observed on dw images for the differential diagnosis of glioma and meningioma . the variation in the signal intensity observed in the area of these brain tumors does not depend on the size / shape and the location of these tumors . however , these details might play an important role in the further planning of management for the tumor subjects . on the other hand , as the signal intensity variations depend on the tumor stage ( higher grade or more aggressive tumors leading to greater signal intensity variations ) , the results obtained in the study largely depend on the tumor stage . the method proposed in the present work is realized with a purpose to overcome the drawbacks [ 18 , 2830 ] associated with the studies reported in literature for measurements using adc methods . the proposed method speeds up the process of evaluating the brain tumor as it can be carried out routinely , without relying on a dedicated workstation to make the measurements , as in adc methods . it adopts a standardized way to place the roi to cover the entire volume of the tumor on the axial dw slice and thereby provides quantitative information about the tissue characteristics and heterogeneity of the whole tumor . further , in our method considering the entire area of the tumor eliminates the requirement of expertise , as selection of a localized area within a tumor can be subjective and prone to sampling bias . also , all the affected axial dw slices are taken into account in order to reach the results , in contrast to the adc methods , wherein , a single axial dw slice ( showing maximum intensity changes ) is considered in evaluating adc values . in our method taking multiple axial dw slices additionally guarantees that there is minimum loss of information , as larger area of the brain is covered for making observations about the tumor . therefore , the work presented here provides a useful method that is both quantitative and user - friendly , with a direct measurement of signal intensity on dw images , based on the entire tumor volume ( all the dw axial slices considered ) that can be positively useful in differentiating between subjects with glioma and subjects with meningioma . analysis of the results suggests that the sig values on dw images in the region of brain tumor can be used to markedly distinguish the tumor subjects quantitatively from the normal subjects . comparing the relative increase in the sig values ( rsig ) on dw images for all the subjects with glioma and meningioma , it is found that they are in the range of ( 10.0828.36 ) times and ( 5.609.86 ) times , respectively , compared to their contralateral normal hemisphere . the quantitative changes in the rsig values based on the entire tumor volume can be assessed and can be positively useful in differentiating between subjects with glioma and subjects with meningioma . therefore the adoption of the quantitative method indicated here , in the clinical diagnosis of brain tumor , could be useful and informative .

the purpose of this study was to evaluate the role of diffusion weighted - magnetic resonance imaging ( dw - mri ) in the examination and classification of brain tumors , namely , glioma and meningioma . our hypothesis was that as signal intensity variations on diffusion weighted ( dw ) images depend on histology and cellularity of the tumor , analysing the signal intensity characteristics on dw images may allow differentiating between the tumor types . towards this end the signal intensity variations on dw images of the entire tumor volume data of 20 subjects with glioma and 12 subjects with meningioma were investigated and quantified using signal intensity gradient ( sig ) parameter . the relative increase in the sig values ( rsig ) for the subjects with glioma and meningioma was in the range of 10.0828.36 times and 5.609.86 times , respectively , compared to their corresponding sig values on the contralateral hemisphere . the rsig values were significantly different between the subjects with glioma and meningioma ( p < 0.01 ) , with no overlap between rsig values across the two tumors . the results indicate that the quantitative changes in the rsig values could be applied in the differential diagnosis of glioma and meningioma , and their adoption in clinical diagnosis and treatment could be helpful and informative .

1. Introduction 2. Materials and Methods 3. Results and Discussions 4. Conclusions

brain tumors are generally classified as either primary ( originating from within the brain cavity ) or secondary ( originating elsewhere in the body , and then spread to the brain ) . the most common types of primary brain tumors are gliomas and meningiomas , constituting , respectively , 60% and 20% of all intracranial tumors ( tumors within the brain ) in adults . hence it is very essential to differentiate between the two tumor types especially when gliomas are peripherally placed and imitate a meningioma [ 4 , 5 ] . the introduction of imaging techniques into clinical practice has been among the most important of all advances in the care of patients with brain tumors . the potential advantage of advanced magnetic resonance imaging ( mri ) methods is that they permit more uniform sampling than heterogeneous biopsies , and that , they can be applied repeatedly to monitor therapy success . diffusion weighted - magnetic resonance imaging ( dw - mri ) is an imaging technique based on the sensitivity of magnetic resonance ( mr ) to microscopic mobility of water molecules within tissues , and provides image contrast that is dependent on the molecular motion of water , which may be substantially altered by disease . dw - mri probes water molecular diffusion over distances that correspond to typical cell sizes , and this diffusion is impeded by membranes ( structures that are an integral part of the cell - architecture ) . of the histologic features for tumor analysis , cellularity has been the target of quantitative assessment with dw - mri . the rationale of using diffusion imaging to quantify cellularity is based on the principle that water diffusivity within the extracellular compartment is inversely related to the content and attenuation of the constituents of the intracellular space . with increasing cell density therefore , tumors with higher cellularity show increased signal intensity on diffusion weighted ( dw ) images , in contrast to the tumors with low cellularity that presents greater signal intensity loss . quantification of the amount of diffusion of water molecules in terms of signal intensity variations found within the brain tumors can thereby provide information about the histologic composition and biologic aggressiveness of the tumor . as a result , the variation in the signal intensity observed on dw images for the different types of brain tumors makes the technique helpful in the differential diagnosis of these tumors . quantification of the amount of diffusion of water molecules found within and adjacent to brain tumors in the biological tissues has been carried out in the past by means of an apparent diffusion coefficient ( adc ) that provides information about the tumor cellularity . although the adc is thought to be inversely correlated with tumor cellularity , and hence tumor grade , its clinical effect remains limited as studies indicate a substantial overlap in the regional adcs between gliomas of differing grades [ 11 , 12 ] . investigators have correlated the adc values with the cellularity of glial and nonglial brain tumors , such as meningioma . others have found that in the case of intracranial tumors , high signal intensity on dw - mri had low adc values , and further , signal intensities and adc values appeared significantly different among the tumor types . additionally , studies have found that adc values are useful for the differentiation of some human brain tumors , and that water diffusivity and the adc values could be reliably used for tumor differentiation . most of the previous studies [ 18 , 2830 ] are based on selected region of interests ( rois ) placed on a representative section of the tumor for analysis . this sampling bias may explain the discrepant results among previous articles [ 30 , 32 , 33 ] for the ability of dw - mri to distinguish high- from low - grade gliomas . moreover , these methods depend on correct identification of the measured structure by the operator , and the choice of location , number , and size of the roi areas is operator dependent . a more objective approach than placement of regional rois would be to analyze the entire volume of the tumor to provide quantitative information about the tissue characteristics and heterogeneity of the whole tumor . by encompassing the whole tumor , potential sampling bias would be largely eliminated , and the tumor areas with different diffusion characteristics would be reflected , meaning that all elements of the tumor that contribute to group differences would be analyzed . the adcs of glioma and meningioma are related to tumor cellularity and thereby dw - mri and adcs can provide information useful to diagnose these brain tumors that can not be obtained with conventional mri . although there are reports that qualitatively describe the signal intensity characteristics of glioma and meningioma on dw images [ 12 , 13 , 36 ] , quantifying the signal intensity on dw images with whole tumor volume for these tumors is not much reported in literature . however , visual assessment of the relative tissue signal attenuation on dw images is being applied in the clinical practice for tumor detection , tumor characterization , and the evaluation of treatment response in subjects . the purpose of the present work was to evaluate the role of dw - mri in the examination and classification of brain tumors , namely , glioma and meningioma . an attempt is made to explore the usefulness of the signal intensity characteristics on dw images to differentiate between subjects with gliomas and subjects with meningiomas , based on the entire tumor volume data . the signal intensity variations on dw images are quantified to identify the different levels of changes taking place in the subjects with tumor and further applied in the differential diagnosis of the tumors . brain tumors are a heterogeneous group of neoplasms , each with its own biology , treatment , and prognosis . dw - mri allows noninvasive examination of tumor cellularity since cells constitute barriers that restrict microscopically the motion of the water molecules within the tissue . increased cellularity in the tumor tissues restricts the regular water movement compared to normal brain tissues and vice versa . in this sense , the diffusion of water molecules across the tumor as compared to the diffusion in normal brain is expected be different , depicting different diffusivity , depending on the tumor cellularity . thereby , a homogeneous signal intensity variation is observed in the normal region of the brain as compared to the region of tumor , wherein there is a heterogeneous signal intensity variation . understanding the heterogeneity of individual tumors may prove to be useful in the diagnosis and classification of tumors . the variation in the signal intensity of different types of brain tumors on dw images yields qualitative and quantitative information that provides unique insight into tumor characteristics . it is observed that the signal intensity of gliomas on dw images is variable ( hyper- , iso- , or hypointense ) . in the tissue microenvironment , water diffusion is more constrained , and tissues consequently display greater signal intensity on dw images and low signal intensity on adc maps [ 18 , 20 ] . thereby by carrying out dw - mri studies and determining signal intensity variations across the brain tumor area it is possible to distinguish different regions within the tumor and also differentiate between the tumor types . the study group includes 20 cases ( 14 males , 06 females ; mean age , 55 years ; age range , 3686 years ) with clinically proven glioma and 12 cases ( 04 males , 08 females ; mean age , 59 years ; age range , 5078 years ) with clinically proven meningioma . dw - mri was performed by using a multisection , single - shot , spin - echo , echo - planar pulse sequence with the following parameters : repetition time [ tr ] = 3200 ms , echo time [ te ] = 94 ms , acquisition matrix = 128 128 , field of view [ fov ] = 230 mm 230 mm , and diffusion gradient value of b = 1000 s / m along 19 axial slices , 5 mm thick slice , and intersection gap of 1.5 mm . for all the subjects with tumor considered in the study , we confirmed the appearance of tumor by ruling out the possibility of bright intensity due to t2 shine through effects . this was done by verifying the appearance of hyperintensities on dw images and concomitant - reduced adcs relative to the contralateral normal brain in their initial mri studies . dw - mri technique makes it to visualize the altered rates of water diffusion , by producing a high signal intensity appearance in the area of tumor , compared to the normal brain tissues . consequently , on examining the spatial intensity variation distribution on dw images for the subjects with tumors , it is observed that the signal intensity is not uniformly distributed over the entire dw image . there are abrupt jumps in signal intensity in the area of tumor in contrast to the other healthy areas of the brain , where the signal intensity distribution is almost uniform . the magnitude of the gradient at a particular point within the image provides information on how the image intensity is changing at that point , leading to a higher value for the higher rate of intensity changes and a smaller value for uniform intensity changes . dw images of the subjects with brain tumors , showing bright imaging appearance in the areas of tumor , would thereby lead to higher values for intensity gradients in contrast to the other healthy areas of the brain . in the present work , we have made an attempt to investigate the water molecule diffusion patterns producing signal intensity variations on dw images in the area of tumor and , further , quantify the amount of signal intensity variations using sig parameter . the axial dw images of the subjects with brain tumor , obtained in dicom format , are converted to bmp format with intensities scaled to fit the conventional range of 0255 . the subject with brain tumor presents abrupt changes in the signal intensities in one ( or more ) of the six areas on the affected axial slices . the axial slices that indicate such abrupt changes in the signal intensities are selected for image analysis . for each axial slice selected from the tumor subject , to carry out image analysis an roi , i(x , y ) , is manually drawn to cover the bright region in the particular brain area , representing the region of tumor . as a control for signal intensity , another roi of the same size is placed in the same ( mirror image ) location in the contralateral hemisphere . the typical values for m and n additionally , extra care is taken while choosing the subregions ( especially on the tumor side ) to avoid any errors that may be caused due to edge effects ( change in intensity along the periphery of tumor ) . the roi and the corresponding subregions on the tumor side and on the contralateral normal side for a subject with glioma ( area 6 , axial slice 13 ) are shown in figure 2 . each and every subregion of the roi on the tumor side is analysed for the variation in signal intensities observed in that subregion . the nature of signal intensity changes in the subregion provides information about the water diffusion characteristics and thereby provides information about cellular density and tumor behavior . given that water diffusion has directionality , in order to take care of the orientation dependent contrast ( diffusion anisotropy ) , the sig value for each subregion , f(x , y ) , is evaluated using the robinson compass operator , comprising eight kernels , as shown in figure 3 . gs , gse , ge , gne , gn , gnw , gw , and gsw , represent gradients along south , south - east , east , north - east , north , north - west , west , and south - west directions , respectively . the magnitude of the maximum change in signal intensities can be evaluated as the maximum value gained for the sig parameter , subsequent to applying all the eight kernels to the pixel neighborhood [ 37 , 38 ] . the above procedure is repeated for all the subregions of the roi on the tumor side . the subregion of the roi that results in the highest sigmax value is the subregion that exhibits highest cellular density and is chosen as the resultant sig value for that axial dw slice . the resultant sig value so obtained for the axial dw slice indicates whether there is any sudden change in the signal intensity level in any of the subregions of the roi for that axial slice . subsequently , the subregions of the ( mirror ) roi on the contralateral hemisphere are considered for image analysis . however in case of contralateral normal hemisphere , by taking the maximum response from the 8 kernels ( as on tumor side ) , we observed that the sig values were irregular over the normal hemisphere . they were considerably high especially at the regions of sulci ( depression or fissure in the surface of the brain ) and gyri ( elevated convolutions on the surfaces of the cerebral hemispheres ) . as an alternative , the minimum response from all the 8 kernels ( sigmin ) is taken as the resulting sig value for the subregion on the contralateral hemisphere , as it best represents the normal side of the brain ( region with uniform signal intensity variation ) . eventually , sigmin values are obtained for all the subregions of the roi on the contralateral hemisphere , and the lowest of all the sigmin values is chosen as the resultant sig value for the axial dw slice . to take into consideration the effect of the presence of tumor on multiple axial dw slices of the subject , the average of the resultant sig values evaluated across all the axial dw slices similarly , the average of the resultant sig values from the contralateral hemisphere is evaluated . the decisive sig value on the tumor side is compared with the corresponding decisive sig value on the contralateral hemisphere , of the same subject , for quantification of the results using relative sig ( rsig ) value . the results are tested for statistical significant difference between the sig values obtained from the subjects with tumor and their contralateral counterpart and also for the rsig values across the two tumor types . the analysis is carried out at 99% confidence level ( p < 0.01 ) using the significance of difference between the means using student 's t - distribution . the plot of the spatial variation of image intensity distribution of the subregion of the roi ( area 5 , axial slice 12 ) resulting in maximum sig value ( resultant sig on tumor side ) , for the subject shown in figure 4 , is shown in figure 5 . the plot of the spatial variation of image intensity distribution of the subregion on the contralateral hemisphere resulting in minimum sig value ( resultant sig on contralateral side ) for the same subject is shown in figure 6 . it is observed from figure 5 that the spatial intensity variation distribution of the subregion on the tumor side has abrupt jumps in contrast to the spatial intensity variation distribution of the subregion on the contralateral hemisphere , which is almost unvarying as shown in figure 6 . the nonuniform image intensity distribution in the subregion on the tumor side results in a higher value for the sig parameter ( 108 ) . in contrast , the uniform intensity distribution in the subregion on the contralateral hemisphere results in a considerable smaller value for the sig parameter ( 8) . the resultant sig value evaluated from the subregion of the roi on the tumor side indicates the maximum change in the signal intensity variation across the roi on the axial dw slice . the plot of the variation in the maximum sig values ( decisive sig value on tumor side ) on the glioma side and the minimum sig values ( decisive sig values on contralateral side ) on the contralateral hemisphere , for all the 20 subjects with glioma , is shown in figure 7 . similarly , the plot of the variation in the decisive sig values on the meningioma side and the corresponding decisive sig values on the contralateral hemisphere , for all the 12 subjects with meningioma , is shown in figure 8 . the analysis is performed at 99% confidence level ( p < 0.01 ) using the significance of difference between the means using student 's t distribution . the decisive sig values for the subjects with glioma and meningioma are statistically compared to the corresponding values from the contralateral hemisphere . the difference in the decisive sig values for the tumor subjects compared to their contralateral hemisphere are highly significant ( p < 0.01 ) in the regions of the brain , where there was a high incidence of tumor ( glioma / meningioma ) . results suggest that the decisive sig values obtained from dw images for the subjects with glioma or meningioma can be used to clearly differentiate the tumor subjects from the normal subjects . the results of analysis for the subjects with glioma and meningioma considered in the present study are summarized in table 1 . the range of the decisive sig values obtained for the subjects with glioma and meningioma and their corresponding decisive sig values on the contralateral hemisphere are listed . also the range of the rsig values evaluated across the subjects with glioma and meningioma is listed . the plot of the variation in the mean rsig values across the subjects with glioma and meningioma ( last column of table 1 ) is shown in figure 9 . it is observed that the mean rsig value obtained for the subjects with glioma ( 16.87 4.69 ) is notably higher in comparison to the mean rsig value for the subjects with meningioma ( 7.94 1.54 ) . the statistical significant difference between the rsig values obtained across the two tumor types is tested . statistical study is carried out at 99% confidence level ( p < 0.01 ) using the significance of difference between the means using student 's t distribution . it is observed that the mean rsig value is significantly different ( p < 0.01 ) across the subjects with glioma and meningioma ( as listed in the last column of table 1 ) . our study is primarily based on quantifying the signal intensity variations observed on dw images for the differential diagnosis of glioma and meningioma . the variation in the signal intensity observed in the area of these brain tumors does not depend on the size / shape and the location of these tumors . however , these details might play an important role in the further planning of management for the tumor subjects . on the other hand , as the signal intensity variations depend on the tumor stage ( higher grade or more aggressive tumors leading to greater signal intensity variations ) , the results obtained in the study largely depend on the tumor stage . it adopts a standardized way to place the roi to cover the entire volume of the tumor on the axial dw slice and thereby provides quantitative information about the tissue characteristics and heterogeneity of the whole tumor . further , in our method considering the entire area of the tumor eliminates the requirement of expertise , as selection of a localized area within a tumor can be subjective and prone to sampling bias . in our method taking multiple axial dw slices additionally guarantees that there is minimum loss of information , as larger area of the brain is covered for making observations about the tumor . therefore , the work presented here provides a useful method that is both quantitative and user - friendly , with a direct measurement of signal intensity on dw images , based on the entire tumor volume ( all the dw axial slices considered ) that can be positively useful in differentiating between subjects with glioma and subjects with meningioma . analysis of the results suggests that the sig values on dw images in the region of brain tumor can be used to markedly distinguish the tumor subjects quantitatively from the normal subjects . comparing the relative increase in the sig values ( rsig ) on dw images for all the subjects with glioma and meningioma , it is found that they are in the range of ( 10.0828.36 ) times and ( 5.609.86 ) times , respectively , compared to their contralateral normal hemisphere . the quantitative changes in the rsig values based on the entire tumor volume can be assessed and can be positively useful in differentiating between subjects with glioma and subjects with meningioma . therefore the adoption of the quantitative method indicated here , in the clinical diagnosis of brain tumor , could be useful and informative .

gamma amino butyric acid type a receptors ( gabaars ) are a major source of fast inhibitory synaptic transmission in the cns and thus play a crucial role in regulating neuronal networks . altering the number of gabaars in a synapse through rapid receptor trafficking trafficking of gabaars to and from the plasma membrane has been shown to depend on neuronal activity [ 47 ] . gabaar redistribution is also mediated by a variety of neuromodulatory substances such as hormones and cytokines . for example , insulin and pi3 kinase cause rapid insertion of gabaars into the plasma membrane increasing the amplitude of miniature inhibitory postsynaptic currents ( mipscs ) [ 8 , 9 ] . tumor necrosis factor alpha ( tnf ) , a proinflammatory cytokine that is constitutively expressed following spinal cord injury ( sci ) , has been shown to alter receptor trafficking of gabaars to and from the cell surface in vitro . in vivo , tnf levels are increased as part of the inflammatory response following sci and various other nervous system disorders , contributing to secondary cell death [ 1113 ] . tnf has been shown to specifically promote trafficking of glutamate - receptor-2-(glua2- ) lacking ampa receptors ( ampars ) to the plasma membrane of spinal neurons inducing excitotoxicity in vivo . blocking tnf action pharmacologically reduces ampar trafficking and cell death after sci , suggesting that modulation of ampars is a major mechanism by which tnf promotes cell death in cns disease . the aim of the current study is to assess in vivo the effect of tnf on gabaar trafficking to the plasma membrane of spinal neurons . we modeled the inflammatory response associated with sci by tnf nanoinjection into the spinal cord to establish whether gabaars are exocytosed onto or endocytosed from the plasma membrane . by determining the direction of trafficking , we can then infer whether trafficking of gabaars is exacerbating or attenuating tnf-mediated excitotoxicity . gabaars were identified and quantified based on the presence of the gamma-2 ( 2 ) subunit as studies show that this subunit is critical for postsynaptic clustering of gabaars and the vast majority of gabaars are composed of subunits and combined with 2 [ 14 , 15 ] . we used a combination of biochemical fractionation and laser scanning confocal microscopy followed by iterative deconvolution and automated image analysis to evaluate gabaar levels in the plasma membrane and at synapses after tnf nanoinjection into the spinal parenchyma . whole membrane fractionation and quantitative western blotting showed a trend towards increased membrane gabaar protein levels within 60 min of tnf nanoinjection delivery , but these results did not reach significance . confocal data revealed increased gabaars at synaptic sites following tnf nanoinjection , underscoring the subcellular specificity of gabaar localization . confocal image findings also suggest that there is a nonlinear dose - dependent relationship between tnf and gabaar trafficking . female long - evans rats , 77- to 87-d - old , were housed in pairs ( n = 30 ) with ad libitum access to food and water . all experimental procedures followed the national institutes of health guidelines and were approved by the institutional animal care and use committees at the ohio state university and the university of california , san francisco . nanoinjections ( 35 nl ) were delivered stereotactically into the t9 - 10 ventral horn as described in hermann et al . , 2001 , by applying compressed air micropressure to pulled glass pipettes ( tip diameter , 30 m ; 30 bevel ; radnoti ) . an albumin injection served as a control due to its similarity in molecular weight to rat recombinant tnf ( r&d systems ) . for imaging experiments , subjects ( n = 4 ) received a dose of tnf ( 0.01 , 0.1 , or 1 m ) and an injection of albumin on the contralateral ventral horn to serve as a within - subject control . these doses of tnf have been found sufficient to increase glutamate - mediated excitotoxicity in the spinal cord , but insufficient to cause cell death . the dye fluororuby ( invitrogen ) was included in each injection solution in order to localize injection sites for high - resolution confocal analyses ( figure 1 ) . subjects were sacrificed 60 minutes following nanoinjection by transcardiac perfusion with 0.9% saline followed by 4% paraformaldehyde . for biochemical experiments , subjects received four evenly spaced nanoinjections of either tnf ( 1 m ) or albumin along a 750 m length of spinal cord ( tnf , n = 8 ; vehicle , n = 10 ) . one hour after nanoinjection , the spinal cord was extracted under deep anesthesia ; 7.5 mm of the cord was snap - frozen with dry ice and stored at 80c for later processing . the snap - frozen spinal cord was thawed on ice and homogenized with 30 passes of a type b pestle in a dounce homogenizer ( kontes ) with 500 l of homogenization buffer ( 10 mm tris , 300 mm sucrose , roche minicomplete protease inhibitor , ph = 7.5 ) . the resulting suspension was then passed through a 22 gauge needle five times and centrifuged at 5,000 rcf for 5 min at 4c . the supernatant ( s1 ) was transferred to a new tube and centrifuged again at 13,000 rcf for 30 min at 4c . the supernatant ( s2 ) was transferred to a new tube , and the membrane - enriched pellet ( p2 ) was resuspended in 50 l of pbs containing protease inhibitor . p2 fractions were vortexed and sonicated , and all sample fractions were stored at 80c . sample protein concentration was assayed using bca ( pierce ) and quantified with a plate reader ( tecan ; genios ) . p2 fractions from each subject were diluted 1 : 2 with cold laemmli sample buffer containing 5% -mercaptoethanol ( biorad ) , and 20 g of protein per lane was immediately loaded onto a precast 10% tris - hcl polyacrylamide gel ( biorad ) . sample loading on the gel was performed so that each subject had their own lane , loading order was counterbalanced across injection condition to account for regional variability within the gel , and the experimenter was blind to subject condition . a kaleidoscope ladder ( 3 l ; biorad ) was loaded to confirm molecular weights . the loaded gel was electrophoresed in sds running buffer ( biorad ; 25 mm tris , 192 mm glycine , 0.1% sds , ph = 8.3 ) for 1 h at 100 volts . the protein was then transferred to nitrocellulose membrane in cold tris - glycine buffer ( 25 mm tris , 192 mm glycine , 20% methanol , ph = 8.3 ) . the membrane was blocked for 1 h in odyssey blocking buffer ( li - cor ) containing 0.1% tween 20 and then incubated overnight ( 18 h ) in the dark at 4c in a primary antibody solution containing odyssey blocking buffer , 0.05% tween 20 , and a rabbit polyclonal anti- gabaa receptor 2 primary antibody ( 1 : 500 ; chemicon , ab5559 ) . the membrane was washed 4 5 min with tbs containing 0.1% tween 20 ( ttbs ) then incubated for 1 h in a fluorescently labeled secondary antibody solution containing odyssey blocking buffer , 0.2% tween 20 , and 1 : 30,000 irdye 680 goat anti - rabbit secondary antibody ( li - cor ) and subsequently washed 4 5 min in ttbs , 1 5 min in tbs . the membrane was immediately scanned for protein bands using the corresponding 680 nm laser at a scanning intensity of 4 on the odyssey infrared imaging system ( li - cor ) . the membrane was then reblocked and reincubated in a primary antibody solution containing 1 : 800 mouse anti - n - cadherin primary antibody ( bd biosciences , 610920 ) . the membrane was washed and reincubated in a secondary antibody solution containing 1 : 30,000 irdye 800 goat anti - mouse secondary antibody ( li - cor ) . the membrane was washed again and rescanned for protein bands in the 800 nm channel at a scanning intensity of 3 . although traditional western blot analysis using chemiluminescence and densitometry measurements is considered to be merely semiquantitative , we used an established near - infrared labeling and detection technique ( odyssey infrared imaging system , li - cor ) to definitively quantify the intensity of fluorescently labeled protein bands . to ensure that our intensity measurements were truly quantitative , we generated linear ranges for each antibody by plotting band intensity measurement relative to the concentration of protein loaded for a protein dilution curve . laser scanning intensities for each antibody were selected by determining the laser intensity which yielded the highest linear range , r , of protein band fluorescent intensity from a protein dilution curve of a control sample ( figures 2(a ) and 2(b ) ) . the intensity of each fluorescently labeled protein band was quantified using the odyssey application software version 3.0 ( li - cor ) . background fluorescence was assessed and corrected for using odyssey software which determined median pixel densities above and below each protein band and normalized these bands of interest accordingly . prior experiments have used the plasma membrane protein n - cadherin ( ncad ) to characterize the degree of membrane enrichment in each fraction generated by spinal cord subcellular fractionation [ 13 , 16 ] . in these studies , western blotting revealed that subcellular fractionation generates p2 samples with a modest enrichment of the plasma membrane as evidenced by the presence of ncad . neither ncad ( p = 0.883 ) nor actin ( p = 0.610 ) intensity varied between conditions . we therefore used ncad as a control both for variability in protein loading and in plasma membrane enrichment through subcellular fractionation . gabaar band intensities from each sample were normalized with respect to ncad by dividing gabaar intensity by ncad intensity . two independent replications were performed , and the normalized densitometry results were averaged across runs . the 30 mm length of spinal cord centered on the injection site was isolated and postfixed overnight ( < 18 h ) in 4% paraformaldehyde followed by cryoprotection of the tissue in 30% sucrose for 2d . the tissue was then cut into 10 mm blocks , flash - frozen on dry ice , embedded in oct , and sectioned into 20 m thick horizontal slices . fixed tissue sections from a full set of experimental conditions were antibody - labeled using a high - throughput staining station ( sequenza ; thermo scientific ) . tissue was blocked and permeabilized with 5% normal goat serum and 0.3% triton x-100 for 1 h. sections were incubated in a solution consisting of mouse monoclonal antibody against presynaptic synaptophysin ( 1 : 200 ; millipore mab5258 - 50ug ) and rabbit polyclonal antibody against gabaa receptor 2 primary antibody ( 1 : 200 ; chemicon , ab5559 ) overnight at room temperature . slides were washed with 2 ml pbs then incubated for 1 h at room temperature in a solution containing 1 : 100 alexa 488 goat anti - rabbit and 1 : 100 alexa 633 goat anti - mouse secondary antibodies . after washing with 2 ml pbs , slides were coverslipped with vectashield containing dapi ( 4 , 6-diamidino-2-phenylindole ; vector laboratories ) . negative immunolabel control conditions consisted of no primary antibody , and each individual primary with the incorrect secondary . large ventral motor neurons ( characterized by a diameter > 40 m ) were selected using wide - field fluorescence based on the distinctive synaptophysin outline surrounding the plasma membrane ( figure 1 ) . one motor neuron was sampled every 100 m following the rostrocaudal axis of each ventral horn centered around the fluororuby - labeled injection sites . this sampling procedure was performed through horizontal sections of the spinal cord up to 600 m rostral and caudal to the center of each injection site . in a sampling region of multiple motor neurons , a single motor neuron was chosen at random . a zeiss 510 meta laser scanning confocal microscope ( 63x objective ; na = 1.4 ; 2x zoom ) was used to generate confocal stacks for large motor neurons . control tissue was used to optimize filter and laser settings , which were then held constant throughout the experiment . these settings allowed for virtually complete distinction between immunolabels gabaars ( alexa 488 ) , synaptophysin ( alexa 633 ) , and fluororuby ( texas red ) . confocal z - stacks consisted of 1 m slices which were oversampled at 0.5 m z - intervals . autoquant software was used to deblur these confocal stacks through the process of 3d blind iterative deconvolution . an iteration number of 3 was determined for gabaar labeling based on a random subset of images and held constant during the experiment . performing deconvolution on confocal image stacks allowed for greater resolution of receptor puncta than was possible with either technique alone . images underwent automated image analysis to quantify the number of fluorescently labeled receptor puncta on the plasma membrane exceeding a predetermined pixel threshold based on control tissue . one macro was designed to measure the amount of total gabaar receptor pixels ( intra- and extracellular puncta ) as well as colocalization of synaptophysin and gabaar pixels ( synaptically localized gabaar puncta ) in each field of the z - stack . first , the macro identified the plane in the z - series with the highest amount of gabaar / synaptophysin colocalization . a blinded researcher supervised the automated plane selection to prevent selection based on staining artifacts . once a single plane was selected , a blinded researcher identified the plasma membrane of the motor neuron by tracing the synaptophysin - labeled outline of the cell . a 2 m - thick image - based subcellular fraction was produced containing the plasma membrane of the cell from the single plane . from the plasma membrane subcellular image fraction , metamorph quantified gabaar pixels ( extrasynaptic receptors ) and colocalized gabaar / synaptophysin pixels ( synaptic receptors ) . immunofluorescence data were analyzed using anova , and the experiment was a mixed design ( injection side and distance from injection site served as within - subject variables ) . when appropriate , tukey 's post hoc analyses were used to determine significant differences amongst multiple outcomes within a variable . total gabaar protein levels were accounted for by ancova , which allowed for the distinction between receptor trafficking versus an increase in total gabaar puncta as a result of receptor synthesis . quantitative western blotting was used to generate normalized gabaar intensity ratios ( gabaar : ncad ) for the membrane - enriched fraction of spinal cord homogenate ( figure 3(a ) ) . these ratios for each subject were generated by running all subjects across two counterbalanced gels . anova revealed no significant difference in total membrane gabaar between injection condition ( p = 0.315 ) , yet group means reflect a nonsignificant trend towards a higher concentration of gabaars in tnf-treated subjects ( figure 3(b ) ) . a crude subcellular fractionation of samples , which in the past has been sufficient to reveal changed receptor levels from tnf-mediated trafficking of ampars , did not show a significant distinction in gabaar between injection conditions perhaps due to a lack of membrane resolution . therefore , the high - resolution , low - throughput method of quantitative confocal microscopy was used to elucidate changes in localized receptor trafficking . automated image analysis of the 3-dimensional neuropil ( defined as the entire set of pixels in a confocal z - stack ) revealed a dose - dependent increase in synaptic gabaar ( colocalized gabaar and synaptophysin pixels ) 60 minutes following tnf nanoinjection ( p < 0.001 ) . total gabaar in the neuropil increased in a dose - dependent manner ( p < 0.001 ) as well ( figure 4 ) . additionally , there was a significant increase in total gabaar puncta from the middle to the lowest tnf dose ( p < 0.02 ) ( figures 4(i ) and 4(j ) ) . studies have shown that gabaars can undergo rapid activity - dependent ubiquitination and lysosomal degradation to maintain homeostatic levels of the receptor [ 15 , 17 ] , which could explain the significant decrease in gabaar associated with the middle dose of tnf relative to the lowest dose . it is possible that the low dose of tnf elicited a modest increase in gabaar undetectable to regulatory systems within the neuron , whereas a middle dose of tnf sufficiently increased gabaars to the point where they would be downregulated . an ancova was used to statistically account for the tnf-mediated increase in total gabaar protein in the neuropil , thus isolating the dose - dependent changes in synaptic gabaar attributable to receptor trafficking . after correcting for changes in total gabaar protein , the dose - dependent effect of tnf on synaptic gabaars in the neuropil was robustly maintained ( p < 0.001 ) indicating that the dose - dependent increase in synaptic gabaar does not depend solely on wholesale protein changes but also relies on receptor trafficking to the synapse . correcting for variance in total gabaar protein in the neuropil with ancova , revealed no significant interaction of dose by side for synaptic gabaar ( p = 0.325 ) . overall , results reveal that tnf dose significantly influenced gabaar receptor trafficking to synaptic sites within the neuropil containing spinal motor neurons . image analysis of the plasma membrane also revealed a dose - dependent increase in synaptic and extrasynaptic gabaar 60 minutes after tnf injection ( figures 5(g ) and 5(h ) ) ( p < 0.001 ) . there was significantly more extrasynaptic gabaars associated with the lowest tnf dose relative to the middle dose ( p < 0.003 ) . after statistically correcting for tnf-mediated changes in total gabaar level with ancova , there remained a significant dose - dependent effect of tnf on synaptic gabaar indicating that tnf increases receptor trafficking to synapses on the motor neurons ' somatic surface in addition to increasing extrasynaptic receptors ( p < 0.05 ) . the effect of tnf was widespread and extended onto the contralateral side of the spinal cord ( the site of vehicle injection ) eliciting a dose - dependent increase in synaptic and total gabaars at the level of the plasma membrane and the neuropil , which spanned both sides of the cord ( all measures , p < 0.001 ) . the extensive effect of tnf on spinal tissue is also evidenced by the fact that there was no main effect of side of the cord in synaptic , nor total gabaar measures ( figure 6 ) ( all measures , p > 0.05 ) . there was an interaction of dose by side for extrasynaptic gabaar on the plasma membrane ( p = 0.037 ) , but there was not a significant interaction for any other gabaar measures ( p > 0.05 ) ( figure 6 ) . accounting for changes in total extrasynaptic gabaar in the plasma membrane with ancova showed no higher order interactions between dose and side for synaptic gabaar ( p > 0.05 ) . taken together , results indicate that there is a significant effect of tnf dose that is driving gabaar trafficking to synapses on the plasma membrane and that this effect was widespread throughout the tissue . our present findings suggest that there is an increase in synaptic gabaar that occurs in spinal cord neurons in response to tnf. tnf nanoinjection in vivo allowed us to model rapid changes in synaptic efficacy that result from the inflammatory response following sci . biochemical fractionation methods that have previously been shown to be sufficient to measure rapid trafficking of the excitatory ampa receptors [ 13 , 16 ] did not appear to have sufficient resolution to detect changes in inhibitory gabaars . however , quantitative high - resolution confocal microscopy on morphologically preserved tissue sections revealed significant changes in gabaars 60 minutes after tnf injection . analysis of the total neuropil in confocal z - series revealed significant increases in gabaar positive puncta . by statistically correcting confocal images for total gabaar puncta , we were able to isolate the effect of tnf on synaptic gabaar trafficking , revealing a specific increase in synaptic gabaar levels in the neuropil . by applying algorithmic postprocessing of confocal images , we were able to generate image - based subcellular fractionation , isolating the plasma membrane of large ventral motor neurons . analysis of this confocal subcellular fraction revealed a u - shaped tnf dose - response effect on gabaar levels at both extrasynaptic and synaptic sites on the motor neuron plasma membrane . the present results contribute to a more complete understanding of tnf-mediated , dynamic receptor changes at synapses following the inflammatory response in sci . this increase in tnf has potential to contribute to excitotoxic cell death in the spinal cord . in vivo studies have shown that tnf causes glua2-lacking ampars to be trafficked to synapses of spinal neurons , thereby contributing to excitotoxicity by increasing neural permeability to ca . inhibitory synaptic strength is directly correlated to the number of synaptic gabaars , thus any mechanism that controls the quantity of synaptic gabaars can have a profound effect on neuronal excitability [ 13 ] . given this , our study suggests that gabaar trafficking to the synapse may serve as a homeostatic mechanism that combats the excitotoxic effect of tnf following sci in vivo . an increase in extrasynaptic gabaar as well as total gabaar is consistent with research showing that gabaar are first exocytosed onto the plasma membrane before trafficking laterally to synapses [ 7 , 19 ] . an upregulation of extrasynaptic gabaar may be necessary in order to ensure a sufficient availability of gabaars for trafficking to synaptic sites . increased gabaar synthesis may occur in this system to meet the demands placed on intracellular receptor stores imposed by trafficking . an unexpected finding was that extrasynaptic and total gabaars decreased following the middle dose of tnf. this phenomenon could be due to a compensatory homeostatic degradation of gabaar at the middle tnf dose . at a low dose , gabaar synthesis could increase to maintain cellular reserves of the receptor for trafficking to the membrane , yet an increase in gabaar at a low dose could be slight enough to go undetected by regulatory mechanisms that would otherwise decrease gabaar accumulation [ 15 , 17 ] . however , at the medium tnf dose , gabaar synthesis could increase to a level that would elicit a rapid homeostatic degradation or ubiquitination of these receptors so that there is a drop in total gabaar protein following the middle dose . a large and rapid gabaar accumulation following a high dose of tnf could conceivably overwhelm homeostatic compensatory degradation of gabaar resulting in a global increase in the protein . results also showed that at high doses of tnf , there was a widespread effect of synaptic and total gabaar protein that extended onto the contralateral side of the spinal cord into the region of albumin nanoinjection , as previously reported for ampar receptor trafficking effects . at a high dose , there was not a significant difference between gabaar measures on the tnf-injected side and the contralateral albumin side . yet , at lower doses , tnf does not influence gabaar measures at the site of albumin injection . this result could be explained by evidence that tnf promotes a heightened inflammatory response , which causes an increase in glial activation leading to the release of a variety of inflammatory cytokines including tnf [ 2022 ] . therefore , higher doses of tnf could elicit a greater immune response leading to further release of tnf through a positive feedback loop , which has been demonstrated in vitro [ 23 , 24 ] . additionally , at higher doses , tnf elicits a higher excitatory effect in the neural circuitry at the injection site that could be transmitted across the midline of the spinal cord by commissural fibers [ 25 , 26 ] . higher doses of tnf also are known to elicit a greater localized excitation which is propagated further relative to lower doses of tnf . our results suggest that gabaar trafficking may counteract this tnf-induced excitotoxicity , so increasing the radius of excitation would likewise increase the tissue area affected by gabaar trafficking . at first glance , taken together with our prior publications , we have now found in vivo that gabaars and ampars are both trafficked to the membrane after tnf nanoinjection ( figures 46 ) . although we found an increase in synaptic gabaar following the highest tnf dose , our experiment also showed that tnf elicited a nonlinear dose - response effect on gabaar levels ( total gabaar in neuropil and extrasynaptic gabaar ) . an interesting finding was that the middle dose of tnf used in our study elicited a lower amount of total gabaar relative to our highest and lowest drug doses ( figure 7 ) . this complex , nonlinear dose - dependent relationship of tnf on gabaar trafficking , actually replicates the finding of stellwagen et al . when examined in the context of the study by ferguson et al . , which demonstrated that , at 0.1 m tnf , the very same intermediate dose used in our experiment , there was an increase in extrasynaptic glua1 coinciding with our decrease in gabaar ( figure 7 ) . our experiment expands upon these in vitro findings , recontextualizing them in vivo and revealing a potentially nonlinear dependence of gabaar trafficking on tnf. an in vivo account of tnf-mediated gabaar trafficking simulates the microenvironmental tissue changes following injury . while cell culture enables greater control over experimental variables and outcomes , a limitation of in vitro studies is their inability to replicate the compensatory homeostatic mechanisms present in a whole organism . in analyzing the effect of a range of tnf doses on gabaar trafficking , we can glean time - emergent effects following injury since tnf is constitutively expressed following sci . thus , there appear to be both dose and time components to tnf receptor trafficking in vivo . further studies are necessary in order to corroborate our current findings and determine their functional consequences . it has been shown that mipsc strength is directly correlated with the number of postsynaptic gabaars indicating that trafficking receptors to the synapse greatly affects neuronal excitability . electrophysiological studies are critical in order to determine whether tnf-mediated gabaar trafficking translates into differences in synaptic current transmission . aside from gabaar trafficking , other modulatory mechanisms regulate receptor functionality and distribution including changes in gabaar phosphorylation , half - life , ubiquitination , lysosomal degradation , and interactions with other glutamate receptor subtypes such as the nmda receptor [ 6 , 7 , 17 , 27 , 28 ] . an additional physiological variable to consider is that , under certain conditions , gabaergic synapses can be excitatory . gabaars are permeable to both cl and hco3 and these currents have reversal potentials ( egaba ) close to the neuronal resting potential . physiological conditions that cause the membrane potential to become more negative than egaba reverse the direction of ion flow through gabaars . for example , a heightened level of neuronal activity has been shown to transiently alter egaba in hippocampal ca1 pyramidal cells in vitro . gabaergic synapses become depolarizing after a high - frequency train of stimulation causing an accumulation of cl inside the cell and high k outside , in the interstitial fluid [ 30 , 31 ] . in the spinal cord , excitatory effects of gabaars have been implicated in pathological pain conditions and maladaptive spinal plasticity [ 3234 ] . in light of these caveats , it is essential to determine the electrophysiological consequences of tnf-mediated gabaar trafficking . a better understanding of the link between tnf-induced synaptic gabaar trafficking and neural excitability is a clear target for further research that may lead to a novel therapeutic target for combating the spread of excitotoxicity after sci and other cns diseases .

the proinflammatory cytokine tnf contributes to cell death in central nervous system ( cns ) disorders by altering synaptic neurotransmission . tnf contributes to excitotoxicity by increasing glua2-lacking ampa receptor ( ampar ) trafficking to the neuronal plasma membrane . in vitro , increased ampar on the neuronal surface after tnf exposure is associated with a rapid internalization of gabaa receptors ( gabaars ) , suggesting complex timing and dose dependency of the cns 's response to tnf. however , the effect of tnf on gabaar trafficking in vivo remains unclear . we assessed the effect of tnf nanoinjection on rapid gabaar changes in rats ( n = 30 ) using subcellular fractionation , quantitative western blotting , and confocal microscopy . gabaar protein levels in membrane fractions of tnf and vehicle - treated subjects were not significantly different by western blot , yet high - resolution quantitative confocal imaging revealed that tnf induces gabaar trafficking to synapses in a dose - dependent manner by 60 min . tnf-mediated gabaar trafficking represents a novel target for cns excitotoxicity .

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

gamma amino butyric acid type a receptors ( gabaars ) are a major source of fast inhibitory synaptic transmission in the cns and thus play a crucial role in regulating neuronal networks . altering the number of gabaars in a synapse through rapid receptor trafficking trafficking of gabaars to and from the plasma membrane has been shown to depend on neuronal activity [ 47 ] . for example , insulin and pi3 kinase cause rapid insertion of gabaars into the plasma membrane increasing the amplitude of miniature inhibitory postsynaptic currents ( mipscs ) [ 8 , 9 ] . tumor necrosis factor alpha ( tnf ) , a proinflammatory cytokine that is constitutively expressed following spinal cord injury ( sci ) , has been shown to alter receptor trafficking of gabaars to and from the cell surface in vitro . in vivo , tnf levels are increased as part of the inflammatory response following sci and various other nervous system disorders , contributing to secondary cell death [ 1113 ] . tnf has been shown to specifically promote trafficking of glutamate - receptor-2-(glua2- ) lacking ampa receptors ( ampars ) to the plasma membrane of spinal neurons inducing excitotoxicity in vivo . blocking tnf action pharmacologically reduces ampar trafficking and cell death after sci , suggesting that modulation of ampars is a major mechanism by which tnf promotes cell death in cns disease . the aim of the current study is to assess in vivo the effect of tnf on gabaar trafficking to the plasma membrane of spinal neurons . we modeled the inflammatory response associated with sci by tnf nanoinjection into the spinal cord to establish whether gabaars are exocytosed onto or endocytosed from the plasma membrane . gabaars were identified and quantified based on the presence of the gamma-2 ( 2 ) subunit as studies show that this subunit is critical for postsynaptic clustering of gabaars and the vast majority of gabaars are composed of subunits and combined with 2 [ 14 , 15 ] . we used a combination of biochemical fractionation and laser scanning confocal microscopy followed by iterative deconvolution and automated image analysis to evaluate gabaar levels in the plasma membrane and at synapses after tnf nanoinjection into the spinal parenchyma . whole membrane fractionation and quantitative western blotting showed a trend towards increased membrane gabaar protein levels within 60 min of tnf nanoinjection delivery , but these results did not reach significance . confocal image findings also suggest that there is a nonlinear dose - dependent relationship between tnf and gabaar trafficking . female long - evans rats , 77- to 87-d - old , were housed in pairs ( n = 30 ) with ad libitum access to food and water . for imaging experiments , subjects ( n = 4 ) received a dose of tnf ( 0.01 , 0.1 , or 1 m ) and an injection of albumin on the contralateral ventral horn to serve as a within - subject control . these doses of tnf have been found sufficient to increase glutamate - mediated excitotoxicity in the spinal cord , but insufficient to cause cell death . the dye fluororuby ( invitrogen ) was included in each injection solution in order to localize injection sites for high - resolution confocal analyses ( figure 1 ) . one hour after nanoinjection , the spinal cord was extracted under deep anesthesia ; 7.5 mm of the cord was snap - frozen with dry ice and stored at 80c for later processing . the supernatant ( s2 ) was transferred to a new tube , and the membrane - enriched pellet ( p2 ) was resuspended in 50 l of pbs containing protease inhibitor . p2 fractions were vortexed and sonicated , and all sample fractions were stored at 80c . p2 fractions from each subject were diluted 1 : 2 with cold laemmli sample buffer containing 5% -mercaptoethanol ( biorad ) , and 20 g of protein per lane was immediately loaded onto a precast 10% tris - hcl polyacrylamide gel ( biorad ) . sample loading on the gel was performed so that each subject had their own lane , loading order was counterbalanced across injection condition to account for regional variability within the gel , and the experimenter was blind to subject condition . the membrane was blocked for 1 h in odyssey blocking buffer ( li - cor ) containing 0.1% tween 20 and then incubated overnight ( 18 h ) in the dark at 4c in a primary antibody solution containing odyssey blocking buffer , 0.05% tween 20 , and a rabbit polyclonal anti- gabaa receptor 2 primary antibody ( 1 : 500 ; chemicon , ab5559 ) . the membrane was washed 4 5 min with tbs containing 0.1% tween 20 ( ttbs ) then incubated for 1 h in a fluorescently labeled secondary antibody solution containing odyssey blocking buffer , 0.2% tween 20 , and 1 : 30,000 irdye 680 goat anti - rabbit secondary antibody ( li - cor ) and subsequently washed 4 5 min in ttbs , 1 5 min in tbs . the membrane was immediately scanned for protein bands using the corresponding 680 nm laser at a scanning intensity of 4 on the odyssey infrared imaging system ( li - cor ) . the membrane was washed and reincubated in a secondary antibody solution containing 1 : 30,000 irdye 800 goat anti - mouse secondary antibody ( li - cor ) . although traditional western blot analysis using chemiluminescence and densitometry measurements is considered to be merely semiquantitative , we used an established near - infrared labeling and detection technique ( odyssey infrared imaging system , li - cor ) to definitively quantify the intensity of fluorescently labeled protein bands . prior experiments have used the plasma membrane protein n - cadherin ( ncad ) to characterize the degree of membrane enrichment in each fraction generated by spinal cord subcellular fractionation [ 13 , 16 ] . in these studies , western blotting revealed that subcellular fractionation generates p2 samples with a modest enrichment of the plasma membrane as evidenced by the presence of ncad . we therefore used ncad as a control both for variability in protein loading and in plasma membrane enrichment through subcellular fractionation . two independent replications were performed , and the normalized densitometry results were averaged across runs . the 30 mm length of spinal cord centered on the injection site was isolated and postfixed overnight ( < 18 h ) in 4% paraformaldehyde followed by cryoprotection of the tissue in 30% sucrose for 2d . tissue was blocked and permeabilized with 5% normal goat serum and 0.3% triton x-100 for 1 h. sections were incubated in a solution consisting of mouse monoclonal antibody against presynaptic synaptophysin ( 1 : 200 ; millipore mab5258 - 50ug ) and rabbit polyclonal antibody against gabaa receptor 2 primary antibody ( 1 : 200 ; chemicon , ab5559 ) overnight at room temperature . large ventral motor neurons ( characterized by a diameter > 40 m ) were selected using wide - field fluorescence based on the distinctive synaptophysin outline surrounding the plasma membrane ( figure 1 ) . this sampling procedure was performed through horizontal sections of the spinal cord up to 600 m rostral and caudal to the center of each injection site . these settings allowed for virtually complete distinction between immunolabels gabaars ( alexa 488 ) , synaptophysin ( alexa 633 ) , and fluororuby ( texas red ) . images underwent automated image analysis to quantify the number of fluorescently labeled receptor puncta on the plasma membrane exceeding a predetermined pixel threshold based on control tissue . one macro was designed to measure the amount of total gabaar receptor pixels ( intra- and extracellular puncta ) as well as colocalization of synaptophysin and gabaar pixels ( synaptically localized gabaar puncta ) in each field of the z - stack . once a single plane was selected , a blinded researcher identified the plasma membrane of the motor neuron by tracing the synaptophysin - labeled outline of the cell . a 2 m - thick image - based subcellular fraction was produced containing the plasma membrane of the cell from the single plane . immunofluorescence data were analyzed using anova , and the experiment was a mixed design ( injection side and distance from injection site served as within - subject variables ) . total gabaar protein levels were accounted for by ancova , which allowed for the distinction between receptor trafficking versus an increase in total gabaar puncta as a result of receptor synthesis . quantitative western blotting was used to generate normalized gabaar intensity ratios ( gabaar : ncad ) for the membrane - enriched fraction of spinal cord homogenate ( figure 3(a ) ) . anova revealed no significant difference in total membrane gabaar between injection condition ( p = 0.315 ) , yet group means reflect a nonsignificant trend towards a higher concentration of gabaars in tnf-treated subjects ( figure 3(b ) ) . therefore , the high - resolution , low - throughput method of quantitative confocal microscopy was used to elucidate changes in localized receptor trafficking . automated image analysis of the 3-dimensional neuropil ( defined as the entire set of pixels in a confocal z - stack ) revealed a dose - dependent increase in synaptic gabaar ( colocalized gabaar and synaptophysin pixels ) 60 minutes following tnf nanoinjection ( p < 0.001 ) . total gabaar in the neuropil increased in a dose - dependent manner ( p < 0.001 ) as well ( figure 4 ) . studies have shown that gabaars can undergo rapid activity - dependent ubiquitination and lysosomal degradation to maintain homeostatic levels of the receptor [ 15 , 17 ] , which could explain the significant decrease in gabaar associated with the middle dose of tnf relative to the lowest dose . it is possible that the low dose of tnf elicited a modest increase in gabaar undetectable to regulatory systems within the neuron , whereas a middle dose of tnf sufficiently increased gabaars to the point where they would be downregulated . an ancova was used to statistically account for the tnf-mediated increase in total gabaar protein in the neuropil , thus isolating the dose - dependent changes in synaptic gabaar attributable to receptor trafficking . after correcting for changes in total gabaar protein , the dose - dependent effect of tnf on synaptic gabaars in the neuropil was robustly maintained ( p < 0.001 ) indicating that the dose - dependent increase in synaptic gabaar does not depend solely on wholesale protein changes but also relies on receptor trafficking to the synapse . overall , results reveal that tnf dose significantly influenced gabaar receptor trafficking to synaptic sites within the neuropil containing spinal motor neurons . image analysis of the plasma membrane also revealed a dose - dependent increase in synaptic and extrasynaptic gabaar 60 minutes after tnf injection ( figures 5(g ) and 5(h ) ) ( p < 0.001 ) . there was significantly more extrasynaptic gabaars associated with the lowest tnf dose relative to the middle dose ( p < 0.003 ) . after statistically correcting for tnf-mediated changes in total gabaar level with ancova , there remained a significant dose - dependent effect of tnf on synaptic gabaar indicating that tnf increases receptor trafficking to synapses on the motor neurons ' somatic surface in addition to increasing extrasynaptic receptors ( p < 0.05 ) . the effect of tnf was widespread and extended onto the contralateral side of the spinal cord ( the site of vehicle injection ) eliciting a dose - dependent increase in synaptic and total gabaars at the level of the plasma membrane and the neuropil , which spanned both sides of the cord ( all measures , p < 0.001 ) . the extensive effect of tnf on spinal tissue is also evidenced by the fact that there was no main effect of side of the cord in synaptic , nor total gabaar measures ( figure 6 ) ( all measures , p > 0.05 ) . there was an interaction of dose by side for extrasynaptic gabaar on the plasma membrane ( p = 0.037 ) , but there was not a significant interaction for any other gabaar measures ( p > 0.05 ) ( figure 6 ) . accounting for changes in total extrasynaptic gabaar in the plasma membrane with ancova showed no higher order interactions between dose and side for synaptic gabaar ( p > 0.05 ) . taken together , results indicate that there is a significant effect of tnf dose that is driving gabaar trafficking to synapses on the plasma membrane and that this effect was widespread throughout the tissue . our present findings suggest that there is an increase in synaptic gabaar that occurs in spinal cord neurons in response to tnf. tnf nanoinjection in vivo allowed us to model rapid changes in synaptic efficacy that result from the inflammatory response following sci . biochemical fractionation methods that have previously been shown to be sufficient to measure rapid trafficking of the excitatory ampa receptors [ 13 , 16 ] did not appear to have sufficient resolution to detect changes in inhibitory gabaars . however , quantitative high - resolution confocal microscopy on morphologically preserved tissue sections revealed significant changes in gabaars 60 minutes after tnf injection . by statistically correcting confocal images for total gabaar puncta , we were able to isolate the effect of tnf on synaptic gabaar trafficking , revealing a specific increase in synaptic gabaar levels in the neuropil . by applying algorithmic postprocessing of confocal images , we were able to generate image - based subcellular fractionation , isolating the plasma membrane of large ventral motor neurons . analysis of this confocal subcellular fraction revealed a u - shaped tnf dose - response effect on gabaar levels at both extrasynaptic and synaptic sites on the motor neuron plasma membrane . this increase in tnf has potential to contribute to excitotoxic cell death in the spinal cord . in vivo studies have shown that tnf causes glua2-lacking ampars to be trafficked to synapses of spinal neurons , thereby contributing to excitotoxicity by increasing neural permeability to ca . inhibitory synaptic strength is directly correlated to the number of synaptic gabaars , thus any mechanism that controls the quantity of synaptic gabaars can have a profound effect on neuronal excitability [ 13 ] . given this , our study suggests that gabaar trafficking to the synapse may serve as a homeostatic mechanism that combats the excitotoxic effect of tnf following sci in vivo . an increase in extrasynaptic gabaar as well as total gabaar is consistent with research showing that gabaar are first exocytosed onto the plasma membrane before trafficking laterally to synapses [ 7 , 19 ] . an upregulation of extrasynaptic gabaar may be necessary in order to ensure a sufficient availability of gabaars for trafficking to synaptic sites . at a low dose , gabaar synthesis could increase to maintain cellular reserves of the receptor for trafficking to the membrane , yet an increase in gabaar at a low dose could be slight enough to go undetected by regulatory mechanisms that would otherwise decrease gabaar accumulation [ 15 , 17 ] . however , at the medium tnf dose , gabaar synthesis could increase to a level that would elicit a rapid homeostatic degradation or ubiquitination of these receptors so that there is a drop in total gabaar protein following the middle dose . a large and rapid gabaar accumulation following a high dose of tnf could conceivably overwhelm homeostatic compensatory degradation of gabaar resulting in a global increase in the protein . results also showed that at high doses of tnf , there was a widespread effect of synaptic and total gabaar protein that extended onto the contralateral side of the spinal cord into the region of albumin nanoinjection , as previously reported for ampar receptor trafficking effects . at a high dose , there was not a significant difference between gabaar measures on the tnf-injected side and the contralateral albumin side . this result could be explained by evidence that tnf promotes a heightened inflammatory response , which causes an increase in glial activation leading to the release of a variety of inflammatory cytokines including tnf [ 2022 ] . therefore , higher doses of tnf could elicit a greater immune response leading to further release of tnf through a positive feedback loop , which has been demonstrated in vitro [ 23 , 24 ] . at first glance , taken together with our prior publications , we have now found in vivo that gabaars and ampars are both trafficked to the membrane after tnf nanoinjection ( figures 46 ) . although we found an increase in synaptic gabaar following the highest tnf dose , our experiment also showed that tnf elicited a nonlinear dose - response effect on gabaar levels ( total gabaar in neuropil and extrasynaptic gabaar ) . this complex , nonlinear dose - dependent relationship of tnf on gabaar trafficking , actually replicates the finding of stellwagen et al . , which demonstrated that , at 0.1 m tnf , the very same intermediate dose used in our experiment , there was an increase in extrasynaptic glua1 coinciding with our decrease in gabaar ( figure 7 ) . our experiment expands upon these in vitro findings , recontextualizing them in vivo and revealing a potentially nonlinear dependence of gabaar trafficking on tnf. an in vivo account of tnf-mediated gabaar trafficking simulates the microenvironmental tissue changes following injury . while cell culture enables greater control over experimental variables and outcomes , a limitation of in vitro studies is their inability to replicate the compensatory homeostatic mechanisms present in a whole organism . in analyzing the effect of a range of tnf doses on gabaar trafficking , we can glean time - emergent effects following injury since tnf is constitutively expressed following sci . thus , there appear to be both dose and time components to tnf receptor trafficking in vivo . electrophysiological studies are critical in order to determine whether tnf-mediated gabaar trafficking translates into differences in synaptic current transmission . aside from gabaar trafficking , other modulatory mechanisms regulate receptor functionality and distribution including changes in gabaar phosphorylation , half - life , ubiquitination , lysosomal degradation , and interactions with other glutamate receptor subtypes such as the nmda receptor [ 6 , 7 , 17 , 27 , 28 ] . gabaars are permeable to both cl and hco3 and these currents have reversal potentials ( egaba ) close to the neuronal resting potential . gabaergic synapses become depolarizing after a high - frequency train of stimulation causing an accumulation of cl inside the cell and high k outside , in the interstitial fluid [ 30 , 31 ] . in light of these caveats , it is essential to determine the electrophysiological consequences of tnf-mediated gabaar trafficking . a better understanding of the link between tnf-induced synaptic gabaar trafficking and neural excitability is a clear target for further research that may lead to a novel therapeutic target for combating the spread of excitotoxicity after sci and other cns diseases .

excessive sexual behaviors have been described and studied by clinicians and sexologists since the 19th century ( e.g. , von krafft - ebing , 1965 ) . at that time , excessive and uncontrolled sexual behaviors were often considered as satyriasis or don juanism in males and nymphomania in females . initial research took the form of observations and reports made by clinicians who described a wide range of persistent and socially deviant sexual behaviors as well as non - paraphilic excessive sexual disorders associated with functional impairment and psychological distress ( kafka , 2010 ) . since the early 2000s , both research and mass media have contributed to the popularization of the label sexual addict , generally used to describe someone involved in a variety of sexual behaviors that occur in excess and that significantly impact on everyday life in a negative way ( levine , 2010 ) . excessive sexual behaviors have been extensively studied in recent years , coinciding with the resurgence in these behaviors since the development and expansion of online sexual activities ( wry & billieux , 2016a ) . some of the structural characteristics of sexual online activities , including the low cost , easy access , and almost infinite variety of activities and content available , have been proposed to fuel their excessive use ( beyens & eggermont , 2014 ; cooper , scherer , boies , & gordon , 1999 ; rosenberg & kraus , 2014 ) . despite the many studies that have explored excessive sexual behaviors , little is known about socio - demographic , psychiatric , and psychosocial background of treatment - seeking self - identified sexually addicted individuals . the fact that few studies are available on clinical samples contributes to the lack of consensus among scholars regarding the conceptualization of the disorder . this absence of conceptual consensus , along with the massive variation in estimated prevalence rates and the lack of data regarding the course of the disorder ( karila et al . , 2014 ; wry & billieux , 2016a ) , contributed to the american psychiatric association s decision not to include hypersexuality as a potential new psychiatric condition in the 5th edition of the diagnostic and statistical manual of mental disorders ( american psychiatric association , 2013 ) . previous research referred to multiple definitions , and various terms have been used to describe the disorder , including tends to be the most commonly used term ( karila et al . , 2014 ) . indeed , excessive sexual behaviors are increasingly described as a behavioral ( i.e. , non - chemical ) addiction and aligned with psychiatric conditions such as gambling disorder or internet gaming disorder ( billieux , schimmenti , khazaal , maurage , & heeren , 2015 ) . this current trend finds its roots in the fact that symptoms constituting a hallmark of addictive behaviors have been consistently associated with excessive sexual behaviors ( carnes , 1991 , 2000 ; goodman , 1998 ; orzack & ross , 2000 ) . accordingly , sexual addiction has generally been defined as an uncontrolled and excessive involvement in sexual activities characterized by a persistent desire or unsuccessful efforts to stop , reduce , or control sexual behaviors , as well as by cognitive salience , mood regulation , withdrawal , and functional impairment ( carnes , 2000 ; corley & hook , 2012 ) . alternatively , sexual addiction - like symptoms have been proposed to reflect a dysfunctional emotion regulation strategy whereby sexual behaviors are displayed to regulate or relieve negative affect , to cope with another psychiatric disorder ( e.g. , depression and anxiety ) , or to face adverse life events ( carnes , 2000 ; cooper , delmonico , griffin - shelley , & mathy , 2004 ; ross , mnsson , & daneback , 2012 ) . various criteria have been proposed to diagnose sexual addiction ( see wry & billieux , 2016a for a review ) , the most recognized in the scientific literature being those formulated by carnes ( 1991 ) , goodman ( 1998 ) , and kafka ( 2010 , 2013 ) . these sets of criteria differ substantially , yet they all include the following three criteria : loss of control , excessive time dedicated to sexual behaviors , and functional impairment . although no consensus currently exists in the literature , we decided for clarity to systematically use the term sexual addiction throughout this paper to describe the condition whereby sexual behaviors are excessive and uncontrolled and are associated with the above - mentioned symptoms . most research conducted during the last decades explored the characteristics associated with sexual addiction , with a particular emphasis on socio - demographics and comorbidities . first , studies highlighted a prevalence of sexual addiction that is three to five times higher in men than in women ( ballester - arnal , castro - calvo , gil - llario , & gimnez - garca , 2014 ; carnes , 2000 ; kafka , 2010 ) . second , higher education was consistently associated with sexual addiction ( cooper et al . , 1999 ; daneback , cooper , & mnsson , 2005 ; ross et al . , 2012 ) . however , it is worth noting that these findings were obtained in samples of self - selected undergraduate students or individuals interested in scientific research , which raises doubts regarding their generalizability . regarding relationship status , sexual addiction equally concerns persons who are involved or not involved in a stable relationship ( cooper et al . , 1999 ; 2005 ) , yet recent data nuanced such a finding by emphasizing that men involved in a stable relationship tend to be recreational cybersex users , whereas single men tend to be addicted users ( ballester - arnal et al . , 2014 ) . third , elevated rates of comorbid psychiatric disorders were systematically shown in samples of sexual addicts . these comorbidities include mood disorders , anxiety disorders , attention deficit hyperactivity disorder ( adhd ; berberovic , 2013 ; garcia & thibaut , 2010 ; mick & hollander , 2006 ; philaretou , mahfouz , & allen , 2005 ; semaille , 2009 ) , and substance use disorders ( berberovic , 2013 ; carnes , 1991 ; kalichman & cain , 2004 ) . sexual addiction has also been linked to sexual abuse during childhood ( carnes , 1991 ; ferree , 2003 ; giugliano , 2006 ) and has been associated with a variety of unhealthy and risky behaviors , including risky sexual practices ( carroll et al . , 2008 ; hggstrm - nordin , hanson , & tyden , 2005 ; kalichman & cain , 2004 ) . most evidence about sexual addiction has , however , been obtained in convenience and self - selected samples , which questions the representativeness of these samples and limits the current knowledge regarding the characteristics of clinical samples . moreover , little information is available regarding the sexual behaviors ( e.g. , sexual preferences and their consequences , sexual dysfunctions ) displayed by treatment - seeking sexual addicts . the aim of this report was to describe the characteristics ( i.e. , socio - demographic background , sexual behaviors , and comorbid psychopathology ) of a cohort of patients self - identified as sexual addicts who enrolled in a behavioral addiction outpatient program . participants were recruited at the department of addictology and psychiatry of the nantes university hospital ( france ) . to meet the inclusion criteria for this study , each participant had to be ( a ) a treatment - seeking person , ( b ) a native or fluent french speaker , and ( c ) 16 years or older ( 16 years is the minimum legal age to be treated at the center where the study was conducted ) . moreover , to be included in the study , participants had to reach a defined threshold in the sex addiction screening test ( sast ; carnes , 1989 ) and/or to endorse the diagnostic criteria of a modified version of kafka s criteria ( kafka , 2010 ) as described below . between april 2011 and december 2014 , 90 treatment - seeking individuals were considered for potential inclusion in this study . of these , 14 did not meet the inclusion criteria for the study , three refused to participate , and one was not able to participate because of visual impairment . accordingly , 72 individuals ( i.e. , 80% of the patients having attended the outpatient behavioral addiction clinic regarding sexual problems ) were enrolled in the study . the mean age was 40.33 years ( sd : 10.93 ; range : 2076 ) . patients were predominantly males ( 94.4% ) involved in a stable relationship ( 63.9% ) , highly educated ( 55.6% held a university degree ) , and active workers ( 73.6% ) . data were obtained through a combination of structured interviewing conducted by psychologists of the outpatient behavioral addiction clinic and self - reports completed by the patients themselves . age , gender , relationship status , educational level , and professional status of the sample ( n = 72 ) in the absence of formal consensus in the literature , we decided to rely on the three most used and recognized diagnostic tools , which were translated into french for the purpose of this study . first , the sast ( carnes , 1989 ) was used , which consists of a 25-item scale with dichotomous answers ( examples of items : have you made efforts to quit a type of sexual activity and failed ? ; do you feel controlled by your sexual desire ? ) . a score of 13 or higher has been suggested to reflect sexual addiction . in this study , this cutoff was used to diagnose sexual addiction , whereas a score of > 10 was used as an inclusion criterion in order to avoid rejecting individuals with subthreshold symptoms who were nonetheless susceptible to experiencing subjective distress or functional impairment . the internal reliability ( cronbach s ) of the sast in the current sample is 0.64 . second , a modified version of kafka s criteria ( kafka , 2010 ) was used . kafka s original proposal formulated three criteria that must be endorsed , on the basis of dichotomous answers , for a diagnosis of hypersexuality . for endorsement of criterion a , three of the following five symptoms must be fulfilled : ( a ) sexual behaviors engender interference in daily life , ( b ) sexual behaviors are displayed to regulate negative affect , ( c ) sexual behaviors are displayed to cope with adverse life events , ( d ) there is a loss of control over sexual behaviors , and ( e ) continuous involvement occurs despite negative outcomes . for endorsement of criterion b , excessive sexual behaviors must imply subjective psychological distress or functional impairment . for endorsement of criterion c , the possibility that excessive sexual behaviors are caused by the effect of a psychoactive substance must be excluded . in this study , dichotomous scoring was replaced by dimensional scoring on a 5-point likert scale from 0 ( never ) to 4 ( almost always ) to allow more nuanced answering and to reduce the likelihood of false - negative answers , given that most questions addressed highly stigmatized behaviors . moreover , criterion b was divided into two questions ( one for subjective psychological distress and one for functional impairment ) , and criterion c was removed ( this criterion was addressed by a psychologist during structured interviewing ) . this modified version of kafka s proposal contains seven items , and the following inclusion criteria were used : endorsement of at least four items for criterion a and at least one item for criterion b , with a score of 3 ( often ) or 4 ( almost always ) . cronbach s of the modified kafka s criteria in the current sample is 0.64 . the scale contains 14 items , and for a diagnosis of sexual addiction , criteria a d must be endorsed : ( a ) failure to resist impulses to engage in sexual behaviors , ( b ) tension prior to sexual behaviors , ( c ) pleasure / relief during sexual behaviors , and ( d ) loss of control over sexual behaviors . for endorsement of criterion e , five of nine symptoms must be fulfilled : ( e1 ) preoccupation with sexual behaviors , ( e2 ) engaging in sexual behaviors for longer than intended , ( e3 ) repeated efforts to reduce or stop sexual behaviors , ( e4 ) excessive time spent on sexual behaviors , ( e5 ) engaging in sexual behaviors when expected to fulfill other life obligations , ( e6 ) giving up or reducing important activities , ( e7 ) continuous involvement despite negative outcomes , ( e8 ) tolerance , and ( e9 ) irritability if unable to engage in sexual behaviors . finally , for endorsement of criterion f , symptoms must be shown to have persisted for at least 1 month . the variables measured were as follows : ( a ) types and frequency of sexual behaviors and interests ; ( b ) sexual disorders , including paraphilic disorders , sexual dysfunctions , and sexual identity disorders ; ( c ) risky sexual behaviors ; and ( d ) conjugal problems . the reasons considered by the patients to promote the identified problematic sexual behaviors were also questioned , along with the negative outcomes associated with the addictive sexual behaviors . psychiatric comorbidities were assessed with the french version of the mini - international neuropsychiatric interview ( m.i.n.i . ; lecrubier et al . , the m.i.n.i . is a structured diagnostic interview that assesses the main psychiatric disorders according to axis 1 of the dsm - iv and the icd-10 . to assess adhd , we used the following two scales:1.the french version of the wender utah rating scale - child ( wurs - c ; romo et al . , 2010 ) , a 25-item test scored on a 5-point likert scale that evaluates the presence of adhd in childhood ( examples of items : as a child i was inattentive , daydreaming ; as a child i was moody , i had ups and downs ) . cronbach s of the wurs - c in the current sample is 0.92.2.the french version of the adult adhd self - report scale ( asrs-1.1 ; kessler et al . , 2005 ) , a 6-item test scored on a 5-point likert scale to measure adult adhd ( examples of items : how often do you have problems remembering appointments or obligations ? ; how often do you fidget or squirm with your hands or feet when you have to sit down for a long time ? ) . the french version of the wender utah rating scale - child ( wurs - c ; romo et al . , 2010 ) , a 25-item test scored on a 5-point likert scale that evaluates the presence of adhd in childhood ( examples of items : as a child i was inattentive , daydreaming ; as a child i was moody , i had ups and downs ) . cronbach s of the wurs - c in the current sample is 0.92 . the french version of the adult adhd self - report scale ( asrs-1.1 ; kessler et al . , 2005 ) , a 6-item test scored on a 5-point likert scale to measure adult adhd ( examples of items : how often do you have problems remembering appointments or obligations ? ; how often do you fidget or squirm with your hands or feet when you have to sit down for a long time ? ) . following current recommendations on the diagnosis of adhd ( rsler et al . , 2006 ) , its prevalence was established from positive scores on both assessments ( i.e. , both childhood and adulthood adhd must be endorsed ) . participants were recruited at the department of addictology and psychiatry of the nantes university hospital ( france ) . to meet the inclusion criteria for this study , each participant had to be ( a ) a treatment - seeking person , ( b ) a native or fluent french speaker , and ( c ) 16 years or older ( 16 years is the minimum legal age to be treated at the center where the study was conducted ) . moreover , to be included in the study , participants had to reach a defined threshold in the sex addiction screening test ( sast ; carnes , 1989 ) and/or to endorse the diagnostic criteria of a modified version of kafka s criteria ( kafka , 2010 ) as described below . between april 2011 and december 2014 , 90 treatment - seeking individuals were considered for potential inclusion in this study . of these , 14 did not meet the inclusion criteria for the study , three refused to participate , and one was not able to participate because of visual impairment . accordingly , 72 individuals ( i.e. , 80% of the patients having attended the outpatient behavioral addiction clinic regarding sexual problems ) were enrolled in the study . the mean age was 40.33 years ( sd : 10.93 ; range : 2076 ) . patients were predominantly males ( 94.4% ) involved in a stable relationship ( 63.9% ) , highly educated ( 55.6% held a university degree ) , and active workers ( 73.6% ) . data were obtained through a combination of structured interviewing conducted by psychologists of the outpatient behavioral addiction clinic and self - reports completed by the patients themselves . age , gender , relationship status , educational level , and professional status of the sample ( n = 72 ) in the absence of formal consensus in the literature , we decided to rely on the three most used and recognized diagnostic tools , which were translated into french for the purpose of this study . first , the sast ( carnes , 1989 ) was used , which consists of a 25-item scale with dichotomous answers ( examples of items : have you made efforts to quit a type of sexual activity and failed ? ; do you feel controlled by your sexual desire ? ) . a score of 13 or higher has been suggested to reflect sexual addiction . in this study , this cutoff was used to diagnose sexual addiction , whereas a score of > 10 was used as an inclusion criterion in order to avoid rejecting individuals with subthreshold symptoms who were nonetheless susceptible to experiencing subjective distress or functional impairment . the internal reliability ( cronbach s ) of the sast in the current sample is 0.64 . second , a modified version of kafka s criteria ( kafka , 2010 ) was used . kafka s original proposal formulated three criteria that must be endorsed , on the basis of dichotomous answers , for a diagnosis of hypersexuality . for endorsement of criterion a , three of the following five symptoms must be fulfilled : ( a ) sexual behaviors engender interference in daily life , ( b ) sexual behaviors are displayed to regulate negative affect , ( c ) sexual behaviors are displayed to cope with adverse life events , ( d ) there is a loss of control over sexual behaviors , and ( e ) continuous involvement occurs despite negative outcomes . for endorsement of criterion b , excessive sexual behaviors must imply subjective psychological distress or functional impairment . for endorsement of criterion c , the possibility that excessive sexual behaviors are caused by the effect of a psychoactive substance must be excluded . in this study , dichotomous scoring was replaced by dimensional scoring on a 5-point likert scale from 0 ( never ) to 4 ( almost always ) to allow more nuanced answering and to reduce the likelihood of false - negative answers , given that most questions addressed highly stigmatized behaviors . moreover , criterion b was divided into two questions ( one for subjective psychological distress and one for functional impairment ) , and criterion c was removed ( this criterion was addressed by a psychologist during structured interviewing ) . this modified version of kafka s proposal contains seven items , and the following inclusion criteria were used : endorsement of at least four items for criterion a and at least one item for criterion b , with a score of 3 ( often ) or 4 ( almost always ) . cronbach s of the modified kafka s criteria in the current sample is 0.64 . the scale contains 14 items , and for a diagnosis of sexual addiction , criteria a d must be endorsed : ( a ) failure to resist impulses to engage in sexual behaviors , ( b ) tension prior to sexual behaviors , ( c ) pleasure / relief during sexual behaviors , and ( d ) loss of control over sexual behaviors . for endorsement of criterion e , five of nine symptoms must be fulfilled : ( e1 ) preoccupation with sexual behaviors , ( e2 ) engaging in sexual behaviors for longer than intended , ( e3 ) repeated efforts to reduce or stop sexual behaviors , ( e4 ) excessive time spent on sexual behaviors , ( e5 ) engaging in sexual behaviors when expected to fulfill other life obligations , ( e6 ) giving up or reducing important activities , ( e7 ) continuous involvement despite negative outcomes , ( e8 ) tolerance , and ( e9 ) irritability if unable to engage in sexual behaviors . finally , for endorsement of criterion f , symptoms must be shown to have persisted for at least 1 month . the variables measured were as follows : ( a ) types and frequency of sexual behaviors and interests ; ( b ) sexual disorders , including paraphilic disorders , sexual dysfunctions , and sexual identity disorders ; ( c ) risky sexual behaviors ; and ( d ) conjugal problems . the reasons considered by the patients to promote the identified problematic sexual behaviors were also questioned , along with the negative outcomes associated with the addictive sexual behaviors . psychiatric comorbidities were assessed with the french version of the mini - international neuropsychiatric interview ( m.i.n.i . ; lecrubier et al . , is a structured diagnostic interview that assesses the main psychiatric disorders according to axis 1 of the dsm - iv and the icd-10 . to assess adhd , we used the following two scales:1.the french version of the wender utah rating scale - child ( wurs - c ; romo et al . , 2010 ) , a 25-item test scored on a 5-point likert scale that evaluates the presence of adhd in childhood ( examples of items : as a child i was inattentive , daydreaming ; as a child i was moody , i had ups and downs ) . cronbach s of the wurs - c in the current sample is 0.92.2.the french version of the adult adhd self - report scale ( asrs-1.1 ; kessler et al . , 2005 ) , a 6-item test scored on a 5-point likert scale to measure adult adhd ( examples of items : how often do you have problems remembering appointments or obligations ? ; how often do you fidget or squirm with your hands or feet when you have to sit down for a long time ? ) . the french version of the wender utah rating scale - child ( wurs - c ; romo et al . , 2010 ) , a 25-item test scored on a 5-point likert scale that evaluates the presence of adhd in childhood ( examples of items : as a child i was inattentive , daydreaming ; as a child i was moody , i had ups and downs ) . the french version of the adult adhd self - report scale ( asrs-1.1 ; kessler et al . , 2005 ) , a 6-item test scored on a 5-point likert scale to measure adult adhd ( examples of items : how often do you have problems remembering appointments or obligations ? ; how often do you fidget or squirm with your hands or feet when you have to sit down for a long time ? ) . following current recommendations on the diagnosis of adhd ( rsler et al . , 2006 ) , its prevalence was established from positive scores on both assessments ( i.e. , both childhood and adulthood adhd must be endorsed ) . in the absence of formal consensus in the literature , we decided to rely on the three most used and recognized diagnostic tools , which were translated into french for the purpose of this study . first , the sast ( carnes , 1989 ) was used , which consists of a 25-item scale with dichotomous answers ( examples of items : have you made efforts to quit a type of sexual activity and failed ? ; do you feel controlled by your sexual desire ? ) . a score of 13 or higher has been suggested to reflect sexual addiction . in this study , this cutoff was used to diagnose sexual addiction , whereas a score of > 10 was used as an inclusion criterion in order to avoid rejecting individuals with subthreshold symptoms who were nonetheless susceptible to experiencing subjective distress or functional impairment . the internal reliability ( cronbach s ) of the sast in the current sample is 0.64 . second , a modified version of kafka s criteria ( kafka , 2010 ) was used . kafka s original proposal formulated three criteria that must be endorsed , on the basis of dichotomous answers , for a diagnosis of hypersexuality . for endorsement of criterion a , three of the following five symptoms must be fulfilled : ( a ) sexual behaviors engender interference in daily life , ( b ) sexual behaviors are displayed to regulate negative affect , ( c ) sexual behaviors are displayed to cope with adverse life events , ( d ) there is a loss of control over sexual behaviors , and ( e ) continuous involvement occurs despite negative outcomes . for endorsement of criterion b , excessive sexual behaviors must imply subjective psychological distress or functional impairment . for endorsement of criterion c , the possibility that excessive sexual behaviors are caused by the effect of a psychoactive substance must be excluded . in this study , dichotomous scoring was replaced by dimensional scoring on a 5-point likert scale from 0 ( never ) to 4 ( almost always ) to allow more nuanced answering and to reduce the likelihood of false - negative answers , given that most questions addressed highly stigmatized behaviors . moreover , criterion b was divided into two questions ( one for subjective psychological distress and one for functional impairment ) , and criterion c was removed ( this criterion was addressed by a psychologist during structured interviewing ) . this modified version of kafka s proposal contains seven items , and the following inclusion criteria were used : endorsement of at least four items for criterion a and at least one item for criterion b , with a score of 3 ( often ) or 4 ( almost always ) . cronbach s of the modified kafka s criteria in the current sample is 0.64 . the scale contains 14 items , and for a diagnosis of sexual addiction , criteria a d must be endorsed : ( a ) failure to resist impulses to engage in sexual behaviors , ( b ) tension prior to sexual behaviors , ( c ) pleasure / relief during sexual behaviors , and ( d ) loss of control over sexual behaviors . for endorsement of criterion e , five of nine symptoms must be fulfilled : ( e1 ) preoccupation with sexual behaviors , ( e2 ) engaging in sexual behaviors for longer than intended , ( e3 ) repeated efforts to reduce or stop sexual behaviors , ( e4 ) excessive time spent on sexual behaviors , ( e5 ) engaging in sexual behaviors when expected to fulfill other life obligations , ( e6 ) giving up or reducing important activities , ( e7 ) continuous involvement despite negative outcomes , ( e8 ) tolerance , and ( e9 ) irritability if unable to engage in sexual behaviors . finally , for endorsement of criterion f , symptoms must be shown to have persisted for at least 1 month . the variables measured were as follows : ( a ) types and frequency of sexual behaviors and interests ; ( b ) sexual disorders , including paraphilic disorders , sexual dysfunctions , and sexual identity disorders ; ( c ) risky sexual behaviors ; and ( d ) conjugal problems . the reasons considered by the patients to promote the identified problematic sexual behaviors were also questioned , along with the negative outcomes associated with the addictive sexual behaviors . psychiatric comorbidities were assessed with the french version of the mini - international neuropsychiatric interview ( m.i.n.i . ; lecrubier et al . , 1997 ) . the m.i.n.i . is a structured diagnostic interview that assesses the main psychiatric disorders according to axis 1 of the dsm - iv and the icd-10 . to assess adhd , we used the following two scales:1.the french version of the wender utah rating scale - child ( wurs - c ; romo et al . , 2010 ) , a 25-item test scored on a 5-point likert scale that evaluates the presence of adhd in childhood ( examples of items : as a child i was inattentive , daydreaming ; as a child i was moody , i had ups and downs ) . cronbach s of the wurs - c in the current sample is 0.92.2.the french version of the adult adhd self - report scale ( asrs-1.1 ; kessler et al . , 2005 ) , a 6-item test scored on a 5-point likert scale to measure adult adhd ( examples of items : how often do you have problems remembering appointments or obligations ? ; how often do you fidget or squirm with your hands or feet when you have to sit down for a long time ? ) . cronbach s of the asrs-1.1 in the current sample is 0.72 . the french version of the wender utah rating scale - child ( wurs - c ; romo et al . , 2010 ) , a 25-item test scored on a 5-point likert scale that evaluates the presence of adhd in childhood ( examples of items : as a child i was inattentive , daydreaming ; as a child i was moody , i had ups and downs ) . the french version of the adult adhd self - report scale ( asrs-1.1 ; kessler et al . , 2005 ) , a 6-item test scored on a 5-point likert scale to measure adult adhd ( examples of items : how often do you have problems remembering appointments or obligations ? ; how often do you fidget or squirm with your hands or feet when you have to sit down for a long time ? ) . following current recommendations on the diagnosis of adhd ( rsler et al . , 2006 ) , its prevalence was established from positive scores on both assessments ( i.e. , both childhood and adulthood adhd must be endorsed ) . results showed that 95.8% of the patients were diagnosed with sexual addiction according to the sast , 56.9% according to goodman s criteria , and 52.8% according to the modified kafka s criteria . as reported in table 2 , the most ubiquitous sexual behaviors identified were masturbation ( 100% of the sample was concerned , among whom 31% considered this behavior as problematic ) , followed by watching pornography ( 90.1% of the sample was concerned , among whom 29.7% considered this behavior as problematic ) , then using cybersex ( 77.5% of the sample was concerned , among whom 43.6% considered this behavior as problematic ) , and being involved in sexual intercourse with multiple partners ( 70.4% of the sample was concerned , among whom 56% considered this behavior as problematic ) . an important proportion of patients ( 53.5% ) indicated that the internet is their favorite medium for sexual behaviors . a comprehensive description of the identified paraphilia and sexual dysfunctions is reported in table 2 . the most frequent sexual dysfunctions identified were erectile disorder ( 16.7% ) and premature ejaculation ( 12.5% ) . results also showed that 31.9% of the participants displayed risky sexual behaviors , 27.8% had couple disharmony , and 8.3% had a gender identity disorder . proportion of sexual behaviors , paraphilia , sexual dysfunctions , risky sexual behaviors , couple disharmony , and gender identity disorder note . the main reasons considered by the patients to promote addictive sexual disorders are reported in table 3 , the most prevalent being searching for pleasure or arousal . a comprehensive list of the negative outcomes resulting from sexual addiction is provided in table 4 . the most frequent negative outcomes reported were family life disruption ( in particular loss of confidence between partners ) and health disruption ( in particular experiences of depressive or anxious symptoms ) . factors promoting addictive sexual behaviors ( n = 72 ) sexual addiction consequences ( n = 72 ) psychiatric and addictive comorbidities are reported in table 5 . the most frequent psychiatric comorbidities identified in the sample were major depressive disorder ( 63.9% ) , followed by social phobia ( 41.7% ) and generalized anxiety disorder ( 33.3% ) . the most frequent addictive comorbidities were nicotine ( 38.9% ) and alcohol ( 27.8% ) dependencies . the patients generally reported these psychiatric and addictive comorbidities to be present before the onset of sexual addiction ( 79.2% ) . adhd = attention deficit hyperactivity disorder ; wurs - c = wender utah rating scale - child ; asrs-1.1 = adult adhd self - report scale ; na = not assessed . results showed that 95.8% of the patients were diagnosed with sexual addiction according to the sast , 56.9% according to goodman s criteria , and 52.8% according to the modified kafka s criteria . as reported in table 2 , the most ubiquitous sexual behaviors identified were masturbation ( 100% of the sample was concerned , among whom 31% considered this behavior as problematic ) , followed by watching pornography ( 90.1% of the sample was concerned , among whom 29.7% considered this behavior as problematic ) , then using cybersex ( 77.5% of the sample was concerned , among whom 43.6% considered this behavior as problematic ) , and being involved in sexual intercourse with multiple partners ( 70.4% of the sample was concerned , among whom 56% considered this behavior as problematic ) . an important proportion of patients ( 53.5% ) indicated that the internet is their favorite medium for sexual behaviors . a comprehensive description of the identified paraphilia and sexual dysfunctions is reported in table 2 . the most frequent sexual dysfunctions identified were erectile disorder ( 16.7% ) and premature ejaculation ( 12.5% ) . results also showed that 31.9% of the participants displayed risky sexual behaviors , 27.8% had couple disharmony , and 8.3% had a gender identity disorder . proportion of sexual behaviors , paraphilia , sexual dysfunctions , risky sexual behaviors , couple disharmony , and gender identity disorder note . the main reasons considered by the patients to promote addictive sexual disorders are reported in table 3 , the most prevalent being searching for pleasure or arousal . a comprehensive list of the negative outcomes resulting from sexual addiction is provided in table 4 . the most frequent negative outcomes reported were family life disruption ( in particular loss of confidence between partners ) and health disruption ( in particular experiences of depressive or anxious symptoms ) . factors promoting addictive sexual behaviors ( n = 72 ) sexual addiction consequences ( n = 72 ) the most frequent psychiatric comorbidities identified in the sample were major depressive disorder ( 63.9% ) , followed by social phobia ( 41.7% ) and generalized anxiety disorder ( 33.3% ) . the most frequent addictive comorbidities were nicotine ( 38.9% ) and alcohol ( 27.8% ) dependencies . the patients generally reported these psychiatric and addictive comorbidities to be present before the onset of sexual addiction ( 79.2% ) . adhd = attention deficit hyperactivity disorder ; wurs - c = wender utah rating scale - child ; asrs-1.1 = adult adhd self - report scale ; na = not assessed . the aim of this study was to describe the characteristics , sexual habits , and psychiatric comorbidities of a cohort of self - identified sexually addicted patients seeking treatment in a dedicated outpatient unit . on the whole , this study emphasized a strong heterogeneity within the patients , especially in terms of sexual behaviors displayed and psychiatric comorbidities . in contrast , when considering the socio - demographic characteristics , some similarities appeared with most of these patients being active male workers of about 40 years who were involved in a stable relationship . the socio - demographic profile of the patients included in this study is consistent with that reported in previous research conducted in clinical ( carnes , 2000 ) and non - clinical participants ( cooper et al . one striking result is the fact that in this sample of self - defined sexual addicts , the prevalence of sexual addiction based on criteria such as those of the modified kafka ( 2010 ) or goodman ( 1998 ) is relatively low ( about 50% ) . although this finding questions the validity of these types of diagnostic criteria , it also supports the view that excessive sexual behaviors are frequently the consequence of a coping strategy displayed to face psychological suffering or adverse life events ( cooper et al . , 2004 ; deleuze et al . , 2015 ; ross et al . , 2012 ) . this latter argument is also supported by the elevated psychiatric comorbidity rate ( about 90% ) found in the sample . in contrast , the self - reported sast identified a larger proportion of patients as presenting sexual addiction symptoms ( 95.8% ) . this may be due to the polythetic nature of the sast ( 13 positive answers are required on a questionnaire comprising 25 items ) , whereas the goodman and modified kafka have a monothetic nature ( all criteria must be endorsed to reach the diagnosis ) . another explanation for the higher scores on sast is that , in contrast to goodman s and kafka s criteria , the sast criteria include three symptoms that have been shown in a recent study ( grubbs , stauner , exline , pargament , & lindberg , 2015 ) to be a hallmark of online pornography overuse ( one of the most frequent types of sexual addiction ) : ( a ) compulsive and uncontrolled use , ( b ) continuous use despite negative outcomes , and ( c ) emotional distress . regarding sexual behaviors per se , this study emphasized that the patients were involved in a wide range of problematic and non - problematic sexual behaviors . the heterogeneity of these sexual behaviors is further confirmed by the fact they can either be solitary ( e.g. , compulsive masturbation ) or interpersonal ( e.g. , sex chat and sex with multiple partners ) , online or offline , involving risky sexual practices or not , and associated with sexual dysfunctions or not . it is worth noting that in the current sample , the prevalence of sexual dysfunctions is comparable to that typically found in the general population ( laumann , paik , & rosen , 1999 ) . moreover , for the majority of these patients , the internet was reported to be the preferred location for sexual behaviors . this latter finding is probably due to the specific structural characteristics of online sexual activities , more specifically , their convenience ( i.e. , free website and permanently accessible service ) and the fact that an infinite variety of sexual activities and content are available online ( beyens & eggermont , 2014 ; cooper et al . , 1999 ; riemersma & sytsma , 2013 ; rosenberg & kraus , 2014 ; ross et al . , 2012 ) . these characteristics are especially attractive to patients with paraphilia , who can easily find sexual materials on the internet that fit their sexual preferences ( ross et al . , 2012 ; wry & billieux , 2016b ) . indeed , an important part of the patients ( 60.6% ) included in this study presented paraphilia . voyeurism ( i.e. , the desire to spy on a non - consenting person who is naked , in the process of disrobing , or engaging in sexual activity ) and exhibitionism ( i.e. , the need to expose one s genitals to strangers in order to gain sexual satisfaction ) were the most frequent , which are consistent with the previous data obtained in the male psychiatric samples ( marsh et al . , 2010 ) . our results also matched those reported in the previous research that identified exhibitionism , pedophilia , and voyeurism as being the three most frequent paraphilia in men seeking help for paraphilic disorders ( kafka & hennen , 2003 ) . a potential explanation for the coexistence of sexual addiction and paraphilia is that they may share similar functions . some authors have indeed suggested that one of the main functions of sexual behaviors in individuals characterized by sexual addiction is to alleviate intolerable affect through the experience of pleasurable sensations associated with sexual behaviors ( carnes , 2000 ; coleman , 1995 ; goodman , 1993 , 1998 ) . similarly , some authors have proposed that the function of paraphilia might be to change childhood traumatic experiences ( e.g. , emotional and sexual abuse , neglect , and humiliation ) into pleasure and triumph - related experiences in adulthood ( money , 1986 ; person , 1999 ; stoller , 1975 ) . in other words , both addictive - like sexual behaviors and paraphilia could have a similar function related to the conversion of an aversive or intolerable emotional state into a more tolerable and pleasurable one ( kahr , 2007 ; krueger & kaplan , 2001 ) . such a hypothesis is also supported by the fact that a large proportion of the patients included in this study presented a psychiatric disorder prior to the development of sexual addiction . thus , our results sustain the view that for a large proportion of patients presenting a sexual addiction , the condition is better conceptualized as a dysfunctional coping strategy displayed to face psychological distress and aversive emotional states ( carnes , 2000 ; cooper et al . , 2004 ; ross et al . , 2012 ) . a nuance to our interpretation of the comorbidity - related results is provided by the finding that escape real life / cope ( i.e. , negative reinforcement ) was the second factor promoting addictive sexual behaviors after search for pleasure / arousal this latter result supports the importance of sexual gratification in the sexual addiction process and highlights the value of the conjoint role of positive ( i.e. , sexual gratification ) and negative reinforcement ( i.e. , reducing aversive emotions or states ) in excessive sexual activities ( laier & brand , 2014 ) first , despite its clinical relevance , the sample used is not necessarily representative in terms of generalizing our findings to other cultures or to non - treatment - seeking patients . second , some of the measures were self - reported questionnaires that presume respondents are aware of and willing to report their behaviors honestly . despite these limitations , this study is among the first to investigate the characteristics and psychiatric comorbidities of a large sample of self - identified sexual addicts . the aim of the study was to investigate the characteristics , habits , and psychiatric comorbidities in a sample of treatment - seeking self - identified sexual addicts . on the whole , our results further support the position that current conceptualizations of sexual addiction lack clinical validity and probably constitute an oversimplification of a heterogeneous and multi - determined phenomenon ( drew & firestone , 2008 ; wry & billieux , 2016a ) . this study also emphasized the elevated psychiatric comorbidity rate in patients who nonetheless consulted the treatment center regarding their sexual addiction . this further calls for paying attention to the fact that these conditions can often be conceptualized as the consequences of pre - existing psychiatric disorders ( deleuze et al . , 2015 ) . such findings have important clinical implications and support the relevance of tailored intervention by taking into account the complexity of this disorder and the multiple potential psychological and sexual factors involved in its etiology . gc - b , f - xp , jc , dl , and mg - b created and organized the study and also conducted the structured interviewing in the department of addictology and psychiatry of the nantes university hospital . aw , kv , and jb realized the data analysis and the redaction of this manuscript . aw and kv contributed equally to this work and are willing to share first authorship .

background and aimsresearch on sexual addiction flourished during the last decade , promoted by the development of an increased number of online sexual activities . despite the accumulation of studies , however , evidence collected in clinical samples of treatment - seeking people remains scarce . the aim of this study was to describe the characteristics ( socio - demographics , sexual habits , and comorbidities ) of self - identified sexual addicts.methodsthe sample was composed of 72 patients who consulted an outpatient treatment center regarding their sexual behaviors . data were collected through a combination of structured interviewing and self - report measures.resultsmost patients were males ( 94.4% ) aged 2076 years ( mean 40.3 10.9 ) . endorsement of sexual addiction diagnosis varied from 56.9% to 95.8% depending on the criteria used . the sexual behaviors reported to have the highest degree of functional impairment were having multiple sexual partners ( 56% ) , having unprotected sexual intercourse ( 51.9% ) , and using cybersex ( 43.6% ) . ninety percent of patients endorsed a comorbid psychiatric diagnosis , and 60.6% presented at least one paraphilia.conclusionsresults showed highly different profiles in terms of sexual preferences and behaviors , as well as comorbidities involved . these findings highlight the need to develop tailored psychotherapeutic interventions by taking into account the complexity and heterogeneity of the disorder .

Introduction Methods Participants and procedure Main outcome measures Diagnosis of sexual addiction Sexual behaviors Psychiatric disorders Ethics Results Diagnosis of sexual addiction Sexual behaviors Comorbid psychiatric and addictive disorders Discussion Conclusions Authors contribution Conflict of interest

initial research took the form of observations and reports made by clinicians who described a wide range of persistent and socially deviant sexual behaviors as well as non - paraphilic excessive sexual disorders associated with functional impairment and psychological distress ( kafka , 2010 ) . since the early 2000s , both research and mass media have contributed to the popularization of the label sexual addict , generally used to describe someone involved in a variety of sexual behaviors that occur in excess and that significantly impact on everyday life in a negative way ( levine , 2010 ) . excessive sexual behaviors have been extensively studied in recent years , coinciding with the resurgence in these behaviors since the development and expansion of online sexual activities ( wry & billieux , 2016a ) . some of the structural characteristics of sexual online activities , including the low cost , easy access , and almost infinite variety of activities and content available , have been proposed to fuel their excessive use ( beyens & eggermont , 2014 ; cooper , scherer , boies , & gordon , 1999 ; rosenberg & kraus , 2014 ) . despite the many studies that have explored excessive sexual behaviors , little is known about socio - demographic , psychiatric , and psychosocial background of treatment - seeking self - identified sexually addicted individuals . the fact that few studies are available on clinical samples contributes to the lack of consensus among scholars regarding the conceptualization of the disorder . previous research referred to multiple definitions , and various terms have been used to describe the disorder , including tends to be the most commonly used term ( karila et al . accordingly , sexual addiction has generally been defined as an uncontrolled and excessive involvement in sexual activities characterized by a persistent desire or unsuccessful efforts to stop , reduce , or control sexual behaviors , as well as by cognitive salience , mood regulation , withdrawal , and functional impairment ( carnes , 2000 ; corley & hook , 2012 ) . alternatively , sexual addiction - like symptoms have been proposed to reflect a dysfunctional emotion regulation strategy whereby sexual behaviors are displayed to regulate or relieve negative affect , to cope with another psychiatric disorder ( e.g. these sets of criteria differ substantially , yet they all include the following three criteria : loss of control , excessive time dedicated to sexual behaviors , and functional impairment . although no consensus currently exists in the literature , we decided for clarity to systematically use the term sexual addiction throughout this paper to describe the condition whereby sexual behaviors are excessive and uncontrolled and are associated with the above - mentioned symptoms . most research conducted during the last decades explored the characteristics associated with sexual addiction , with a particular emphasis on socio - demographics and comorbidities . however , it is worth noting that these findings were obtained in samples of self - selected undergraduate students or individuals interested in scientific research , which raises doubts regarding their generalizability . third , elevated rates of comorbid psychiatric disorders were systematically shown in samples of sexual addicts . most evidence about sexual addiction has , however , been obtained in convenience and self - selected samples , which questions the representativeness of these samples and limits the current knowledge regarding the characteristics of clinical samples . , sexual preferences and their consequences , sexual dysfunctions ) displayed by treatment - seeking sexual addicts . the aim of this report was to describe the characteristics ( i.e. , socio - demographic background , sexual behaviors , and comorbid psychopathology ) of a cohort of patients self - identified as sexual addicts who enrolled in a behavioral addiction outpatient program . to meet the inclusion criteria for this study , each participant had to be ( a ) a treatment - seeking person , ( b ) a native or fluent french speaker , and ( c ) 16 years or older ( 16 years is the minimum legal age to be treated at the center where the study was conducted ) . patients were predominantly males ( 94.4% ) involved in a stable relationship ( 63.9% ) , highly educated ( 55.6% held a university degree ) , and active workers ( 73.6% ) . data were obtained through a combination of structured interviewing conducted by psychologists of the outpatient behavioral addiction clinic and self - reports completed by the patients themselves . age , gender , relationship status , educational level , and professional status of the sample ( n = 72 ) in the absence of formal consensus in the literature , we decided to rely on the three most used and recognized diagnostic tools , which were translated into french for the purpose of this study . in this study , this cutoff was used to diagnose sexual addiction , whereas a score of > 10 was used as an inclusion criterion in order to avoid rejecting individuals with subthreshold symptoms who were nonetheless susceptible to experiencing subjective distress or functional impairment . for endorsement of criterion a , three of the following five symptoms must be fulfilled : ( a ) sexual behaviors engender interference in daily life , ( b ) sexual behaviors are displayed to regulate negative affect , ( c ) sexual behaviors are displayed to cope with adverse life events , ( d ) there is a loss of control over sexual behaviors , and ( e ) continuous involvement occurs despite negative outcomes . for endorsement of criterion c , the possibility that excessive sexual behaviors are caused by the effect of a psychoactive substance must be excluded . moreover , criterion b was divided into two questions ( one for subjective psychological distress and one for functional impairment ) , and criterion c was removed ( this criterion was addressed by a psychologist during structured interviewing ) . this modified version of kafka s proposal contains seven items , and the following inclusion criteria were used : endorsement of at least four items for criterion a and at least one item for criterion b , with a score of 3 ( often ) or 4 ( almost always ) . the scale contains 14 items , and for a diagnosis of sexual addiction , criteria a d must be endorsed : ( a ) failure to resist impulses to engage in sexual behaviors , ( b ) tension prior to sexual behaviors , ( c ) pleasure / relief during sexual behaviors , and ( d ) loss of control over sexual behaviors . for endorsement of criterion e , five of nine symptoms must be fulfilled : ( e1 ) preoccupation with sexual behaviors , ( e2 ) engaging in sexual behaviors for longer than intended , ( e3 ) repeated efforts to reduce or stop sexual behaviors , ( e4 ) excessive time spent on sexual behaviors , ( e5 ) engaging in sexual behaviors when expected to fulfill other life obligations , ( e6 ) giving up or reducing important activities , ( e7 ) continuous involvement despite negative outcomes , ( e8 ) tolerance , and ( e9 ) irritability if unable to engage in sexual behaviors . the variables measured were as follows : ( a ) types and frequency of sexual behaviors and interests ; ( b ) sexual disorders , including paraphilic disorders , sexual dysfunctions , and sexual identity disorders ; ( c ) risky sexual behaviors ; and ( d ) conjugal problems . cronbach s of the wurs - c in the current sample is 0.92.2.the french version of the adult adhd self - report scale ( asrs-1.1 ; kessler et al . the french version of the adult adhd self - report scale ( asrs-1.1 ; kessler et al . to meet the inclusion criteria for this study , each participant had to be ( a ) a treatment - seeking person , ( b ) a native or fluent french speaker , and ( c ) 16 years or older ( 16 years is the minimum legal age to be treated at the center where the study was conducted ) . patients were predominantly males ( 94.4% ) involved in a stable relationship ( 63.9% ) , highly educated ( 55.6% held a university degree ) , and active workers ( 73.6% ) . data were obtained through a combination of structured interviewing conducted by psychologists of the outpatient behavioral addiction clinic and self - reports completed by the patients themselves . age , gender , relationship status , educational level , and professional status of the sample ( n = 72 ) in the absence of formal consensus in the literature , we decided to rely on the three most used and recognized diagnostic tools , which were translated into french for the purpose of this study . in this study , this cutoff was used to diagnose sexual addiction , whereas a score of > 10 was used as an inclusion criterion in order to avoid rejecting individuals with subthreshold symptoms who were nonetheless susceptible to experiencing subjective distress or functional impairment . for endorsement of criterion a , three of the following five symptoms must be fulfilled : ( a ) sexual behaviors engender interference in daily life , ( b ) sexual behaviors are displayed to regulate negative affect , ( c ) sexual behaviors are displayed to cope with adverse life events , ( d ) there is a loss of control over sexual behaviors , and ( e ) continuous involvement occurs despite negative outcomes . for endorsement of criterion b , excessive sexual behaviors must imply subjective psychological distress or functional impairment . moreover , criterion b was divided into two questions ( one for subjective psychological distress and one for functional impairment ) , and criterion c was removed ( this criterion was addressed by a psychologist during structured interviewing ) . this modified version of kafka s proposal contains seven items , and the following inclusion criteria were used : endorsement of at least four items for criterion a and at least one item for criterion b , with a score of 3 ( often ) or 4 ( almost always ) . the scale contains 14 items , and for a diagnosis of sexual addiction , criteria a d must be endorsed : ( a ) failure to resist impulses to engage in sexual behaviors , ( b ) tension prior to sexual behaviors , ( c ) pleasure / relief during sexual behaviors , and ( d ) loss of control over sexual behaviors . for endorsement of criterion e , five of nine symptoms must be fulfilled : ( e1 ) preoccupation with sexual behaviors , ( e2 ) engaging in sexual behaviors for longer than intended , ( e3 ) repeated efforts to reduce or stop sexual behaviors , ( e4 ) excessive time spent on sexual behaviors , ( e5 ) engaging in sexual behaviors when expected to fulfill other life obligations , ( e6 ) giving up or reducing important activities , ( e7 ) continuous involvement despite negative outcomes , ( e8 ) tolerance , and ( e9 ) irritability if unable to engage in sexual behaviors . the variables measured were as follows : ( a ) types and frequency of sexual behaviors and interests ; ( b ) sexual disorders , including paraphilic disorders , sexual dysfunctions , and sexual identity disorders ; ( c ) risky sexual behaviors ; and ( d ) conjugal problems . the french version of the adult adhd self - report scale ( asrs-1.1 ; kessler et al . in this study , this cutoff was used to diagnose sexual addiction , whereas a score of > 10 was used as an inclusion criterion in order to avoid rejecting individuals with subthreshold symptoms who were nonetheless susceptible to experiencing subjective distress or functional impairment . for endorsement of criterion a , three of the following five symptoms must be fulfilled : ( a ) sexual behaviors engender interference in daily life , ( b ) sexual behaviors are displayed to regulate negative affect , ( c ) sexual behaviors are displayed to cope with adverse life events , ( d ) there is a loss of control over sexual behaviors , and ( e ) continuous involvement occurs despite negative outcomes . for endorsement of criterion b , excessive sexual behaviors must imply subjective psychological distress or functional impairment . for endorsement of criterion c , the possibility that excessive sexual behaviors are caused by the effect of a psychoactive substance must be excluded . moreover , criterion b was divided into two questions ( one for subjective psychological distress and one for functional impairment ) , and criterion c was removed ( this criterion was addressed by a psychologist during structured interviewing ) . this modified version of kafka s proposal contains seven items , and the following inclusion criteria were used : endorsement of at least four items for criterion a and at least one item for criterion b , with a score of 3 ( often ) or 4 ( almost always ) . the scale contains 14 items , and for a diagnosis of sexual addiction , criteria a d must be endorsed : ( a ) failure to resist impulses to engage in sexual behaviors , ( b ) tension prior to sexual behaviors , ( c ) pleasure / relief during sexual behaviors , and ( d ) loss of control over sexual behaviors . for endorsement of criterion e , five of nine symptoms must be fulfilled : ( e1 ) preoccupation with sexual behaviors , ( e2 ) engaging in sexual behaviors for longer than intended , ( e3 ) repeated efforts to reduce or stop sexual behaviors , ( e4 ) excessive time spent on sexual behaviors , ( e5 ) engaging in sexual behaviors when expected to fulfill other life obligations , ( e6 ) giving up or reducing important activities , ( e7 ) continuous involvement despite negative outcomes , ( e8 ) tolerance , and ( e9 ) irritability if unable to engage in sexual behaviors . finally , for endorsement of criterion f , symptoms must be shown to have persisted for at least 1 month . the variables measured were as follows : ( a ) types and frequency of sexual behaviors and interests ; ( b ) sexual disorders , including paraphilic disorders , sexual dysfunctions , and sexual identity disorders ; ( c ) risky sexual behaviors ; and ( d ) conjugal problems . cronbach s of the wurs - c in the current sample is 0.92.2.the french version of the adult adhd self - report scale ( asrs-1.1 ; kessler et al . the french version of the adult adhd self - report scale ( asrs-1.1 ; kessler et al . results showed that 95.8% of the patients were diagnosed with sexual addiction according to the sast , 56.9% according to goodman s criteria , and 52.8% according to the modified kafka s criteria . as reported in table 2 , the most ubiquitous sexual behaviors identified were masturbation ( 100% of the sample was concerned , among whom 31% considered this behavior as problematic ) , followed by watching pornography ( 90.1% of the sample was concerned , among whom 29.7% considered this behavior as problematic ) , then using cybersex ( 77.5% of the sample was concerned , among whom 43.6% considered this behavior as problematic ) , and being involved in sexual intercourse with multiple partners ( 70.4% of the sample was concerned , among whom 56% considered this behavior as problematic ) . results also showed that 31.9% of the participants displayed risky sexual behaviors , 27.8% had couple disharmony , and 8.3% had a gender identity disorder . proportion of sexual behaviors , paraphilia , sexual dysfunctions , risky sexual behaviors , couple disharmony , and gender identity disorder note . results showed that 95.8% of the patients were diagnosed with sexual addiction according to the sast , 56.9% according to goodman s criteria , and 52.8% according to the modified kafka s criteria . as reported in table 2 , the most ubiquitous sexual behaviors identified were masturbation ( 100% of the sample was concerned , among whom 31% considered this behavior as problematic ) , followed by watching pornography ( 90.1% of the sample was concerned , among whom 29.7% considered this behavior as problematic ) , then using cybersex ( 77.5% of the sample was concerned , among whom 43.6% considered this behavior as problematic ) , and being involved in sexual intercourse with multiple partners ( 70.4% of the sample was concerned , among whom 56% considered this behavior as problematic ) . results also showed that 31.9% of the participants displayed risky sexual behaviors , 27.8% had couple disharmony , and 8.3% had a gender identity disorder . proportion of sexual behaviors , paraphilia , sexual dysfunctions , risky sexual behaviors , couple disharmony , and gender identity disorder note . factors promoting addictive sexual behaviors ( n = 72 ) sexual addiction consequences ( n = 72 ) the most frequent psychiatric comorbidities identified in the sample were major depressive disorder ( 63.9% ) , followed by social phobia ( 41.7% ) and generalized anxiety disorder ( 33.3% ) . the aim of this study was to describe the characteristics , sexual habits , and psychiatric comorbidities of a cohort of self - identified sexually addicted patients seeking treatment in a dedicated outpatient unit . on the whole , this study emphasized a strong heterogeneity within the patients , especially in terms of sexual behaviors displayed and psychiatric comorbidities . the socio - demographic profile of the patients included in this study is consistent with that reported in previous research conducted in clinical ( carnes , 2000 ) and non - clinical participants ( cooper et al . one striking result is the fact that in this sample of self - defined sexual addicts , the prevalence of sexual addiction based on criteria such as those of the modified kafka ( 2010 ) or goodman ( 1998 ) is relatively low ( about 50% ) . in contrast , the self - reported sast identified a larger proportion of patients as presenting sexual addiction symptoms ( 95.8% ) . another explanation for the higher scores on sast is that , in contrast to goodman s and kafka s criteria , the sast criteria include three symptoms that have been shown in a recent study ( grubbs , stauner , exline , pargament , & lindberg , 2015 ) to be a hallmark of online pornography overuse ( one of the most frequent types of sexual addiction ) : ( a ) compulsive and uncontrolled use , ( b ) continuous use despite negative outcomes , and ( c ) emotional distress . regarding sexual behaviors per se , this study emphasized that the patients were involved in a wide range of problematic and non - problematic sexual behaviors . these characteristics are especially attractive to patients with paraphilia , who can easily find sexual materials on the internet that fit their sexual preferences ( ross et al . indeed , an important part of the patients ( 60.6% ) included in this study presented paraphilia . some authors have indeed suggested that one of the main functions of sexual behaviors in individuals characterized by sexual addiction is to alleviate intolerable affect through the experience of pleasurable sensations associated with sexual behaviors ( carnes , 2000 ; coleman , 1995 ; goodman , 1993 , 1998 ) . such a hypothesis is also supported by the fact that a large proportion of the patients included in this study presented a psychiatric disorder prior to the development of sexual addiction . , negative reinforcement ) was the second factor promoting addictive sexual behaviors after search for pleasure / arousal this latter result supports the importance of sexual gratification in the sexual addiction process and highlights the value of the conjoint role of positive ( i.e. , reducing aversive emotions or states ) in excessive sexual activities ( laier & brand , 2014 ) first , despite its clinical relevance , the sample used is not necessarily representative in terms of generalizing our findings to other cultures or to non - treatment - seeking patients . despite these limitations , this study is among the first to investigate the characteristics and psychiatric comorbidities of a large sample of self - identified sexual addicts . the aim of the study was to investigate the characteristics , habits , and psychiatric comorbidities in a sample of treatment - seeking self - identified sexual addicts . on the whole , our results further support the position that current conceptualizations of sexual addiction lack clinical validity and probably constitute an oversimplification of a heterogeneous and multi - determined phenomenon ( drew & firestone , 2008 ; wry & billieux , 2016a ) . this study also emphasized the elevated psychiatric comorbidity rate in patients who nonetheless consulted the treatment center regarding their sexual addiction . such findings have important clinical implications and support the relevance of tailored intervention by taking into account the complexity of this disorder and the multiple potential psychological and sexual factors involved in its etiology . gc - b , f - xp , jc , dl , and mg - b created and organized the study and also conducted the structured interviewing in the department of addictology and psychiatry of the nantes university hospital .

the term osteoimmunology and the study of osteoimmunology have developed due to the close interrelationship between the immune system and bone metabolism . this is evident in immune - mediated diseases , such as rheumatoid arthritis and periodontal disease ( periodontitis ) , where there are local bone erosion and inflammation as reviewed in detail in multiple publications [ 24 ] . similarities in the mechanisms of bone loss in disease are likely related to the inflammatory cytokines expressed in a number of bone loss diseases . these cytokines are known to upregulate osteoclast activity via increased expression levels of receptor activator nf kappa b ligand ( rankl ) relative to osteoprotegerin ( opg ) ( as explored below ) and increase localized bone loss in diseases such as ra , periodontal disease , and periprosthetic osteolysis [ 510 ] . this review highlights the role of immune - related cells and factors in modulating bone loss , particularly in these diseases . while the importance of the rankl - rank - opg axis has been appreciated for nearly two decades [ 1113 ] , more recent studies have highlighted the importance of factors associated with immunoreceptor tyrosine - based activation motif ( itam ) signalling . this review will briefly discuss the rankl - rank - opg axis but its major focus will be on the role of itam - associated factors , the more recently investigated pathway , and how it relates to inflammatory bone loss diseases , in particular osteoclast - associated receptor ( oscar ) . rheumatoid arthritis ( ra ) affects 1 - 2% of the population and involves an autoimmune reaction with an autoantibody response to citrullinated proteins ( and others such as rheumatoid factor and collagen type ii ) . ra is characterized by synovitis involving angiogenesis , synovial proliferation , increased infiltration , survival , and decreased apoptosis of inflammatory cells . further to this there is an increase in osteoclast number and activity leading to focal bone erosions , juxta - articular osteopenia , and joint destruction [ 1720 ] . animal models suggest that there may also be suppression of localised osteoblast formation of bone . periodontitis is a chronic inflammatory disease of the gingival tissues , with an associated loss of the supporting structures including the periodontal ligament and alveolar bone . the aetiology involves an inflammatory response to bacterial infection such as p. gingivalis and possibly an autoimmune reaction , as reviewed . periodontitis is the most common and widespread bone loss pathology in humans with 64% of the us population aged 65 years and older reported as having moderate or severe periodontitis . despite the prevalence of this disease inevitably , in the absence of effective treatment , support structures ( periodontium ) are compromised and the affected teeth will loosen and fall out . ra and periodontitis have a similar pathophysiology , characterized by destructive inflammation that culminates in localized bone loss . the citrullination of proteins by p. gingivalis and the subsequent generation of autoantigens that drive autoimmunity in ra have been proposed as a possible mechanism linking these two diseases . similarities in ra and periodontitis may relate to citrullinated enolase as the specific antigen involved as well as cross - reaction between the antibodies directed towards the immunodominant epitope of human citrullinated alpha - enolase and a conserved sequence on citrullinated p. gingivalis enolase . new evidence suggests a relationship between the extent and severity of chronic periodontitis and ra [ 24 , 25 ] . individuals with advanced ra are more likely to experience more significant periodontal problems compared to their non - ra counterparts and vice versa . this is supported by findings in a study using a combined animal model of ra and periodontitis , which demonstrated more severe development of arthritis in mice with periodontitis . further to this , mice in which periodontitis alone was induced had evidence of radiocarpal bone loss in the absence of arthritic disease . this suggests presence of either ra or periodontitis places the individual at risk of developing the other disease . both conditions involve an imbalance between proinflammatory and anti - inflammatory cytokines and increased bone - resorbing activity . cantley et al . ( 2011 ) thus proposed that these two diseases are related through a common underlying dysfunction of fundamental inflammatory mechanisms . new treatment strategies are needed for both diseases that target the inhibition of proinflammatory cytokines , destructive enzymes , and bone - resorbing activity . the clinical implications of the current research strongly suggest that patients with ra should be carefully screened for their periodontal status , as reviewed . joint replacement surgery is used as a last resort in osteoarthritis ( oa ) and ra patients and is a relatively successful operation ; however , a large proportion of implants fail within 1020 years as a result of bone loss and implant loosening [ 4 , 10 , 28 ] . the pathogenesis behind prosthetic implant failure involves wear of prosthetic alloys , such as polyethylene ( pe ) , cobalt chrome , and titanium liberated from the implant surface [ 2931 ] . these particles stimulate a chronic inflammatory response , which increases bone - resorbing activity of the osteoclasts and suppresses bone formation by the osteoblast [ 33 , 34 ] resulting in bone loss . periprosthetic tissues contain granulomatous lesions dominated by inflammatory cells , particularly macrophages , and foreign - body giant cells [ 2931 , 35 , 36 ] . it is believed that an inflammatory reaction is initiated within the tissues in an attempt at particle clearance . this granulomatous lesion in periprosthetic osteolysis often leads to the formation of pseudosynovium - like structure , in which cells are organized into lining layer , in the membranous tissues adjacent to the failed implant surface . juxtaposed to this pseudosynovium are fibrous and collagenous regions , possibly scar tissues , which could be indicative of late stage periprosthetic osteolysis . the plethora of factors release in this inflammatory reaction within the tissues contributes towards the promotion of osteoclast formation [ 9 , 37 ] . higher numbers of t lymphocytes have been observed in the periprosthetic tissues of human and mouse models containing polyethylene and metal particles compared with normal tissues [ 3840 ] and osteoarthritic tissues . ( 1998 ) proposed that t cells are indirectly affected by the inflammatory cascade induced by wear particles . it is however important to note that t cells make up less than 10% of total cell population in periprosthetic tissues . given the low levels of t lymphocytes , the general belief held is that lymphocyte infiltrates are not normally associated with wear - particle induced periprosthetic osteolysis , in particular , in the granulomatous region [ 29 , 32 , 42 , 43 ] . the role of t cells may however be more pertinent in ra patients with implants . the prevalence of foreign - body giant cells in response to implant - derived wear debris in ra patients and non - ra patients does not differ but appears to be linked to the amount of polyethylene wear debris . as would be expected , this study reported a high prevalence of plasma cells in lymphocytic infiltrates in untreated ra patients compared with non - ra patients with a different distribution . whether implant wear is inducing a different reaction in ra versus non - ra patients may potentially have implications for combination and immune targeted treatment of inflammation and osteolysis in these patients . receptor activator nf kappa b ligand- ( rankl- ) rank signalling has several important roles in the immune system and bone [ 13 , 4446 ] . physiologically , rankl is required for normal development of lymph nodes , as evident in knockout mice . in bone , rankl interaction with its receptor , rank , expressed by the osteoclast , induces the transcription factor nuclear factor of activated t cells-1 ( nfatc1 ) [ 4749 ] . nfatc1 is an essential factor for differentiation , multi - nucleation and activation [ 4749 ] . nfatc1 binds directly to and regulates osteoclast differentiation genes such as tartrate resistant acid phosphatase ( trap ) , cathepsin k ( cath k ) , osteoclast - associated receptor ( oscar ) , 3-integrin [ 53 , 54 ] , and calcitonin receptor ( ctr ) . nfatc1 is also involved in autoregulation of itself , further enhancing gene expression and osteoclast differentiation . rank - rankl interaction is inhibited by the decoy receptor opg and thus the ratio of rankl to opg has a crucial influence on bone resorption . interactions between rank expressing cells of the lamina propria and t cells expressing rankl also play a role in intestinal inflammation . in the vasculature , further to this , in an inflammatory arthritis model reminiscent of ra , activated t cells exacerbate joint destruction via rankl upregulation . the rank - rankl - opg axis is known to regulate not only normal bone physiology but also alterations in rank - rankl - opg interactions that play a role in bone disease . uncoupling in the balance between the level and activity of these molecules culminates in osteoporotic or osteopetrotic phenotypes due to an increase or decrease in osteoclast formation and activity . this is particularly evident in focal bone loss associated with chronic inflammatory diseases such as rheumatoid arthritis , periodontal disease , and periprosthetic bone loss . in active ra , periodontal disease , and prosthetic loosening , elevated levels of rankl relative to opg are observed in the synovial - like soft tissue , gingival tissue , and soft tissues adjacent to sites of osteolysis [ 510 ] . further to this the elevated ratio of rankl : opg expression is associated with increased differentiation and activity of the bone - resorbing osteoclasts [ 5 , 79 , 32 , 60 ] , suggesting rankl : opg ratio as an important indicator for bone erosion . as rankl and opg are key molecules regulating bone loss in diseases , therapeutic interventions targeting these molecules and their signaling cascades are being investigated to treat a wide range of diseases . osteoclasts are the prominent cell eroding bone in inflammatory arthritis . a seminal paper in the field of osteoimmunology used a rankl knockout background to demonstrate that animals developed an osteopetrotic phenotype and a reduction in bone erosion , characterized by the absence of osteoclasts , whilst inflammation did not differ between wild - type and rankl knockout mice . in contrast , cartilage erosion was present in both control littermate and rankl knockout mice , suggesting that the rank - rankl - opg axis does not directly affect cartilage metabolism . in human studies , the levels of soluble rankl have been found to be higher than opg in synovial fluids from patients with ra compared with osteoarthritis ( oa ) patients suggesting a role in increased bone resorption . in support of this , a more recent large center study has reported the ratio of rankl : opg and markers of bone and cartilage degradation ( such as collagen terminal 1 ( ctx-1 ) ) to be predictive of progression of radiological bone damage in ra . in synovial tissues from patients with active ra , rankl expression is predominantly located in sublining regions [ 62 , 63 ] concentrated at focal sites of osteoclastic bone erosion in the pannus- bone interface . in contrast , opg has been described as being in regions of synovium some distance from the sites of bone erosion in ra . we reported opg is associated with endothelial cells and macrophages in the synovial lining layer of oa and normal tissues whilst absent in patients with active ra . rankl expressing cells have been detected in a subset of fibroblast - like synoviocytes and infiltrating mononuclear cells . further to this , activated synoviocytes from ra tissue express rankl and have decreased opg and are capable of supporting osteoclastogenesis in vitro . rankl expressing cells are also seen within areas of lymphocyte infiltration and dual immunostaining by ourselves , and others have shown that many of the rankl - positive cells are a subset of cd3 + and cd4 + cells [ 6 , 62 , 63 ] . activated t cells from ra patients have increased rankl are able to induce osteoclast formation in vitro . this study also reported a higher ratio of soluble rankl relative to opg suggesting t cells as a source of soluble rankl in ra . nfatc1 is a transcription factor crucial to rankl - rank signaling in the osteoclasts and is initially identified as being expressed by t cells and involved in regulation of cytokine transcription . many of the nfatc1-immunostained mononuclear cells observed were single nucleated and thus could be either precursor cells of osteoclasts such as macrophages , or lymphocytes . those with lymphocyte morphology are most likely activated t cells as most of them demonstrated nfatc1 positive staining localized mainly in nucleus . these cells may promote osteoclastogenesis through the rank / rankl pathway because as already mentioned , as activated t - cells demonstrate elevated expression of membrane - bound rankl with the ability to support osteoclastogenesis in vitro . the relative ratio of rankl to opg is also a significant indicator in bone loss associated with periodontal disease [ 5 , 69 ] . soluble rankl is significantly higher in gingival crevicular fluid ( gcf ) of periodontitis patients than in healthy gcf , while opg is not . similar to ra , b and t lymphocytes express rankl in gingival tissues associated with periodontitis [ 5 , 69 ] with expression of more than 50 and 90% of t cells and b cells , respectively . consistent with a role in osteoclast regulation , lymphocytes isolated from gingival tissues of patients induced differentiation of mature osteoclast cells in a rankl - dependent manner in vitro . these results suggest that activated t and b cells can be the cellular source of rankl and an inducer of bone resorption in periodontal disease . in a crude mrna analysis of tissue from dental patients , those with periodontitis exhibited significantly higher nfatc1 gene expression , compared with healthy subjects . interestingly , nfatc1 and rankl expression levels strongly correlated with each other and with clinical periodontal parameters . aseptic bone loss adjacent to orthopedic joint implants is a common cause of joint implant failure in humans . rank , rankl , and tumour necrosis factor ( tnf- ) are key modulators of bone turnover and their expression has been reported by ourselves and others in the tissues near periprosthetic osteolysis in patients undergoing revision of total hip prostheses [ 9 , 71 , 72 ] . these factors were strongly expressed by large multinucleated cells containing polyethylene wear debris in revision tissues . more interestingly a strong statistical correlation was found between volume of bone loss , polyethylene wear debris , and rank , rankl , and tnf- expression . ( 2000 ) where immunohistochemical detection of tnf- positively correlated with radiographic scores of osteolysis . in periprosthetic osteolysis , elevated levels of rankl , relative to its competitor opg , are associated with increased differentiation and activity of the bone - resorbing osteoclasts [ 8 , 32 , 73 ] . an earlier study had shown that cells isolated from periprosthetic tissues containing wear particles expressed mrna encoding for the proosteoclastogenic molecules , rankl , its receptor rank , monocyte colony - stimulating factor ( m - csf ) , interleukin- ( il- ) 1 beta , tnf- , il-6 , and soluble il-6 receptor , as well as opg . other studies showed that osteoclasts formed from cells isolated from periprosthetic tissues in the presence and absence of human osteoblastic cells in vitro [ 8 , 74 ] . when osteoclasts formed in the absence of osteoblastic cells , markedly higher levels of rankl mrna relative to opg mrna were expressed . particles of prosthetic materials also stimulated human monocytes to express both osteoclast - associated genes and osteoclast mediating factors in vitro . these findings suggest that ingestion of prosthetic wear particles by macrophages results in expression of osteoclast - differentiating molecules and stimulation of macrophage differentiation into osteoclasts . subsequent immunohistochemical studies demonstrate significantly higher levels of rankl in the periprosthetic tissues of patients with implant failure than in similar tissues from osteoarthritic and healthy subjects . in contrast , opg protein levels were similar in all tissues with the net result of higher rankl : opg ratio . of interest , rankl protein and mrna were predominantly associated with macrophage cells containing wear particles in the periprosthetic tissues . these findings support the contention that high levels of rankl in periprosthetic tissues of patients with prosthetic loosening may significantly contribute to aseptic implant loosening . we observed both mrna and protein expressions of nfatc1 to be higher in periprosthetic osteolysis than in oa tissues although levels did not reach significance . this may provide an explanation why lower than expected nfatc1 protein and mrna levels are found in periprosthetic osteolytic tissues . the itam pathway regulates proliferation , survival , and differentiation of effector immune cells and provides osteoclasts with costimulatory signals [ 14 , 7680 ] . in preosteoclasts and osteoclasts , innate immune receptors , trem2 and oscar , associate with the itam adaptor proteins dap12 and fc receptor gamma - chain ( fcr ) , respectively [ 80 , 81 ] . dap12 and trem2 are required for differentiation into multinucleated , bone - resorbing osteoclasts in vitro via phosphorylation of the syk tyrosine kinase . oscar on the cell surface mediates signal transduction via fcr [ 82 , 83 ] ( figure 1 ) . the induction of intracellular calcium via this pathway is required in conjunction with rankl - rank interaction for nfatc1 induction . in the context of human pathology studies in diseased tissues , particularly in nasu - hakola disease [ 8587 ] and very recently in alzheimer 's disease , have shown that these molecules may be involved . mutations in trem2 or dap12 have been associated with bone pathologies such as bone cysts and increased fractures ( in addition to presenile dementia ) in nasu - hakola disease [ 87 , 89 ] . these studies support a role for dap12 and a relationship between the skeletal and psychotic characteristics observed in nasu - hakola disease and for schizophrenia and presenile dementia . in trem2 . demonstrated that function mutations in dap12 and trem2 result in an inefficient and delayed differentiation of osteoclasts in vitro . in postmenopausal osteoporosis a rare allele ( g allele ) of oscar-2322a&gt ; g ( snp in the 5 flanking region ) has been associated with lower bone mineral density . although animal models are not the focus of this review it is interesting to note the phenotypes of itam related molecules in single and combination knockouts . trem mice have an osteopenic phenotype similar to nasu - hakola disease . in vitro , lack of trem2 impairs proliferation osteoclast precursors and affects the rate of osteoclastogenesis by accelerating differentiation into mature osteoclasts suggesting different effects of knocking out trem2 in vivo and in vitro . in dap12 deficient mice ( / ) there are an increased bone mass ( osteopetrosis ) and impeded development of osteoclasts . mice that are double knockout for the adaptors dap12 and fcr are severely osteopetrotic and bone marrow derived osteoclast precursors from these mice are unable to differentiate into mature osteoclasts in a rankl- and m - csf - mediated culture system [ 79 , 82 ] . although there is some redundancy , findings from these studies suggest that dap12 is the predominant factor responsible for optimal osteoclast differentiation . oscar is an igg - like receptor expressed by monocytes , macrophages monocyte - derived dendritic cells in humans , and is involved in antigen presentation as well as survival , maturation , and activation of dendritic cells [ 14 , 76 , 77 , 83 , 94 , 95 ] . ligation of human oscar on monocytes and neutrophils results in the induction of a proinflammatory cascade and the initiation of downstream immune responses . importantly , cell bound oscar on osteoclast precursors is an essential costimulatory factor in osteoclast formation but does not bypass the requirement of rankl . rankl - rank induction of nfatc1 expression precedes that of oscar and is crucial for induction of oscar gene expression . in addition , ligand - activated oscar interacts with fcr to produce an increase in intracellular calcium that augments nfatc1 expression . this establishes a positive feedback loop that results in marked elevation of both oscar and nfatc1 expressions in terminal stages of osteoclast formation [ 52 , 96 ] . these findings demonstrate a significant role for oscar in immune modulation as well as osteoclastogenesis . in vitro studies demonstrate that addition of oscar - fc to osteoblast - osteoclast cocultures results in the inhibition of osteoclast differentiation with kim et al . ( 2002 ) suggesting that this was due to oscar - fc blocking an osteoblast derived oscar ligand binding to oscar . this may be in addition to the recent identification of the motifs within fibrillar collagens in the extracellular matrix ( ecm ) as oscar ligands . the importance of oscar is further highlighted by the fact that soluble oscar ( s)oscar , in the form of oscar - fc , has also been shown to inhibit osteoclast differentiation from pbmcs in the presence of rankl , m - csf , and tgf- . costimulatory immune pathways may further increase osteoclast differentiation and activity [ 81 , 82 ] , particularly in chronic inflammatory diseases with an immune component such as in ra , periprosthetic osteolysis , and periodontal disease . in fact , our studies [ 67 , 75 ] and those of others suggest that deregulation of itam - associated molecules contributes to the pathogenesis and severity of rheumatoid arthritis , periodontal disease , periprosthetic osteolysis , and osteoporosis [ 10 , 67 , 75 , 92 , 94 , 98 , 99 ] . we , and others , have demonstrated increased levels of itam - related factors , including trem2 , dap12 , oscar , and fcr in human periprosthetic tissues adjacent to sites of osteolysis and in ra synovial tissues [ 67 , 94 ] . additionally , we have observed itam - related factors expressed in periodontitis tissue adjacent to bone loss ( unpublished observations ) . of these factors , soluble and membrane - bound oscar have been more extensively assessed in the context of ra and vascular disease ( expanded on below ) . we observed markedly higher levels of trem2 , dap12 , oscar , and fcr in active ra patients compared to synovial tissues from inactive ra , oa , or control healthy joint . multiple cell types expressed trem2 including mononuclear cells in lymphoid aggregates and fibroblasts . in oa tissues , trem2 immunostaining was noticed in monocyte / macrophage - like cells mainly around perivascular areas and on blood vessels ( unpublished observations ) . the positive trem2 immunostaining on the vasculature was consistent with the finding on expression of trem2 in endothelial cells that has been documented earlier . trem2 immunostaining was also occasionally spotted on lymphocyte - like cells in some oa tissues ; however , to date there has been no study indicating the expression of trem2 in lymphocytes but further investigation is needed for confirmation . interestingly , dap12 appeared predominantly associated with macrophage - like cells in the sublining of the synovial tissue , particularly in the macrophage - like cells in the lining of the oa group . ( 2014 ) reported that mrna expression levels of dap12 in the peripheral blood mononuclear cells of active ra patients were significantly higher in active ra patients than in inactive ra or oa patients . this is consistent with our observations of higher levels of dap12 protein expressed in the synovium in active ra patients than in inactive ra or oa patients . fcr protein associates with fibroblasts and monocytes of the synovial sublining whilst lymphoid aggregates and the vasculature do not express fcr . of note , similar to dap12 , fcr was associated with macrophage - like synoviocytes in the synovial lining with some scattered monocytes in the sublining of the oa tissue . this increased dap12 and fcr expression might indicate a role in the pathogenesis of oa but this is yet to be determined . analysis of human synovial tissue , serum , plasma , and synovial fluid suggests that oscar expression is associated with disease activity in ra [ 67 , 94 , 98 , 99 ] . recent studies show that oscar protein expression is increased in monocytes from ra patients compared with healthy individuals , correlating with inflammatory disease activity ( das28 ) . oscar has also been noted to be expressed by mononuclear cells adjacent to synovial microvessels in ra tissues . consistent with these findings , our immunohistochemical studies show that high levels of oscar are associated with mono- and multinuclear cells in active ra tissues compared to tissues from oa and normal patients ( figure 2 ) . furthermore , semiquantitative analysis confirmed that there is a significant elevation of oscar ( p < 0.05 ) in active ra synovial tissues compared to osteoarthritis synovial tissues . this increased expression of oscar in the synovial tissue of active ra suggests oscar regulation by inflammatory cytokines and supports a role for oscar in the pathogenesis of ra . a study investigating the clinical , radiological , and synovial immunopathological responses following antirheumatic treatment in ra proposed that high synovial tissue vascularity predicted favorable clinical and radiological responses to treatment . similar to this , we previously reported the increased opg staining associated with the vasculature in synovial tissues retrieved from the patient in remission , oa and normal compared with active ra . furthermore , we have reported increased levels of opg associated with vasculature following treatment with dmards . more recently we have detected increased expression of oscar protein associated with the microvasculature of synovial tissue from all inactive and active ra patient tissues ( 9/9 ) compared to none in the normal synovial tissue group ( 0/9 ) . importantly , in diseased tissues oscar was expressed mostly on the luminal side of the microvasculature , consistent with oscar expression by endothelial cells [ 67 , 103 ] . our findings suggest that oscar is associated with the endothelial cells of the microvasculature and is either produced by endothelial cells or secreted by other cells and bound by the endothelial cells in inflammatory states . the marked reduction in the oscar associated with endothelial cells observed in oa and healthy synovial tissues compared with active ra indicates an immune modulatory mechanism , which may also signal back to the osteoclasts and regulate bone resorption . following observations of oscar association with blood vessels the expression of oscar was investigated in endothelial cell lines in vitro . our analysis on bone marrow - derived endothelial cells ( bmecs ) challenged with il-1 and tnf- in vitro demonstrated that the inflammatory cytokines increased oscar expressed as both mrna and secreted and membrane - bound proteins . together with in vivo observations on synovial tissues and serum levels these studies suggest that inflammatory cytokines in ra regulate cleavage or secretion of soscar from preosteoclasts or the microvasculature . the regulation by inflammatory cytokines , such as tnf- , of oscar messenger rna expression has also been observed in monocytes . interestingly , levels of oscar were found to increase in serum from ra patients following anti - tnf treatment . of note , these studies did not investigate gene or the release of protein oscar by human peripheral blood derived osteoclasts in response to tnf- in conjunction with rankl . to our knowledge , very limited descriptive or functional studies have investigated itam factors in periodontitis and normal gingival tissues . an early study however reported that isolated polymorphonuclear neutrophils from gcf of adult periodontitis patients exhibited higher fc alpha ri and fc gamma ri levels and lower fc gamma riia and fc gamma riiib levels than peripheral blood polymorphonuclear neutrophils . they found that gcf derived polymorphonuclear neutrophils had a reduced ability to phagocytose and kill igg1-opsonized p. gingivalis compared to peripheral blood polymorphonuclear neutrophils . our recent unpublished observations have identified oscar colocalizing with trap in cells in serial sections of mildly inflamed gingival tissue ( figure 3 ) . of note , these osteoclast markers are highly expressed in the multinucleated cells on the bone . similar to the previous observations of expression of itam - associated molecules in active and inactive ra patients oscar expression was also noted in the microvasculature . given the similarities in pathogenesis of ra and periodontitis it is worth investigating expression of itam factors in periodontitis , gingivitis , and normal gingival tissues . we have reported a marked increase in the levels of trem2 and dap12 , oscar , and fcr in tissues containing pe particles , compared with oa synovial control tissue when assessed by a semiquantitative scoring system . furthermore , the observed increased levels of these proteins in peri - prosthetic tissues were consistent with the finding that the corresponding mrna levels were also increased . of interest , pe - containing osteoclast - like cells in these tissues were associated with high levels of trem2 and oscar protein and their respective adaptor molecules dap12 and fcr ( figure 4 ) . consistent with these in vivo observations , pe particles added to human peripheral blood derived osteoclast cells in vitro upregulated itam expression . it is important to also understand the role synovial fluid may play in modulating regulators of cartilage and bone destruction in the joint . andersson et al . ( 2007 ) found that synovial fluid from patients with oa stimulated the mrna expression of oscar and nfatc1 in mouse calvarial implants in vitro , while mrna expressions of dap12 and fcr were not affected by synovial fluid from either revision or oa patient groups . the authors suggested that perhaps oscar and nfatc1 mrna might be regulated by soluble factors that are present in oa synovial fluid . however , considering that inflammation recruits osteoclast precursors and can induce the differentiation and activation of osteoclasts the enhanced expression of itam - related molecules in revision tissues could exacerbate bone loss in this disease . previous studies have demonstrated that soluble fusion ( oscar - fc ) protein , comprising the extracellular domain of oscar , could inhibit osteoclastogenesis in murine preosteoclast / osteoblast cocultures and pbmcs cultured in the presence of rankl , m - csf , and tgf- . in this situation soscar the ability of soluble soscar in vitro to impede osteoclast formation may prove useful in inhibiting osteoclast differentiation and may thus prevent bone damage in diseases such as ra . there is conflicting data as to whether soscar increases in healthy individuals or it increases as a result of erosive activity in ra . soluble oscar has been detected in serum and reported to be higher in healthy compared to ra patients [ 94 , 99 ] . serum levels of soscar were shown to inversely correlate with erosion and disease activity . a recent study , however , reported higher levels of soscar in the plasma of ra patients rather than healthy individuals . we have also detected soscar in the synovial fluid of oa and active ra patients with no significant difference between these diseases . we believe that soscar has the potential to act as decoy receptor for oscar ligand within the joint and affect osteoclast development in ra . it is possible that successful treatment results in increased cleavage of cell associated oscar resulting in increased soscar levels in the joint . in this way the biological effect of serum and synovial fluid - derived oscar on osteoclastogenesis is yet to be investigated . synovitis and erosion are not always linked with some patients having progressive erosive disease despite being in remission and it is unclear what factors drive this . the discordance between clinical inflammatory disease activity and radiological outcomes emphasizes the need for a validated marker of bone damage in conjunction with current clinical parameters that are routinely assessed [ 107 , 108 ] . the ability to monitor bone erosion will allow the clinician to make important decisions on therapy earlier and reduce structural joint damage . currently unaddressed ra - induced joint damage affects mobility of patients later in life and predisposes to secondary osteoarthritis . in addition , while we have numerous markers that reflect inflammation and related disease activity there are none that monitor joint erosion , other than x - rays which only indicate damage after it has occurred . therefore , it is essential that an accurate early marker of joint erosion is identified in order to guide effective treatment modalities in order to protect the joint of arthritic patients . while the significance of itam - associated molecules has been largely established in the context of bone biology and an immunological point of view , limited studies have been carried out on osteoclast itam - related molecules in human bone pathologies . the increased levels of itam factors in inflamed tissues adjacent to sites of localized bone loss in ra , periodontal disease , and periprosthetic osteolysis may prove indicative of the disease progression . further to this , levels of the soluble factor , oscar , in serum or local fluid , may provide us with a potential bone destructive marker and potential target for modulation of bone erosion .

the field of osteoimmunology has emerged in response to the range of evidences demonstrating the close interrelationship between the immune system and bone metabolism . this is pertinent to immune - mediated diseases , such as rheumatoid arthritis and periodontal disease , where there are chronic inflammation and local bone erosion . periprosthetic osteolysis is another example of chronic inflammation with associated osteolysis . this may also involve immune mediation when occurring in a patient with rheumatoid arthritis ( ra ) . similarities in the regulation and mechanisms of bone loss are likely to be related to the inflammatory cytokines expressed in these diseases . this review highlights the role of immune - related factors influencing bone loss particularly in diseases of chronic inflammation where there is associated localized bone loss . the importance of the balance of the rankl - rank - opg axis is discussed as well as the more recently appreciated role that receptors and adaptor proteins involved in the immunoreceptor tyrosine - based activation motif ( itam ) signaling pathway play . although animal models are briefly discussed , the focus of this review is on the expression of itam associated molecules in relation to inflammation induced localized bone loss in ra , chronic periodontitis , and periprosthetic osteolysis , with an emphasis on the soluble and membrane bound factor osteoclast - associated receptor ( oscar ) .

1. Introduction 2. Chronic Inflammation-Mediated Bone Loss 3. RANKL-RANK-OPG Axis 4. The ITAM Pathway in Osteoimmunology 5. Conclusion

the term osteoimmunology and the study of osteoimmunology have developed due to the close interrelationship between the immune system and bone metabolism . this is evident in immune - mediated diseases , such as rheumatoid arthritis and periodontal disease ( periodontitis ) , where there are local bone erosion and inflammation as reviewed in detail in multiple publications [ 24 ] . similarities in the mechanisms of bone loss in disease are likely related to the inflammatory cytokines expressed in a number of bone loss diseases . these cytokines are known to upregulate osteoclast activity via increased expression levels of receptor activator nf kappa b ligand ( rankl ) relative to osteoprotegerin ( opg ) ( as explored below ) and increase localized bone loss in diseases such as ra , periodontal disease , and periprosthetic osteolysis [ 510 ] . this review highlights the role of immune - related cells and factors in modulating bone loss , particularly in these diseases . while the importance of the rankl - rank - opg axis has been appreciated for nearly two decades [ 1113 ] , more recent studies have highlighted the importance of factors associated with immunoreceptor tyrosine - based activation motif ( itam ) signalling . this review will briefly discuss the rankl - rank - opg axis but its major focus will be on the role of itam - associated factors , the more recently investigated pathway , and how it relates to inflammatory bone loss diseases , in particular osteoclast - associated receptor ( oscar ) . rheumatoid arthritis ( ra ) affects 1 - 2% of the population and involves an autoimmune reaction with an autoantibody response to citrullinated proteins ( and others such as rheumatoid factor and collagen type ii ) . further to this there is an increase in osteoclast number and activity leading to focal bone erosions , juxta - articular osteopenia , and joint destruction [ 1720 ] . animal models suggest that there may also be suppression of localised osteoblast formation of bone . periodontitis is a chronic inflammatory disease of the gingival tissues , with an associated loss of the supporting structures including the periodontal ligament and alveolar bone . the aetiology involves an inflammatory response to bacterial infection such as p. gingivalis and possibly an autoimmune reaction , as reviewed . periodontitis is the most common and widespread bone loss pathology in humans with 64% of the us population aged 65 years and older reported as having moderate or severe periodontitis . despite the prevalence of this disease inevitably , in the absence of effective treatment , support structures ( periodontium ) are compromised and the affected teeth will loosen and fall out . similarities in ra and periodontitis may relate to citrullinated enolase as the specific antigen involved as well as cross - reaction between the antibodies directed towards the immunodominant epitope of human citrullinated alpha - enolase and a conserved sequence on citrullinated p. gingivalis enolase . new evidence suggests a relationship between the extent and severity of chronic periodontitis and ra [ 24 , 25 ] . this is supported by findings in a study using a combined animal model of ra and periodontitis , which demonstrated more severe development of arthritis in mice with periodontitis . further to this , mice in which periodontitis alone was induced had evidence of radiocarpal bone loss in the absence of arthritic disease . new treatment strategies are needed for both diseases that target the inhibition of proinflammatory cytokines , destructive enzymes , and bone - resorbing activity . joint replacement surgery is used as a last resort in osteoarthritis ( oa ) and ra patients and is a relatively successful operation ; however , a large proportion of implants fail within 1020 years as a result of bone loss and implant loosening [ 4 , 10 , 28 ] . the pathogenesis behind prosthetic implant failure involves wear of prosthetic alloys , such as polyethylene ( pe ) , cobalt chrome , and titanium liberated from the implant surface [ 2931 ] . these particles stimulate a chronic inflammatory response , which increases bone - resorbing activity of the osteoclasts and suppresses bone formation by the osteoblast [ 33 , 34 ] resulting in bone loss . this granulomatous lesion in periprosthetic osteolysis often leads to the formation of pseudosynovium - like structure , in which cells are organized into lining layer , in the membranous tissues adjacent to the failed implant surface . given the low levels of t lymphocytes , the general belief held is that lymphocyte infiltrates are not normally associated with wear - particle induced periprosthetic osteolysis , in particular , in the granulomatous region [ 29 , 32 , 42 , 43 ] . the role of t cells may however be more pertinent in ra patients with implants . the prevalence of foreign - body giant cells in response to implant - derived wear debris in ra patients and non - ra patients does not differ but appears to be linked to the amount of polyethylene wear debris . whether implant wear is inducing a different reaction in ra versus non - ra patients may potentially have implications for combination and immune targeted treatment of inflammation and osteolysis in these patients . receptor activator nf kappa b ligand- ( rankl- ) rank signalling has several important roles in the immune system and bone [ 13 , 4446 ] . nfatc1 binds directly to and regulates osteoclast differentiation genes such as tartrate resistant acid phosphatase ( trap ) , cathepsin k ( cath k ) , osteoclast - associated receptor ( oscar ) , 3-integrin [ 53 , 54 ] , and calcitonin receptor ( ctr ) . the rank - rankl - opg axis is known to regulate not only normal bone physiology but also alterations in rank - rankl - opg interactions that play a role in bone disease . uncoupling in the balance between the level and activity of these molecules culminates in osteoporotic or osteopetrotic phenotypes due to an increase or decrease in osteoclast formation and activity . this is particularly evident in focal bone loss associated with chronic inflammatory diseases such as rheumatoid arthritis , periodontal disease , and periprosthetic bone loss . in active ra , periodontal disease , and prosthetic loosening , elevated levels of rankl relative to opg are observed in the synovial - like soft tissue , gingival tissue , and soft tissues adjacent to sites of osteolysis [ 510 ] . further to this the elevated ratio of rankl : opg expression is associated with increased differentiation and activity of the bone - resorbing osteoclasts [ 5 , 79 , 32 , 60 ] , suggesting rankl : opg ratio as an important indicator for bone erosion . as rankl and opg are key molecules regulating bone loss in diseases , therapeutic interventions targeting these molecules and their signaling cascades are being investigated to treat a wide range of diseases . a seminal paper in the field of osteoimmunology used a rankl knockout background to demonstrate that animals developed an osteopetrotic phenotype and a reduction in bone erosion , characterized by the absence of osteoclasts , whilst inflammation did not differ between wild - type and rankl knockout mice . in contrast , cartilage erosion was present in both control littermate and rankl knockout mice , suggesting that the rank - rankl - opg axis does not directly affect cartilage metabolism . in support of this , a more recent large center study has reported the ratio of rankl : opg and markers of bone and cartilage degradation ( such as collagen terminal 1 ( ctx-1 ) ) to be predictive of progression of radiological bone damage in ra . in synovial tissues from patients with active ra , rankl expression is predominantly located in sublining regions [ 62 , 63 ] concentrated at focal sites of osteoclastic bone erosion in the pannus- bone interface . in contrast , opg has been described as being in regions of synovium some distance from the sites of bone erosion in ra . we reported opg is associated with endothelial cells and macrophages in the synovial lining layer of oa and normal tissues whilst absent in patients with active ra . rankl expressing cells are also seen within areas of lymphocyte infiltration and dual immunostaining by ourselves , and others have shown that many of the rankl - positive cells are a subset of cd3 + and cd4 + cells [ 6 , 62 , 63 ] . nfatc1 is a transcription factor crucial to rankl - rank signaling in the osteoclasts and is initially identified as being expressed by t cells and involved in regulation of cytokine transcription . many of the nfatc1-immunostained mononuclear cells observed were single nucleated and thus could be either precursor cells of osteoclasts such as macrophages , or lymphocytes . the relative ratio of rankl to opg is also a significant indicator in bone loss associated with periodontal disease [ 5 , 69 ] . similar to ra , b and t lymphocytes express rankl in gingival tissues associated with periodontitis [ 5 , 69 ] with expression of more than 50 and 90% of t cells and b cells , respectively . rank , rankl , and tumour necrosis factor ( tnf- ) are key modulators of bone turnover and their expression has been reported by ourselves and others in the tissues near periprosthetic osteolysis in patients undergoing revision of total hip prostheses [ 9 , 71 , 72 ] . more interestingly a strong statistical correlation was found between volume of bone loss , polyethylene wear debris , and rank , rankl , and tnf- expression . in periprosthetic osteolysis , elevated levels of rankl , relative to its competitor opg , are associated with increased differentiation and activity of the bone - resorbing osteoclasts [ 8 , 32 , 73 ] . an earlier study had shown that cells isolated from periprosthetic tissues containing wear particles expressed mrna encoding for the proosteoclastogenic molecules , rankl , its receptor rank , monocyte colony - stimulating factor ( m - csf ) , interleukin- ( il- ) 1 beta , tnf- , il-6 , and soluble il-6 receptor , as well as opg . these findings suggest that ingestion of prosthetic wear particles by macrophages results in expression of osteoclast - differentiating molecules and stimulation of macrophage differentiation into osteoclasts . we observed both mrna and protein expressions of nfatc1 to be higher in periprosthetic osteolysis than in oa tissues although levels did not reach significance . the induction of intracellular calcium via this pathway is required in conjunction with rankl - rank interaction for nfatc1 induction . in the context of human pathology studies in diseased tissues , particularly in nasu - hakola disease [ 8587 ] and very recently in alzheimer 's disease , have shown that these molecules may be involved . although animal models are not the focus of this review it is interesting to note the phenotypes of itam related molecules in single and combination knockouts . mice that are double knockout for the adaptors dap12 and fcr are severely osteopetrotic and bone marrow derived osteoclast precursors from these mice are unable to differentiate into mature osteoclasts in a rankl- and m - csf - mediated culture system [ 79 , 82 ] . oscar is an igg - like receptor expressed by monocytes , macrophages monocyte - derived dendritic cells in humans , and is involved in antigen presentation as well as survival , maturation , and activation of dendritic cells [ 14 , 76 , 77 , 83 , 94 , 95 ] . rankl - rank induction of nfatc1 expression precedes that of oscar and is crucial for induction of oscar gene expression . these findings demonstrate a significant role for oscar in immune modulation as well as osteoclastogenesis . this may be in addition to the recent identification of the motifs within fibrillar collagens in the extracellular matrix ( ecm ) as oscar ligands . the importance of oscar is further highlighted by the fact that soluble oscar ( s)oscar , in the form of oscar - fc , has also been shown to inhibit osteoclast differentiation from pbmcs in the presence of rankl , m - csf , and tgf- . costimulatory immune pathways may further increase osteoclast differentiation and activity [ 81 , 82 ] , particularly in chronic inflammatory diseases with an immune component such as in ra , periprosthetic osteolysis , and periodontal disease . in fact , our studies [ 67 , 75 ] and those of others suggest that deregulation of itam - associated molecules contributes to the pathogenesis and severity of rheumatoid arthritis , periodontal disease , periprosthetic osteolysis , and osteoporosis [ 10 , 67 , 75 , 92 , 94 , 98 , 99 ] . we , and others , have demonstrated increased levels of itam - related factors , including trem2 , dap12 , oscar , and fcr in human periprosthetic tissues adjacent to sites of osteolysis and in ra synovial tissues [ 67 , 94 ] . additionally , we have observed itam - related factors expressed in periodontitis tissue adjacent to bone loss ( unpublished observations ) . of these factors , soluble and membrane - bound oscar have been more extensively assessed in the context of ra and vascular disease ( expanded on below ) . we observed markedly higher levels of trem2 , dap12 , oscar , and fcr in active ra patients compared to synovial tissues from inactive ra , oa , or control healthy joint . the positive trem2 immunostaining on the vasculature was consistent with the finding on expression of trem2 in endothelial cells that has been documented earlier . interestingly , dap12 appeared predominantly associated with macrophage - like cells in the sublining of the synovial tissue , particularly in the macrophage - like cells in the lining of the oa group . this is consistent with our observations of higher levels of dap12 protein expressed in the synovium in active ra patients than in inactive ra or oa patients . of note , similar to dap12 , fcr was associated with macrophage - like synoviocytes in the synovial lining with some scattered monocytes in the sublining of the oa tissue . this increased dap12 and fcr expression might indicate a role in the pathogenesis of oa but this is yet to be determined . analysis of human synovial tissue , serum , plasma , and synovial fluid suggests that oscar expression is associated with disease activity in ra [ 67 , 94 , 98 , 99 ] . oscar has also been noted to be expressed by mononuclear cells adjacent to synovial microvessels in ra tissues . this increased expression of oscar in the synovial tissue of active ra suggests oscar regulation by inflammatory cytokines and supports a role for oscar in the pathogenesis of ra . a study investigating the clinical , radiological , and synovial immunopathological responses following antirheumatic treatment in ra proposed that high synovial tissue vascularity predicted favorable clinical and radiological responses to treatment . more recently we have detected increased expression of oscar protein associated with the microvasculature of synovial tissue from all inactive and active ra patient tissues ( 9/9 ) compared to none in the normal synovial tissue group ( 0/9 ) . our findings suggest that oscar is associated with the endothelial cells of the microvasculature and is either produced by endothelial cells or secreted by other cells and bound by the endothelial cells in inflammatory states . the marked reduction in the oscar associated with endothelial cells observed in oa and healthy synovial tissues compared with active ra indicates an immune modulatory mechanism , which may also signal back to the osteoclasts and regulate bone resorption . following observations of oscar association with blood vessels the expression of oscar was investigated in endothelial cell lines in vitro . our analysis on bone marrow - derived endothelial cells ( bmecs ) challenged with il-1 and tnf- in vitro demonstrated that the inflammatory cytokines increased oscar expressed as both mrna and secreted and membrane - bound proteins . the regulation by inflammatory cytokines , such as tnf- , of oscar messenger rna expression has also been observed in monocytes . of note , these studies did not investigate gene or the release of protein oscar by human peripheral blood derived osteoclasts in response to tnf- in conjunction with rankl . of note , these osteoclast markers are highly expressed in the multinucleated cells on the bone . similar to the previous observations of expression of itam - associated molecules in active and inactive ra patients oscar expression was also noted in the microvasculature . given the similarities in pathogenesis of ra and periodontitis it is worth investigating expression of itam factors in periodontitis , gingivitis , and normal gingival tissues . we have reported a marked increase in the levels of trem2 and dap12 , oscar , and fcr in tissues containing pe particles , compared with oa synovial control tissue when assessed by a semiquantitative scoring system . of interest , pe - containing osteoclast - like cells in these tissues were associated with high levels of trem2 and oscar protein and their respective adaptor molecules dap12 and fcr ( figure 4 ) . it is important to also understand the role synovial fluid may play in modulating regulators of cartilage and bone destruction in the joint . however , considering that inflammation recruits osteoclast precursors and can induce the differentiation and activation of osteoclasts the enhanced expression of itam - related molecules in revision tissues could exacerbate bone loss in this disease . previous studies have demonstrated that soluble fusion ( oscar - fc ) protein , comprising the extracellular domain of oscar , could inhibit osteoclastogenesis in murine preosteoclast / osteoblast cocultures and pbmcs cultured in the presence of rankl , m - csf , and tgf- . in this situation soscar the ability of soluble soscar in vitro to impede osteoclast formation may prove useful in inhibiting osteoclast differentiation and may thus prevent bone damage in diseases such as ra . there is conflicting data as to whether soscar increases in healthy individuals or it increases as a result of erosive activity in ra . we have also detected soscar in the synovial fluid of oa and active ra patients with no significant difference between these diseases . in addition , while we have numerous markers that reflect inflammation and related disease activity there are none that monitor joint erosion , other than x - rays which only indicate damage after it has occurred . while the significance of itam - associated molecules has been largely established in the context of bone biology and an immunological point of view , limited studies have been carried out on osteoclast itam - related molecules in human bone pathologies . the increased levels of itam factors in inflamed tissues adjacent to sites of localized bone loss in ra , periodontal disease , and periprosthetic osteolysis may prove indicative of the disease progression . further to this , levels of the soluble factor , oscar , in serum or local fluid , may provide us with a potential bone destructive marker and potential target for modulation of bone erosion .

the mechanism of action of carbon tetrachloride ( ct)-induced hepatotoxicity has been linked to the metabolism of ct into the trichloromethyl radical and the trichloromethyl peroxy radical , and their subsequent reactions with lipids and proteins in the cell membrane , resulting in mda , where the rate of mda is mediated by reactive oxygen species , such as superoxide radicals and hydroxyl radicals.[112 ] also , it has been suggested that ct - induced hepatotoxicity also involves the disruption of hepatic antioxidant defense systems by the depletion of reduced glutathione ( gsh ) , superoxide dismutase ( sod ) , and catalase . although a number of antioxidants have been evaluated for preventing ct - induced hepatotoxicity , efficacy has not been demonstrated . recent findings indicate that free radicals produced by ct lead to mitochondrial damage and nuclear dna damage , resulting in the activation of their respective repair mechanisms , including induction of the enzyme poly(adp - ribose ) polymerase 1 ( parp-1 ) . parp-1 represents the most abundant isoform of the poly ( adp - ribose ) polymerase family , which responds to single- and double - strand nicks in nuclear dna.[1418 ] upon binding to damaged dna , parp-1 forms homodimers and catalyzes the cleavage of nad into nicotinamide and adp - ribose to form long branches of adp - ribose polymers on glutamic acid residues of a number of target proteins , including histones and the parp enzyme itself . under normal cellular operation , this activity provides protection against damage to nuclear dna ; however , when excessive activation of parp-1 occurs , the result is a rapid depletion of nad and atp , eventually leading to necrotic cell death . previously , we have demonstrated the hepatoprotective effect against ct - induced centrilobular necrosis with concomitant treatment of the parp inhibitor 6(5h)-phenanthridinone . however , due to poor water solubility , 6(5h)-phenanthridinone is required to be dissolved in dimethyl sulfoxide ( dmso ) for effective administration , which may modify both effective delivery and bioavailability , potentially altering the attenuation of ct - induced toxicity . recently , specific water - soluble parp-1 inhibitors have become available , and some have been shown to confer protective effects in various models of toxic exposure . this investigation evaluates the ability of 3 water - soluble parp-1 inhibitors , namely , 3-aminobenzamide ( aba ) , 5-aminoisoquinolinone ( aiq ) , and n-(6-oxo-5,6-dihydro - phenanthridin-2-yl)-n , n - dimethylacetamide hcl ( pj-34 ) to block or attenuate centrilobular hepatotoxicity from ct treatment in imprinting control region ( icr ) mice . ct , malondialdehyde bis ( dimethyl acetal ) , thiobarbituric acid , and 5,5-dithiobis-2-nitrobenzoic acid were obtained from sigma chemical co. ( st . louis , mo ) . the alanine amino transferase ( alt ) kit was obtained from teco diagnostics ( anaheim , ca ) . cleaved caspase-3 ( asp175 ) rabbit monoclonal antibody was from cell signaling technology ( danvers , ma ) . all other chemicals were of analytical grade or higher from merck ( darmstadt , germany ) . adult male icr mice ( 30 5 g ) were used for all experiments . animals were housed under controlled conditions ( 25 2c and a 12 h light / dark cycle ) and allowed free access to food and water . the animals were divided into the following groups : the first group ( basic control group ) received no ct or parp inhibitor , the second group ( negative control ) received no ct and received only individual parp inhibitor , the third group ( treatment ) received ct and no parp inhibitor , and the last group ( experimental group ) received ct and individual parp inhibitors . parp inhibitors were dissolved in normal saline ( ph 7.4 ) and administered intraperitoneally ( i.p . ) . ct was dissolved in corn oil and administered i.p . with doses ranging from 0.3 to 1.2 ml / kg . a time - dependent experiment followed , using 0.6 ml / kg pj-34 in the following administration groups : 1 h before ct treatment , concomitantly with ct treatment , 1 h after ct treatment , and 3 h after ct treatment . all the experiments were conducted under controlled conditions according to the guide to the care and use of experimental animals and the university of south florida institutional animal care and use committee ( iacuc ) . the mice were sacrificed after 24 h and blood samples to determine alanine transaminase ( alt ) activity were withdrawn by cardiac puncture . livers were isolated , perfused with normal saline , and dissected for ( 1 ) histopathology and ( 2 ) for determination of total gsh , thiobarbituric acid - reactive substances ( tbars ) , sod activity , and carbonyl content . the colorimetric assay for parp activity was performed in 96-well plates ( trevigen , inc . , gaithersburg , md ) according to manufacturer 's protocol . serial dilutions of the parp enzyme were distributed into wells to generate a standard curve . the clarified tissue homogenates ( 10 l / well ) were added to triplicate wells to determine cellular parp activity . the plate was washed 4 times with 1 phosphate - buffered saline ( pbs ) and then incubated for 20 min with 50 l / well streptavidin horseradish peroxidase ( strep hrp ) diluted 1:500 in 1 strep - diluent ( trevigen ) . the plate was washed 4 times with 1 pbs prior to the addition of the hrp substrate . for colorimetric readout , 50 l of tacs - sapphire ( trevigen ) was added to each well and incubated in the dark , at room temperature , for 15 min . development of the colorimetric reaction was stopped by the addition of an equal volume of 0.2 m hcl . the results were expressed as units of parp activity calculated per milligram of protein and normalized to equal concentrations of protein . protein determination in all assays described here were determined by the bicinchoninic acid assay using bovine serum albumin as standard ( sigma ) . serum ( 0.2 ml ) was obtained by centrifugation of cardiac puncture blood samples at 4000rpm for 15 min . each serum sample was then transferred to a new sterile microcentrifuge tube and stored at 4c until time of assay . alt activity , expressed as iu / l , was determined by a previously described method using a commercially prepared reagent kit from teco diagnostics ( anaheim , ca ) . individual liver samples ( 100 mg ) were homogenized in 1 ml of 0.2 m pbs ( ph 8.0 ) , and the mixture was centrifuged at 12,000 rpm for 30 min . the supernatant ( 0.5 ml ) was mixed with 0.5 ml of 4% sulfosalicylic acid , allowed to stand for 5 min at 4c , and centrifuged again at 3000 rpm for 10 min . the supernatant obtained ( 0.5 ml ) was mixed with 2 ml of 0.2 m pbs ( ph 8.0 ) and 10 l of 10 mm 5,5-dithiobis-2-nitrobenzoic acid . the absorbance was measured at 412 nm and concentration of gsh was expressed as nmol / mg protein . formation of lipid peroxide derivatives was evaluated by measuring tbars according to a method previously described by cascio et al . liver samples were individually homogenized in ice - cold 1.15% kcl ( w / v ) . homogenates ( 0.4 ml ) were mixed with 1 ml of 0.375% tba , 15% trichloroacetic acid ( tca ) ( w / v ) , 0.25 n hcl , and 6.8 mm butylated - hydroxytoluene , placed in a boiling water bath for 10 min , removed and allowed to cool on ice . following centrifugation at 3000 rpm for 10 min , the absorbance in the supernatants was measured at 532 nm . the amount of tbars produced was expressed as nmol tbars / mg protein using malondialdehyde bis(dimethylacetal ) for calibration . determination of sod activity in mouse liver was based on inhibition of nitrite formation in the reaction of oxidation of hydroxylammonium with superoxide anion radical . the total sod was determined in one aliquot and cytosolic sod was determined in the second aliquot supplemented with 10 mm kcn . the concentration of mitochondrial sod was calculated as the difference between the amount of total and cytosolic fraction of sod . nitrite formation was generated in a mixture that contained 25ml xanthine ( 15 mm ) , 25 ml hydroxylammonium chloride ( 10 mm ) , 250 ml pbs ( 65 mm , ph 7.8 ) , 90 ml distilled water , and 100 ml xanthine oxidase ( 0.1 u / ml ) to initiate the reaction . the inhibitory effect of the extant sod was assayed at 25c after 20 min of incubation with 10 ml of liver tissue extracts . the determination of the resulting nitrite was performed following the reaction ( 20 min at room temperature ) with 0.5 ml sulfanilic acid ( 3.3 mg / ml ) and 0.5 ml -naphthylamine ( 1 mg / ml ) . optical absorbance at 530 nm was measured on ultrospec iii spectrophotometer ( pharmacia lkb , uppsala , sweden ) . the procedure used was similar to that described by levine et al ( 1990 ) with slight modifications . two sets of 250 l sample homogenates were labeled as test and reference . a 1 ml amount of 10 mm 2,4-dinitrophenylhydrazine ( dnph ) prepared in 2.5 m hcl was added to test samples and 2.5 m hcl alone was added to the reference . the contents were mixed and incubated in the dark for 1 h. then 1 ml of 20% tca was added to each tube . the tubes were centrifuged at 3500 rpm for 20 min and protein pellets were washed with 1 ml of 10% tca . the precipitates were washed 3 times with 1 ml of ethyl acetate : ethanol ( 1:1 , each pellet was then dissolved in 1 ml of 6 m guanidine hydrochloride , at 37c , for 10 min . carbonyl content was determined with ultrospec iii spectrophotometer ( pharmacia lkb , uppsala , sweden ) . each test sample was read against the corresponding control at 370 nm using an absorption coefficient of 22,000 m cm . liver samples were fixed in 10% neutral buffered formalin and embedded in paraffin following standard procedures . tissue sections , 4 m in thickness , were stained with hematoxylin and eosin ( h and e ) to assess parenchymal histopathologic changes . histopathologic parameters evaluated in the centrilobular region were proliferation , apoptosis , necrosis , and fibrosis using the methodology described previously by price et al . summary descriptive analysis for all cytotoxicity outcome data is reported as the mean levels of biomarkers of cytotoxicity sem . f - test analysis was performed to evaluate the differences between ct dosage groups in the initial treatment experiment , and again for the select parp inhibitor groups in the experiment that held the ct dosage constant for all the groups ( p < 0.05 was considered to indicate a statistically significant difference ) . f - test analysis was then performed for the experiment that evaluated differences in measures of cytotoxicity between the time of treatment with pj-34 relative to ct treatment among groups that held the ct and pj-34 dosage constant for all groups ( p < 0.05 was considered to indicate a statistically significant difference ) . pearson 's correlation coefficients were calculated for the combined data of pj-34 , aba , and aiq to determine the significance and magnitude of correlation between the degree of parp-1 inhibition and measures of cytotoxicity ( p < 0.05 considered to indicate a statistically significant correlation ) . the analysis was performed with sas statistical software package 9.2 , sas institute cary , nc , usa . ct , malondialdehyde bis ( dimethyl acetal ) , thiobarbituric acid , and 5,5-dithiobis-2-nitrobenzoic acid were obtained from sigma chemical co. ( st . louis , mo ) . the alanine amino transferase ( alt ) kit was obtained from teco diagnostics ( anaheim , ca ) . cleaved caspase-3 ( asp175 ) rabbit monoclonal antibody was from cell signaling technology ( danvers , ma ) . all other chemicals were of analytical grade or higher from merck ( darmstadt , germany ) . adult male icr mice ( 30 5 g ) were used for all experiments . animals were housed under controlled conditions ( 25 2c and a 12 h light / dark cycle ) and allowed free access to food and water . the animals were divided into the following groups : the first group ( basic control group ) received no ct or parp inhibitor , the second group ( negative control ) received no ct and received only individual parp inhibitor , the third group ( treatment ) received ct and no parp inhibitor , and the last group ( experimental group ) received ct and individual parp inhibitors . parp inhibitors were dissolved in normal saline ( ph 7.4 ) and administered intraperitoneally ( i.p . ) . ct was dissolved in corn oil and administered i.p . with doses ranging from 0.3 to 1.2 ml / kg . a time - dependent experiment followed , using 0.6 ml / kg pj-34 in the following administration groups : 1 h before ct treatment , concomitantly with ct treatment , 1 h after ct treatment , and 3 h after ct treatment . all the experiments were conducted under controlled conditions according to the guide to the care and use of experimental animals and the university of south florida institutional animal care and use committee ( iacuc ) . the mice were sacrificed after 24 h and blood samples to determine alanine transaminase ( alt ) activity were withdrawn by cardiac puncture . livers were isolated , perfused with normal saline , and dissected for ( 1 ) histopathology and ( 2 ) for determination of total gsh , thiobarbituric acid - reactive substances ( tbars ) , sod activity , and carbonyl content . the colorimetric assay for parp activity was performed in 96-well plates ( trevigen , inc . , gaithersburg , md ) according to manufacturer 's protocol . serial dilutions of the parp enzyme were distributed into wells to generate a standard curve . the clarified tissue homogenates ( 10 l / well ) were added to triplicate wells to determine cellular parp activity . the plate was washed 4 times with 1 phosphate - buffered saline ( pbs ) and then incubated for 20 min with 50 l / well streptavidin horseradish peroxidase ( strep hrp ) diluted 1:500 in 1 strep - diluent ( trevigen ) . the plate was washed 4 times with 1 pbs prior to the addition of the hrp substrate . for colorimetric readout , 50 l of tacs - sapphire ( trevigen ) was added to each well and incubated in the dark , at room temperature , for 15 min . development of the colorimetric reaction was stopped by the addition of an equal volume of 0.2 m hcl . the results were expressed as units of parp activity calculated per milligram of protein and normalized to equal concentrations of protein . protein determination in all assays described here were determined by the bicinchoninic acid assay using bovine serum albumin as standard ( sigma ) . serum ( 0.2 ml ) was obtained by centrifugation of cardiac puncture blood samples at 4000rpm for 15 min . each serum sample was then transferred to a new sterile microcentrifuge tube and stored at 4c until time of assay . alt activity , expressed as iu / l , was determined by a previously described method using a commercially prepared reagent kit from teco diagnostics ( anaheim , ca ) . individual liver samples ( 100 mg ) were homogenized in 1 ml of 0.2 m pbs ( ph 8.0 ) , and the mixture was centrifuged at 12,000 rpm for 30 min . the supernatant ( 0.5 ml ) was mixed with 0.5 ml of 4% sulfosalicylic acid , allowed to stand for 5 min at 4c , and centrifuged again at 3000 rpm for 10 min . the supernatant obtained ( 0.5 ml ) was mixed with 2 ml of 0.2 m pbs ( ph 8.0 ) and 10 l of 10 mm 5,5-dithiobis-2-nitrobenzoic acid . the absorbance was measured at 412 nm and concentration of gsh was expressed as nmol / mg protein . formation of lipid peroxide derivatives was evaluated by measuring tbars according to a method previously described by cascio et al . liver samples were individually homogenized in ice - cold 1.15% kcl ( w / v ) . homogenates ( 0.4 ml ) were mixed with 1 ml of 0.375% tba , 15% trichloroacetic acid ( tca ) ( w / v ) , 0.25 n hcl , and 6.8 mm butylated - hydroxytoluene , placed in a boiling water bath for 10 min , removed and allowed to cool on ice . following centrifugation at 3000 rpm for 10 min , the absorbance in the supernatants was measured at 532 nm . the amount of tbars produced was expressed as nmol tbars / mg protein using malondialdehyde bis(dimethylacetal ) for calibration . determination of sod activity in mouse liver was based on inhibition of nitrite formation in the reaction of oxidation of hydroxylammonium with superoxide anion radical . the total sod was determined in one aliquot and cytosolic sod was determined in the second aliquot supplemented with 10 mm kcn . the concentration of mitochondrial sod was calculated as the difference between the amount of total and cytosolic fraction of sod . nitrite formation was generated in a mixture that contained 25ml xanthine ( 15 mm ) , 25 ml hydroxylammonium chloride ( 10 mm ) , 250 ml pbs ( 65 mm , ph 7.8 ) , 90 ml distilled water , and 100 ml xanthine oxidase ( 0.1 u / ml ) to initiate the reaction . the inhibitory effect of the extant sod was assayed at 25c after 20 min of incubation with 10 ml of liver tissue extracts . the determination of the resulting nitrite was performed following the reaction ( 20 min at room temperature ) with 0.5 ml sulfanilic acid ( 3.3 mg / ml ) and 0.5 ml -naphthylamine ( 1 mg / ml ) . optical absorbance at 530 nm was measured on ultrospec iii spectrophotometer ( pharmacia lkb , uppsala , sweden ) . the procedure used was similar to that described by levine et al ( 1990 ) with slight modifications . two sets of 250 l sample homogenates were labeled as test and reference . a 1 ml amount of 10 mm 2,4-dinitrophenylhydrazine ( dnph ) prepared in 2.5 m hcl was added to test samples and 2.5 m hcl alone was added to the reference . the contents were mixed and incubated in the dark for 1 h. then 1 ml of 20% tca was added to each tube . the tubes were centrifuged at 3500 rpm for 20 min and protein pellets were washed with 1 ml of 10% tca . the precipitates were washed 3 times with 1 ml of ethyl acetate : ethanol ( 1:1 , each pellet was then dissolved in 1 ml of 6 m guanidine hydrochloride , at 37c , for 10 min . carbonyl content was determined with ultrospec iii spectrophotometer ( pharmacia lkb , uppsala , sweden ) . each test sample was read against the corresponding control at 370 nm using an absorption coefficient of 22,000 m cm . liver samples were fixed in 10% neutral buffered formalin and embedded in paraffin following standard procedures . tissue sections , 4 m in thickness , were stained with hematoxylin and eosin ( h and e ) to assess parenchymal histopathologic changes . histopathologic parameters evaluated in the centrilobular region were proliferation , apoptosis , necrosis , and fibrosis using the methodology described previously by price et al . summary descriptive analysis for all cytotoxicity outcome data is reported as the mean levels of biomarkers of cytotoxicity sem . f - test analysis was performed to evaluate the differences between ct dosage groups in the initial treatment experiment , and again for the select parp inhibitor groups in the experiment that held the ct dosage constant for all the groups ( p < 0.05 was considered to indicate a statistically significant difference ) . f - test analysis was then performed for the experiment that evaluated differences in measures of cytotoxicity between the time of treatment with pj-34 relative to ct treatment among groups that held the ct and pj-34 dosage constant for all groups ( p < 0.05 was considered to indicate a statistically significant difference ) . pearson 's correlation coefficients were calculated for the combined data of pj-34 , aba , and aiq to determine the significance and magnitude of correlation between the degree of parp-1 inhibition and measures of cytotoxicity ( p < 0.05 considered to indicate a statistically significant correlation ) . the analysis was performed with sas statistical software package 9.2 , sas institute cary , nc , usa . ct , malondialdehyde bis ( dimethyl acetal ) , thiobarbituric acid , and 5,5-dithiobis-2-nitrobenzoic acid were obtained from sigma chemical co. ( st . louis , mo ) . the alanine amino transferase ( alt ) kit was obtained from teco diagnostics ( anaheim , ca ) . cleaved caspase-3 ( asp175 ) rabbit monoclonal antibody was from cell signaling technology ( danvers , ma ) . all other chemicals were of analytical grade or higher from merck ( darmstadt , germany ) . adult male icr mice ( 30 5 g ) were used for all experiments . animals were housed under controlled conditions ( 25 2c and a 12 h light / dark cycle ) and allowed free access to food and water . the animals were divided into the following groups : the first group ( basic control group ) received no ct or parp inhibitor , the second group ( negative control ) received no ct and received only individual parp inhibitor , the third group ( treatment ) received ct and no parp inhibitor , and the last group ( experimental group ) received ct and individual parp inhibitors . parp inhibitors were dissolved in normal saline ( ph 7.4 ) and administered intraperitoneally ( i.p . ) . ct was dissolved in corn oil and administered i.p . with doses ranging from 0.3 to 1.2 ml / kg . a time - dependent experiment followed , using 0.6 ml / kg pj-34 in the following administration groups : 1 h before ct treatment , concomitantly with ct treatment , 1 h after ct treatment , and 3 h after ct treatment . all the experiments were conducted under controlled conditions according to the guide to the care and use of experimental animals and the university of south florida institutional animal care and use committee ( iacuc ) . the mice were sacrificed after 24 h and blood samples to determine alanine transaminase ( alt ) activity were withdrawn by cardiac puncture . livers were isolated , perfused with normal saline , and dissected for ( 1 ) histopathology and ( 2 ) for determination of total gsh , thiobarbituric acid - reactive substances ( tbars ) , sod activity , and carbonyl content . the colorimetric assay for parp activity was performed in 96-well plates ( trevigen , inc . , gaithersburg , md ) according to manufacturer 's protocol . serial dilutions of the parp enzyme were distributed into wells to generate a standard curve . the clarified tissue homogenates ( 10 l / well ) were added to triplicate wells to determine cellular parp activity . the plate was washed 4 times with 1 phosphate - buffered saline ( pbs ) and then incubated for 20 min with 50 l / well streptavidin horseradish peroxidase ( strep hrp ) diluted 1:500 in 1 strep - diluent ( trevigen ) . the plate was washed 4 times with 1 pbs prior to the addition of the hrp substrate . for colorimetric readout , 50 l of tacs - sapphire ( trevigen ) was added to each well and incubated in the dark , at room temperature , for 15 min . development of the colorimetric reaction was stopped by the addition of an equal volume of 0.2 m hcl . the results were expressed as units of parp activity calculated per milligram of protein and normalized to equal concentrations of protein . protein determination in all assays described here were determined by the bicinchoninic acid assay using bovine serum albumin as standard ( sigma ) . serum ( 0.2 ml ) was obtained by centrifugation of cardiac puncture blood samples at 4000rpm for 15 min . each serum sample was then transferred to a new sterile microcentrifuge tube and stored at 4c until time of assay . alt activity , expressed as iu / l , was determined by a previously described method using a commercially prepared reagent kit from teco diagnostics ( anaheim , ca ) . individual liver samples ( 100 mg ) were homogenized in 1 ml of 0.2 m pbs ( ph 8.0 ) , and the mixture was centrifuged at 12,000 rpm for 30 min . the supernatant ( 0.5 ml ) was mixed with 0.5 ml of 4% sulfosalicylic acid , allowed to stand for 5 min at 4c , and centrifuged again at 3000 rpm for 10 min . the supernatant obtained ( 0.5 ml ) was mixed with 2 ml of 0.2 m pbs ( ph 8.0 ) and 10 l of 10 mm 5,5-dithiobis-2-nitrobenzoic acid . the absorbance was measured at 412 nm and concentration of gsh was expressed as nmol / mg protein . formation of lipid peroxide derivatives was evaluated by measuring tbars according to a method previously described by cascio et al . liver samples were individually homogenized in ice - cold 1.15% kcl ( w / v ) . homogenates ( 0.4 ml ) were mixed with 1 ml of 0.375% tba , 15% trichloroacetic acid ( tca ) ( w / v ) , 0.25 n hcl , and 6.8 mm butylated - hydroxytoluene , placed in a boiling water bath for 10 min , removed and allowed to cool on ice . following centrifugation at 3000 rpm for 10 min , the absorbance in the supernatants was measured at 532 nm . the amount of tbars produced was expressed as nmol tbars / mg protein using malondialdehyde bis(dimethylacetal ) for calibration . determination of sod activity in mouse liver was based on inhibition of nitrite formation in the reaction of oxidation of hydroxylammonium with superoxide anion radical . the total sod was determined in one aliquot and cytosolic sod was determined in the second aliquot supplemented with 10 mm kcn . the concentration of mitochondrial sod was calculated as the difference between the amount of total and cytosolic fraction of sod . nitrite formation was generated in a mixture that contained 25ml xanthine ( 15 mm ) , 25 ml hydroxylammonium chloride ( 10 mm ) , 250 ml pbs ( 65 mm , ph 7.8 ) , 90 ml distilled water , and 100 ml xanthine oxidase ( 0.1 u / ml ) to initiate the reaction . the inhibitory effect of the extant sod was assayed at 25c after 20 min of incubation with 10 ml of liver tissue extracts . the determination of the resulting nitrite was performed following the reaction ( 20 min at room temperature ) with 0.5 ml sulfanilic acid ( 3.3 mg / ml ) and 0.5 ml -naphthylamine ( 1 mg / ml ) . optical absorbance at 530 nm was measured on ultrospec iii spectrophotometer ( pharmacia lkb , uppsala , sweden ) . the procedure used was similar to that described by levine et al ( 1990 ) with slight modifications . two sets of 250 l sample homogenates were labeled as test and reference . a 1 ml amount of 10 mm 2,4-dinitrophenylhydrazine ( dnph ) prepared in 2.5 m hcl was added to test samples and 2.5 m hcl alone was added to the reference . the contents were mixed and incubated in the dark for 1 h. then 1 ml of 20% tca was added to each tube . the tubes were centrifuged at 3500 rpm for 20 min and protein pellets were washed with 1 ml of 10% tca . the precipitates were washed 3 times with 1 ml of ethyl acetate : ethanol ( 1:1 , v / v ) mixture to remove unreacted dnph and lipids . each pellet was then dissolved in 1 ml of 6 m guanidine hydrochloride , at 37c , for 10 min . carbonyl content was determined with ultrospec iii spectrophotometer ( pharmacia lkb , uppsala , sweden ) . each test sample was read against the corresponding control at 370 nm using an absorption coefficient of 22,000 m cm . liver samples were fixed in 10% neutral buffered formalin and embedded in paraffin following standard procedures . tissue sections , 4 m in thickness , were stained with hematoxylin and eosin ( h and e ) to assess parenchymal histopathologic changes . histopathologic parameters evaluated in the centrilobular region were proliferation , apoptosis , necrosis , and fibrosis using the methodology described previously by price et al . summary descriptive analysis for all cytotoxicity outcome data is reported as the mean levels of biomarkers of cytotoxicity sem . f - test analysis was performed to evaluate the differences between ct dosage groups in the initial treatment experiment , and again for the select parp inhibitor groups in the experiment that held the ct dosage constant for all the groups ( p < 0.05 was considered to indicate a statistically significant difference ) . f - test analysis was then performed for the experiment that evaluated differences in measures of cytotoxicity between the time of treatment with pj-34 relative to ct treatment among groups that held the ct and pj-34 dosage constant for all groups ( p < 0.05 was considered to indicate a statistically significant difference ) . pearson 's correlation coefficients were calculated for the combined data of pj-34 , aba , and aiq to determine the significance and magnitude of correlation between the degree of parp-1 inhibition and measures of cytotoxicity ( p < 0.05 considered to indicate a statistically significant correlation ) . the analysis was performed with sas statistical software package 9.2 , sas institute cary , nc , usa . intraperitoneal injection of ct caused a dose - dependent elevation of alt 24 h after treatment , with a statistically significant elevation at each increasing dose level [ table 1 ] . at a ct dose of 1.2 ml / kg , a statistically significant increase of alt was evident at doses of ct as low as 0.3 ml / kg , suggesting a significantly damaging effect to hepatocytes . hepatic gsh levels were depleted in treated mice , and sod levels ( both cytosolic and total ) were elevated in response to ct treatment . ct treatment initiated substantial dose - dependent activation of the parp-1 enzyme as indicated by a 2.4-fold increase at a ct dose of 1.2 ml / kg [ table 1 ] . histologic examination of the livers from ct - treated mice revealed severe hepatocyte necrosis in the centrilobular area with an influx of inflammatory cells 24 h after ct treatment [ figure 1 ] . summary of cytotoxic indicators affected by select doses of ct ( a ) liver histopathology ( 100 ) following carbon tetrachloride ( ct ) treatment ( 0.6 ml / kg ) . areas of necrosis are recognized by the reddish color of hemorrhage associated with lack of tissue avidity for dyes for hematoxylin due to breakdown of cellular morphology and tissue organization . ( b ) liver histopathology ( 100 ) sample from experimental group treated with n-(6-oxo-5,6-dihydro - phenanthridin- 2-yl)-n , n - dimethylacetamide hcl ( 3 mg / kg ) 1 h before ct treatment ( 0.6 ml / kg ) . three water - soluble parp-1 inhibitors , namely , 3-aminobenzamide ( aba ) , 5-aminoisoquinolinone ( aiq ) , and n-(6-oxo-5,6-dihydro - phenanthridin-2-yl)-n , n - dimethylacetamide hcl ( pj-34 ) were tested to determine their efficacy in attenuating ct - induced hepatotoxicity [ table 2 ] . the greatest reduction of alt was produced by pj-34 at a dose of 3 mg / kg , which resulted in a 2.9-fold decrease in the alt levels compared with the ct - treated group . pj-34 decreased mda concentrations and reduced gsh depletion with greater efficacy compared with aba or aiq ; pj-34 also demonstrated the greatest inhibition of parp-1 activity . indicators of hepatotoxicity and parp activity for parp-1 inhibitor treatment groups and treatment groups to evaluate the effect of differences in potency of aba , aiq , and pj-34 as parp-1 inhibitors and the correlation of the degree of parp-1 inhibition to measures of cytotoxicity , the combined data of aba , aiq , and pj-34 were analyzed to produce pearson 's correlation coefficients . alt was strongly correlated to parp-1 activity ( r = 0.745 , p < 0.001 ) , and a moderate , inverse correlation was observed for gsh levels ( r = 0.522 , p = 0.007 ) . the strongest correlation observed between the degree of parp-1 activity and cytotoxicity was found with mda ( r = 0.840 , p < 0.001 ) . as pj-34 provided improved inhibition of parp and protection against mda at statistically significant levels over aiq and aba , pj-34 was chosen to be used in the time of treatment experiments [ table 2 ] . the time of treatment with pj-34 relative to ct administration was evaluated with the following treatment schedule : 1 h before ct ( ct+pj(1 ) ) ; concomitantly with ct ( ct+pj(0 ) ) ; 1 h after ct ( ct+pj(+1 ) ) ; and 3 h after ct ( ct+pj(+3 ) ) . the attenuation of ct - induced hepatotoxicity was demonstrated by pj-34 treatment at 1 h before , concomitantly , and 1 h after ct treatment as indicated by alt . these treatments reduced alt elevation by more than 50% compared with mice receiving ct treatment alone . the protective effect was not observed with pj-34 treatment 3 h after ct administration [ figure 2 ] . mean alanine transaminase levels at select times following n-(6-oxo- 5,6-dihydro - phenanthridin-2-yl)-n , n - dimethylacetamide hcl ( pj-34 ) treatment of 3 mg / kg and carbon tetrachloride ( ct ) treatment 0.6 ml / kg : ct+pj(1 ) : 1 h before ct administration ; ct+pj(0 ) : concomitant administration with ct ; ct+pj(+1 ) : 1 h after ct administration ; ct+pj(+3 ) : and 3 h after ct administration . ( fnx01indicates a significant difference from ct alone by f - test , p < 0.05 ) substantial protection against mda was conferred from pj-34 treatment at 1 h before and concomitantly with ct treatment at statistically significant levels compared with mice treated with ct alone [ figure 3 ] . f - test analysis indicates that mda levels were not statistically significantly different from untreated mice compared with mice pretreated with pj-34 1 h before ct treatment ( p = 0.332 ) and mice treated with pj-34 and ct concomitantly ( p = 0.168 ) . mean lipid peroxidation levels following n-(6-oxo-5,6-dihydrophenanthridin-2-yl)-n , n - dimethylacetamide hcl ( pj-34 ) treatment of 3 mg / kg and ct treatment 0.6 ml / kg : ct + pj(1 ) : 1 h before ct administration ; ct+pj(0 ) : concomitant administration with ct ; ct+pj(+1 ) : 1 h after ct administration ; and ct+pj(+3 ) : 3 h after ct administration . ( indicates a significant difference from ct alone by f - test , p < 0.05 ) treatment with pj-34 1 h prior to ct treatment and concomitantly with ct treatment displayed the greatest protective effect against gsh depletion , with both treatment groups retaining levels not significantly different than untreated mice ( p = 0.275 and p = 0.198 , respectively ) . a statistically significant reduction in gsh depletion was observed for all treatment time intervals compared with mice treated with ct alone [ figure 4 ] . mean glutathione levels following n-(6-oxo-5,6-dihydro - phenanthridin-2-yl)-n , n - dimethylacetamide hcl ( pj-34 ) treatment of 3 mg / kg and carbon tetrachloride ( ct ) treatment 0.6 ml / kg : ct + pj(1 ) : 1 h before ct administration ; ct+pj(0 ) : concomitant administration with ct ; ct+pj(+1 ) : 1 h after ct administration ; and ct+pj(+3 ) : 3 h after ct administration . ( indicates a significant difference from ct alone by f - test , p < 0.05 ) an observable component of cellular toxicity resulting from ct treatment is the disruption of both lipids and protein complexes , indicated by the observed increase of carbonyl content in hepatocytes . treatment with pj-34 at 1 h before , concomitantly with ct treatment , and 1 h after ct treatment resulted in statistically significant lower carbonyl levels compared with ct treatment alone [ figure 5 ] . the protective effect conferred to these groups kept carbonyl content to levels that did not differ significantly from untreated mice ( p = 0.520 , p = 0.932 , and p = 0.439 , respectively ) . however , treatment with pj-34 did not result in any statistically significant reduction in total , cytosolic , or mitochondrial sod activity ( data not shown ) . mean carbonyl levels following n-(6-oxo-5,6-dihydro - phenanthridin-2-yl)-n , n - dimethylacetamide hcl ( pj-34 ) treatment of 3 mg / kg and carbon tetrachloride ( ct ) treatment 0.6 ml / kg : ct + pj(1 ) : 1 h before ct administration ; ct+pj(0 ) : concomitant administration with ct ; ct+pj(+1 ) : 1 h after ct administration ; and ct+pj(+3 ) : 3 h after ct administration . ( indicates a significant difference from ct alone by f - test , p < 0.05 ) necrosis and apoptosis in the centrilobular regions of the liver are consistent with ct - induced hepatotoxicity . a representative sample from the ct treatment group and the group that received pj-34 1 h before ct treatment are presented for comparison in figure 1 . no necrosis or significant presence of inflammatory cells were observed in centrilobular hepatic tissues from the treatment group that received pj-34 1 h prior to ct treatment , while substantial necrosis and inflammatory cell influx were observed in centrilobular hepatic tissue from mice treated with ct only . the protective effect observed in histopathology specimens is consistent with the protective effect observed in the biochemical indicators of cytotoxicity reported above from pj-34 treatment 1 h prior to ct treatment . in previous investigations , parp-1 activity has been shown to mediate necrosis in centrilobular hepatocytes after treatment with select hepatotoxicants , which is consistent with the findings of the current investigation . under normal cellular conditions when activated in response to oxidative stress , however , parp-1 initiates an excessive transference of adp - ribose units from nad to various nuclear proteins , including histones , several chromatin - binding proteins , and parp-1 itself . the consequence of this response is a rapid depletion of intracellular nad , which prevents atp synthesis and ultimately causes cellular apoptosis or necrosis . excessive parp-1 activation is clearly an important mechanism of hepatic tissue damage under conditions of increased oxidative stress , including chemical - induced toxicity , such as excessive ct exposure . several specific and nonspecific parp inhibitors have been evaluated for their efficacy in blocking cellular necrosis and their ability to attenuate ct - induced hepatotoxicity.[3234 ] most commercially available parp inhibitors ( eg , 3-aminobenzamide or nicotinamide ) have short cellular residence time and low potency in blocking ct - induced hepatotoxicity . it has been demonstrated that specific parp-1 inhibitors , such as 6(5h)-phenanthridinone , may have a greater potency in attenuating ct - induced hepatotoxicity both in animal models and human hepg2 cell models . however , 6(5h)-phenanthridinone is water insoluble , requiring the use of organic solvents , such as dmso , as vehicles for administration . although the use of dmso as a vehicle for parp-1 inhibitors may decrease hepatotoxicity from ct treatment , it has also been shown to increase the acute lethality from ct treatment . as well , several studies have shown that dmso can alter the activity of various isoforms of cytochrome p450 ( cyp ) , including cyp2e1 , the isoform that bioactivates ct to the trichloromethyl radical in human hepatocytes . the use of dmso potentially confounds the effects of water - insoluble parp inhibitors and increases the difficulty in characterizing bioavailability , dose response , and adverse effects . the use of water - soluble parp inhibitors eliminates the experimental issues associated with dmso , providing more effective and transparent results . the current investigation evaluated the efficacy of 3 novel , water - soluble parp-1 inhibitors : the phenanthridinone - based pj-34 , aba , and qia in blocking or attenuating ct - induced hepatotoxicity and cellular necrosis . the results demonstrate that ct substantively increases parp-1 activity in the centrilobular hepatic tissue of icr mice during ct - induced hepatocellular necrosis . the measures of hepatocellular toxicity resulting from ct treatment , such as alt , mda , gsh depletion , and carbonyl content , were all significantly reduced with pj-34 treatment , resulting in the preservation of hepatic tissue as observed through histopathology . ct - induced hepatotoxicity is mediated through a cascade of events , beginning with the biotransformation and bioactivation of ct . an alternative explanation for the protective effect observed from treatment with parp-1 inhibitors against ct - induced hepatotoxicity is that the inhibitors may alter ct metabolism and bioactivation . however , the water - soluble nature of the 3 parp-1 inhibitors tested here makes interaction with a mixed function oxidase metabolism unlikely . also , correlation coefficients demonstrated that measures of hepatocellular toxicity were strongly correlated to parp activity , despite unaffected levels of intracellular free radical activity as indicated by a lack of statistically significant reduction in total , cytosolic , or mitochondrial sod activity . if a significant alteration of metabolism was occurring as a result of parp inhibitor treatment , a substantive difference in free radical production ( and the resulting sod depletion ) would be expected . these results indicate that excessive parp-1 induction is an important , pathologically relevant , event in the cascade of events leading to cellular necrosis from ct treatment , and that reduced parp-1 activity is directly correlated with reduced hepatocellular toxicity . the current investigation provides evidence that select water - soluble parp-1 inhibitors confer potent hepatoprotective effects from the hepatocellular toxicity induced by ct treatment through reduced parp-1 activation and the resultant preservation of cellular atp . future research should be conducted examining other models of chemically induced hepatotoxicity and the effect of water - soluble parp-1 inhibitors to confirm the mechanistic role of excessive parp-1 induction in chemically induced hepatocellular necrosis .

background : inhibitors of the nuclear enzyme poly ( adp - ribose ) polymerase ( parp-1 ) have been demonstrated to attenuate pathophysiologic conditions associated with oxidative stress , specifically with carbon tetrachloride ( ct)-induced hepatotoxicity.settings and design : in this investigation , we evaluated 3 previously untested water - soluble parp-1 inhibitors , namely , 3-aminobenzamide ( aba ) , 5-aminoisoquinolinone ( aiq ) , and n-(6-oxo-5,6-dihydro - phenanthridin-2-yl)-n , n - dimethylacetamide hcl ( pj-34 ) to determine their efficacy in blocking or attenuating ct - induced hepatotoxicity in male imprinting control region ( icr ) mice.statistical analysis : indicators of hepatotoxicity were compared with f - tests among groups to determine statistically significant effects . pearson 's correlation coefficients were used to evaluate the correlation between parp inhibition and the attenuation of hepatotoxicity.results and conclusions : ct treatment resulted in hepatic cytotoxicity , increased serum transaminase ( alt ) , lipid peroxidation ( mda ) , intracellular glutathione ( gsh ) depletion , increased carbonyl content , and substantially increased parp-1 activity . ct treatment also produced profound observable hemorrhagic necrosis in the hepatic centrilobular region of icr mice . pretreatment with pj-34 , aba , and aiq before ct treatment significantly decreased parp-1 activity in hepatocytes after ct treatment by 3.4 , 2.0 , and 1.9 times , respectively . corresponding to this reduction in parp-1 activity , a significant reduction in the alt levels and mda and a reduction in the gsh depletion were observed . also , there were no visible tissue defects in the liver samples from animals pretreated with individual parp-1 inhibitors before ct administration . these results demonstrate the efficacy of the 3 previously untested water - soluble parp-1 inhibitors in attenuating ct - induced hepatocellular toxicity and further characterize the role of parp-1 activation and oxidative stress among the cascade of events in hepatocellular necrosis induced by ct treatment .

INTRODUCTION MATERIALS AND METHODS None Materials Animals and treatment Determination of PARP activity Alanine transaminase activity Glutathione assay Lipid peroxidation assay Superoxide dismutase assay Determination of protein carbonyl content Histopathology Statistical analysis RESULTS DISCUSSION CONCLUSION

the mechanism of action of carbon tetrachloride ( ct)-induced hepatotoxicity has been linked to the metabolism of ct into the trichloromethyl radical and the trichloromethyl peroxy radical , and their subsequent reactions with lipids and proteins in the cell membrane , resulting in mda , where the rate of mda is mediated by reactive oxygen species , such as superoxide radicals and hydroxyl radicals. [112 ] also , it has been suggested that ct - induced hepatotoxicity also involves the disruption of hepatic antioxidant defense systems by the depletion of reduced glutathione ( gsh ) , superoxide dismutase ( sod ) , and catalase . although a number of antioxidants have been evaluated for preventing ct - induced hepatotoxicity , efficacy has not been demonstrated . recent findings indicate that free radicals produced by ct lead to mitochondrial damage and nuclear dna damage , resulting in the activation of their respective repair mechanisms , including induction of the enzyme poly(adp - ribose ) polymerase 1 ( parp-1 ) . parp-1 represents the most abundant isoform of the poly ( adp - ribose ) polymerase family , which responds to single- and double - strand nicks in nuclear dna. [1418 ] upon binding to damaged dna , parp-1 forms homodimers and catalyzes the cleavage of nad into nicotinamide and adp - ribose to form long branches of adp - ribose polymers on glutamic acid residues of a number of target proteins , including histones and the parp enzyme itself . previously , we have demonstrated the hepatoprotective effect against ct - induced centrilobular necrosis with concomitant treatment of the parp inhibitor 6(5h)-phenanthridinone . however , due to poor water solubility , 6(5h)-phenanthridinone is required to be dissolved in dimethyl sulfoxide ( dmso ) for effective administration , which may modify both effective delivery and bioavailability , potentially altering the attenuation of ct - induced toxicity . recently , specific water - soluble parp-1 inhibitors have become available , and some have been shown to confer protective effects in various models of toxic exposure . this investigation evaluates the ability of 3 water - soluble parp-1 inhibitors , namely , 3-aminobenzamide ( aba ) , 5-aminoisoquinolinone ( aiq ) , and n-(6-oxo-5,6-dihydro - phenanthridin-2-yl)-n , n - dimethylacetamide hcl ( pj-34 ) to block or attenuate centrilobular hepatotoxicity from ct treatment in imprinting control region ( icr ) mice . a time - dependent experiment followed , using 0.6 ml / kg pj-34 in the following administration groups : 1 h before ct treatment , concomitantly with ct treatment , 1 h after ct treatment , and 3 h after ct treatment . the mice were sacrificed after 24 h and blood samples to determine alanine transaminase ( alt ) activity were withdrawn by cardiac puncture . livers were isolated , perfused with normal saline , and dissected for ( 1 ) histopathology and ( 2 ) for determination of total gsh , thiobarbituric acid - reactive substances ( tbars ) , sod activity , and carbonyl content . individual liver samples ( 100 mg ) were homogenized in 1 ml of 0.2 m pbs ( ph 8.0 ) , and the mixture was centrifuged at 12,000 rpm for 30 min . histopathologic parameters evaluated in the centrilobular region were proliferation , apoptosis , necrosis , and fibrosis using the methodology described previously by price et al . f - test analysis was performed to evaluate the differences between ct dosage groups in the initial treatment experiment , and again for the select parp inhibitor groups in the experiment that held the ct dosage constant for all the groups ( p < 0.05 was considered to indicate a statistically significant difference ) . f - test analysis was then performed for the experiment that evaluated differences in measures of cytotoxicity between the time of treatment with pj-34 relative to ct treatment among groups that held the ct and pj-34 dosage constant for all groups ( p < 0.05 was considered to indicate a statistically significant difference ) . pearson 's correlation coefficients were calculated for the combined data of pj-34 , aba , and aiq to determine the significance and magnitude of correlation between the degree of parp-1 inhibition and measures of cytotoxicity ( p < 0.05 considered to indicate a statistically significant correlation ) . a time - dependent experiment followed , using 0.6 ml / kg pj-34 in the following administration groups : 1 h before ct treatment , concomitantly with ct treatment , 1 h after ct treatment , and 3 h after ct treatment . the mice were sacrificed after 24 h and blood samples to determine alanine transaminase ( alt ) activity were withdrawn by cardiac puncture . livers were isolated , perfused with normal saline , and dissected for ( 1 ) histopathology and ( 2 ) for determination of total gsh , thiobarbituric acid - reactive substances ( tbars ) , sod activity , and carbonyl content . individual liver samples ( 100 mg ) were homogenized in 1 ml of 0.2 m pbs ( ph 8.0 ) , and the mixture was centrifuged at 12,000 rpm for 30 min . histopathologic parameters evaluated in the centrilobular region were proliferation , apoptosis , necrosis , and fibrosis using the methodology described previously by price et al . f - test analysis was performed to evaluate the differences between ct dosage groups in the initial treatment experiment , and again for the select parp inhibitor groups in the experiment that held the ct dosage constant for all the groups ( p < 0.05 was considered to indicate a statistically significant difference ) . f - test analysis was then performed for the experiment that evaluated differences in measures of cytotoxicity between the time of treatment with pj-34 relative to ct treatment among groups that held the ct and pj-34 dosage constant for all groups ( p < 0.05 was considered to indicate a statistically significant difference ) . pearson 's correlation coefficients were calculated for the combined data of pj-34 , aba , and aiq to determine the significance and magnitude of correlation between the degree of parp-1 inhibition and measures of cytotoxicity ( p < 0.05 considered to indicate a statistically significant correlation ) . a time - dependent experiment followed , using 0.6 ml / kg pj-34 in the following administration groups : 1 h before ct treatment , concomitantly with ct treatment , 1 h after ct treatment , and 3 h after ct treatment . the mice were sacrificed after 24 h and blood samples to determine alanine transaminase ( alt ) activity were withdrawn by cardiac puncture . livers were isolated , perfused with normal saline , and dissected for ( 1 ) histopathology and ( 2 ) for determination of total gsh , thiobarbituric acid - reactive substances ( tbars ) , sod activity , and carbonyl content . individual liver samples ( 100 mg ) were homogenized in 1 ml of 0.2 m pbs ( ph 8.0 ) , and the mixture was centrifuged at 12,000 rpm for 30 min . f - test analysis was performed to evaluate the differences between ct dosage groups in the initial treatment experiment , and again for the select parp inhibitor groups in the experiment that held the ct dosage constant for all the groups ( p < 0.05 was considered to indicate a statistically significant difference ) . f - test analysis was then performed for the experiment that evaluated differences in measures of cytotoxicity between the time of treatment with pj-34 relative to ct treatment among groups that held the ct and pj-34 dosage constant for all groups ( p < 0.05 was considered to indicate a statistically significant difference ) . pearson 's correlation coefficients were calculated for the combined data of pj-34 , aba , and aiq to determine the significance and magnitude of correlation between the degree of parp-1 inhibition and measures of cytotoxicity ( p < 0.05 considered to indicate a statistically significant correlation ) . histologic examination of the livers from ct - treated mice revealed severe hepatocyte necrosis in the centrilobular area with an influx of inflammatory cells 24 h after ct treatment [ figure 1 ] . ( b ) liver histopathology ( 100 ) sample from experimental group treated with n-(6-oxo-5,6-dihydro - phenanthridin- 2-yl)-n , n - dimethylacetamide hcl ( 3 mg / kg ) 1 h before ct treatment ( 0.6 ml / kg ) . three water - soluble parp-1 inhibitors , namely , 3-aminobenzamide ( aba ) , 5-aminoisoquinolinone ( aiq ) , and n-(6-oxo-5,6-dihydro - phenanthridin-2-yl)-n , n - dimethylacetamide hcl ( pj-34 ) were tested to determine their efficacy in attenuating ct - induced hepatotoxicity [ table 2 ] . the greatest reduction of alt was produced by pj-34 at a dose of 3 mg / kg , which resulted in a 2.9-fold decrease in the alt levels compared with the ct - treated group . pj-34 decreased mda concentrations and reduced gsh depletion with greater efficacy compared with aba or aiq ; pj-34 also demonstrated the greatest inhibition of parp-1 activity . indicators of hepatotoxicity and parp activity for parp-1 inhibitor treatment groups and treatment groups to evaluate the effect of differences in potency of aba , aiq , and pj-34 as parp-1 inhibitors and the correlation of the degree of parp-1 inhibition to measures of cytotoxicity , the combined data of aba , aiq , and pj-34 were analyzed to produce pearson 's correlation coefficients . alt was strongly correlated to parp-1 activity ( r = 0.745 , p < 0.001 ) , and a moderate , inverse correlation was observed for gsh levels ( r = 0.522 , p = 0.007 ) . the time of treatment with pj-34 relative to ct administration was evaluated with the following treatment schedule : 1 h before ct ( ct+pj(1 ) ) ; concomitantly with ct ( ct+pj(0 ) ) ; 1 h after ct ( ct+pj(+1 ) ) ; and 3 h after ct ( ct+pj(+3 ) ) . the attenuation of ct - induced hepatotoxicity was demonstrated by pj-34 treatment at 1 h before , concomitantly , and 1 h after ct treatment as indicated by alt . the protective effect was not observed with pj-34 treatment 3 h after ct administration [ figure 2 ] . mean alanine transaminase levels at select times following n-(6-oxo- 5,6-dihydro - phenanthridin-2-yl)-n , n - dimethylacetamide hcl ( pj-34 ) treatment of 3 mg / kg and carbon tetrachloride ( ct ) treatment 0.6 ml / kg : ct+pj(1 ) : 1 h before ct administration ; ct+pj(0 ) : concomitant administration with ct ; ct+pj(+1 ) : 1 h after ct administration ; ct+pj(+3 ) : and 3 h after ct administration . ( fnx01indicates a significant difference from ct alone by f - test , p < 0.05 ) substantial protection against mda was conferred from pj-34 treatment at 1 h before and concomitantly with ct treatment at statistically significant levels compared with mice treated with ct alone [ figure 3 ] . f - test analysis indicates that mda levels were not statistically significantly different from untreated mice compared with mice pretreated with pj-34 1 h before ct treatment ( p = 0.332 ) and mice treated with pj-34 and ct concomitantly ( p = 0.168 ) . mean lipid peroxidation levels following n-(6-oxo-5,6-dihydrophenanthridin-2-yl)-n , n - dimethylacetamide hcl ( pj-34 ) treatment of 3 mg / kg and ct treatment 0.6 ml / kg : ct + pj(1 ) : 1 h before ct administration ; ct+pj(0 ) : concomitant administration with ct ; ct+pj(+1 ) : 1 h after ct administration ; and ct+pj(+3 ) : 3 h after ct administration . ( indicates a significant difference from ct alone by f - test , p < 0.05 ) treatment with pj-34 1 h prior to ct treatment and concomitantly with ct treatment displayed the greatest protective effect against gsh depletion , with both treatment groups retaining levels not significantly different than untreated mice ( p = 0.275 and p = 0.198 , respectively ) . a statistically significant reduction in gsh depletion was observed for all treatment time intervals compared with mice treated with ct alone [ figure 4 ] . mean glutathione levels following n-(6-oxo-5,6-dihydro - phenanthridin-2-yl)-n , n - dimethylacetamide hcl ( pj-34 ) treatment of 3 mg / kg and carbon tetrachloride ( ct ) treatment 0.6 ml / kg : ct + pj(1 ) : 1 h before ct administration ; ct+pj(0 ) : concomitant administration with ct ; ct+pj(+1 ) : 1 h after ct administration ; and ct+pj(+3 ) : 3 h after ct administration . ( indicates a significant difference from ct alone by f - test , p < 0.05 ) an observable component of cellular toxicity resulting from ct treatment is the disruption of both lipids and protein complexes , indicated by the observed increase of carbonyl content in hepatocytes . treatment with pj-34 at 1 h before , concomitantly with ct treatment , and 1 h after ct treatment resulted in statistically significant lower carbonyl levels compared with ct treatment alone [ figure 5 ] . however , treatment with pj-34 did not result in any statistically significant reduction in total , cytosolic , or mitochondrial sod activity ( data not shown ) . mean carbonyl levels following n-(6-oxo-5,6-dihydro - phenanthridin-2-yl)-n , n - dimethylacetamide hcl ( pj-34 ) treatment of 3 mg / kg and carbon tetrachloride ( ct ) treatment 0.6 ml / kg : ct + pj(1 ) : 1 h before ct administration ; ct+pj(0 ) : concomitant administration with ct ; ct+pj(+1 ) : 1 h after ct administration ; and ct+pj(+3 ) : 3 h after ct administration . ( indicates a significant difference from ct alone by f - test , p < 0.05 ) necrosis and apoptosis in the centrilobular regions of the liver are consistent with ct - induced hepatotoxicity . in previous investigations , parp-1 activity has been shown to mediate necrosis in centrilobular hepatocytes after treatment with select hepatotoxicants , which is consistent with the findings of the current investigation . under normal cellular conditions when activated in response to oxidative stress , however , parp-1 initiates an excessive transference of adp - ribose units from nad to various nuclear proteins , including histones , several chromatin - binding proteins , and parp-1 itself . excessive parp-1 activation is clearly an important mechanism of hepatic tissue damage under conditions of increased oxidative stress , including chemical - induced toxicity , such as excessive ct exposure . several specific and nonspecific parp inhibitors have been evaluated for their efficacy in blocking cellular necrosis and their ability to attenuate ct - induced hepatotoxicity. [3234 ] most commercially available parp inhibitors ( eg , 3-aminobenzamide or nicotinamide ) have short cellular residence time and low potency in blocking ct - induced hepatotoxicity . it has been demonstrated that specific parp-1 inhibitors , such as 6(5h)-phenanthridinone , may have a greater potency in attenuating ct - induced hepatotoxicity both in animal models and human hepg2 cell models . the use of water - soluble parp inhibitors eliminates the experimental issues associated with dmso , providing more effective and transparent results . the current investigation evaluated the efficacy of 3 novel , water - soluble parp-1 inhibitors : the phenanthridinone - based pj-34 , aba , and qia in blocking or attenuating ct - induced hepatotoxicity and cellular necrosis . the results demonstrate that ct substantively increases parp-1 activity in the centrilobular hepatic tissue of icr mice during ct - induced hepatocellular necrosis . the measures of hepatocellular toxicity resulting from ct treatment , such as alt , mda , gsh depletion , and carbonyl content , were all significantly reduced with pj-34 treatment , resulting in the preservation of hepatic tissue as observed through histopathology . ct - induced hepatotoxicity is mediated through a cascade of events , beginning with the biotransformation and bioactivation of ct . an alternative explanation for the protective effect observed from treatment with parp-1 inhibitors against ct - induced hepatotoxicity is that the inhibitors may alter ct metabolism and bioactivation . however , the water - soluble nature of the 3 parp-1 inhibitors tested here makes interaction with a mixed function oxidase metabolism unlikely . also , correlation coefficients demonstrated that measures of hepatocellular toxicity were strongly correlated to parp activity , despite unaffected levels of intracellular free radical activity as indicated by a lack of statistically significant reduction in total , cytosolic , or mitochondrial sod activity . if a significant alteration of metabolism was occurring as a result of parp inhibitor treatment , a substantive difference in free radical production ( and the resulting sod depletion ) would be expected . these results indicate that excessive parp-1 induction is an important , pathologically relevant , event in the cascade of events leading to cellular necrosis from ct treatment , and that reduced parp-1 activity is directly correlated with reduced hepatocellular toxicity . the current investigation provides evidence that select water - soluble parp-1 inhibitors confer potent hepatoprotective effects from the hepatocellular toxicity induced by ct treatment through reduced parp-1 activation and the resultant preservation of cellular atp . future research should be conducted examining other models of chemically induced hepatotoxicity and the effect of water - soluble parp-1 inhibitors to confirm the mechanistic role of excessive parp-1 induction in chemically induced hepatocellular necrosis .